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Studies on the effect of radiation on 3T3 cell motility Thurston, Gavin O. 1988

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STUDIES ON THE EFFECT OF RADIATION ON 3T3 CELL MOTILITY By Gavin Thurston B.Sc, Yale University, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Physics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1988 © G a v i n Thurston, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 •ate r e * > s - f t ( m% DE-6(3/81) ABSTRACT The a b i l i t y of mammalian c e l l s to locomote i s important i n a v a r i e t y of normal and pathological processes. Previous work has suggested that low doses of x - i r r a d i a t i o n may perturb c e l l m o t i l i t y , a f i n d i n g that may have important consequences i n embryogenesis, cancer metastasis, and immune response. This thesis has sought to study i n more d e t a i l the e f f e c t of r a d i a t i o n on mammalian c e l l m o t i l i t y . Work performed i n other laboratories used the c o l l o i d a l gold assay and time lapse cinemicroscopy to study x - i r r a d i a t i o n induced changes to 3T3 f i b r o b l a s t m o t i l i t y i n tissue culture. These studies were repeated here, with q u a l i t a t i v e r e s u l t s s i m i l a r to those reported e a r l i e r . However, these methods were not amenable to a d e t a i l e d quantitative a n a l y s i s . For t h i s , s p a t i a l and temporal information on the m o t i l i t y and dynamic morphology of a large number of c e l l s i s required. Such a task would be impossible to perform manually, thus an automated microscope system was developed that used a computer-driven p r e c i s i o n stage and a s o l i d state o p t i c a l sensor to track i n d i v i d u a l c e l l s i n tis s u e culture. Information on m o t i l i t y and morphology was concurrently extracted from many c e l l s . As part of the thes i s , several techniques were developed to analyze and display these data, and to co r r e l a t e m o t i l i t y and morphology observations. These techniques were di r e c t e d at preserving the actual process of 3T3 c e l l m o t i l i t y , and parameters were measured to quantify the short term persistence of c e l l movement (on a time scale of 0.5 to 2 hours), and the long term persistence of c e l l s i n maintaining c e r t a i n c h a r a c t e r i s t i c behaviour (on a time scale of 3 to 12 hours). The response of 3T3 f i b r o b l a s t s to x - i r r a d i a t i o n was characterized by a number of parameters. The population average c e l l speed was measured following treatment, and a dose response and time response was determined i n the range of 1.5 Gy. Other m o t i l i t y parameters indicate that the normal process of c e l l m o t i l i t y , evidenced by a seri e s of motile segments, was disrupted by x-rays. This was thought to r e f l e c t perturbation to the co n t r o l mechanisms of c e l l m o t i l i t y . The morphology of 3T3 c e l l s stained with Coomassie blue was examined i n an e f f o r t to c o r r e l a t e the observed m o t i l i t y changes with changes i n the f i x e d c e l l morphology. This s t a i n i s a general s t r u c t u r a l p r o t e i n s t a i n with higher a f f i n i t y toward microfilaments. High doses of x-rays were required to produce s i g n i f i c a n t perturbation to c e l l morphology, and i n the dose regime of i n t e r e s t , the morphology of i r r a d i a t e d c e l l s was not i d e n t i f i a b l y d i f f e r e n t from c o n t r o l . Of note i s that i t was the well spread, quiescent c e l l s that seemed l e a s t perturbed by large doses of i r r a d i a t i o n . In summary, x-rays apparently disrupt the normal process of c e l l m o t i l i t y . Several l i n e s of evidence suggest that a c t i v e l y migrating c e l l s are the most perturbed by i r r a d i a t i o n . This work has developed techniques to quantify c e l l m o t i l i t y i n a meaningful way, and to characterize the x-ray induced perturbations. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v ABBREVIATIONS v i i i DEFINITION OF TERMS ix LIST OF FIGURES x i 1. INTRODUCTION 1 1.1 Preliminaries 1.2 C e l l M o t i l i t y - General 2 1.2.1 Examples of c e l l m o t i l i t y i n biology 1.2.2 In v i t r o model systems of c e l l m o t i l i t y 1.3 How C e l l s Move 4 1.3.1 Basic m o t i l i t y functions 1.3.2 Structure and function of cytoplasm 1.3.3 C e l l membrane 1.3.4 Phenomenological d e s c r i p t i o n 1.3.5 Biophysical model of c e l l m o t i l i t y 1.3.6 Control of m o t i l i t y 1.4 General Aspects of the Interaction of 14 Ionizing Radiation with L i v i n g Matter 1.5 Radiation as a Perturbation of C e l l M o t i l i t y and C e l l 17 Morphology 1.5.1 In vivo 1.5.2 In v i t r o 1.6 Techniques to Measure C e l l M o t i l i t y and C e l l Morphology 20 V 1.7 Analysis of Dynamic C e l l Behaviour 22 1.7.1 C e l l m o t i l i t y 1.7.2 C e l l morphology 1.8 Thesis Project 26 MATERIALS AND METHODS 27 2.1 C e l l s and Culture 27 2.1.1 C e l l s and growth c h a r a c t e r i s t i c s 2.1.2 Measurement of p r o l i f e r a t i o n rates 2.1.3 C e l l cycle times with FACS 2.2 Gold Dust Assay 33 2.3 C e l l Analyzer and C e l l Tracking 37 2.3.1 General information 2.3.2 C e l l m o t i l i t y measurements 2.3.3 Measuring c e l l morphology 2.3.4 Choosing RSCAN parameters 2.3.5 Program control experiments 2.3.6 Display and manipulation of m o t i l i t y data 2.4 Stained C e l l s 56 2.5 Dosimetry 57 RESULTS 60 3.1 Gold Dust Assay 60 3.1.1 Control c e l l s 3.1.2 C e l l s i r r a d i a t e d before p l a t i n g 3.1.3 C e l l s i r r a d i a t e d i n the p e t r i e dish 3.2 Time Lapse Records 72 3.2.1 Control c e l l s 3.2.1.a Low magnification and long time span v i 3.2.1. b High magnification and short time scale 3.2.2 Irr a d i a t e d c e l l s 3.2.2. a Low magnification and long time span 3.2.2.a High magnification and short time scale 3.3 Automated Measurements of M o t i l i t y and Morphology 89 3.3.1 Control c e l l s : m o t i l i t y 3.3.1.a Individual c e l l s 3.3.1. b Populations of c e l l s 3.3.2 Control c e l l s : morphology 3.3.2. a Individual c e l l s 3.3.2. D Populations of c e l l s 3.3.2.C M o t i l i t y and morphology 3.3.3 Irr a d i a t e d c e l l s : m o t i l i t y and morphology 3.3.3. a Individual c e l l s 3.3.3.b Populations of c e l l s 3.4 Stained C e l l s 135 3.4.1 Control c e l l s 3.4.2 Ir r a d i a t e d c e l l s 4. DISCUSSION 143 4.1 Repeat of Published Experiments 143 4.2 Design of an Automated System f o r C e l l M o t i l i t y 145 4.3 Average Rate of C e l l Movement following I r r a d i a t i o n 145 4.4 Development of C e l l M o t i l i t y Parameters 147 4.5 Further Characterization of X-ray E f f e c t on C e l l M o t i l i t y 148 4.6 Comparison with Published Reports 149 4.7 Mechanisms 150 v i i 4.8 Further Studies 156 5. BIBLIOGRAPHY 159 6. APPENDIX 168 6.1 D e t a i l s on I r r a d i a t i o n and Dosimetry 168 6.1.1 I r r a d i a t i o n procedure 6.1.2 Control procedures f o r Fricke dosimetry 6.1.3 Absorption and dose f or Fricke dosimetry 6.1.4 B i o l o g i c a l dosimetry 6,2 De t a i l s on DMIPS Components 174 6.2.1 CCD o p t i c a l sensor 6.2.2 Microscope stage 6.2.3 Incubator 6.2.4 D i g i t a l s i g n a l processor v i i i LIST OF FIGURES Figure T i t l e Page 1.1 Schematic diagram of a motile f i b r o b l a s t 11 1.2 Time sequence of events following i r r a d i a t i o n of 16 l i v i n g organisms 1.3 D e f i n i t i o n of dynamic morphology features 25 2.1 P r o l i f e r a t i o n rate of 3T3 c e l l s i n various serum 28 concentrations 2.2 Doubling time of 3T3 c e l l s as a function of temperature 29 2.3 Micrograph of 3T3 c e l l s showing c e l l morphology 30 2.4 Micrograph of 3T3 c e l l s at higher density 31 2.5 DNA d i s t r i b u t i o n s of 3T3 c e l l s 34 2.6 Schematic diagram of C e l l Analyzer 38 2.7 Schematic diagram of c e l l l o c a t i n g routine 41 2.8 Flow chart of RSCAN program 42 2.9 Flow chart of c e l l detection routine 44 2.10 Chain code of 3T3 c e l l and morphological 45 features 2.11 A s e r i e s of l i n e scans across a c e l l at d i f f e r e n t 49 focus settings 2.12 A s e r i e s of chain codes of a c e l l at d i f f e r e n t 50 focus settings 2.13 Two walk patterns from a single c e l l 52 2.14 D e f i n i t i o n of various c e l l m o t i l i t y parameters 55 3.1 Gold dust tracks of untreated c e l l s 61,62 3.2 Gold dust tracks of i r r a d i a t e d c e l l s 64-67 3.3 Gold dust tracks of c e l l s i r r a d i a t e d while i n 69-71 p e t r i e dish ix Figure T i t l e Page 3.4 Series of images of motile 3T3 c e l l 75-77 3.5 I l l u s t r a t i o n of f i r s t mode of 3T3 c e l l morphology 79 3.6 I l l u s t r a t i o n of second mode of 3T3 c e l l morphology 81 3.7 Image of an active 3T3 c e l l 85 3.8 Walk patterns of untreated 3T3 c e l l s 91 3.9 C e l l speed vs. scan number f o r untreated i n d i v i d u a l 93 c e l l s 3.10 D i s t r i b u t i o n of c e l l speed for untreated i n d i v i d u a l c e l l s 94 3.11 Displacement analysis for untreated c e l l s 96 3.12 Average c e l l speed vs. scan number f o r untreated c e l l s 98 3.13 D i s t r i b u t i o n of speed for untreated c e l l s 100 3.14 D i s t r i b u t i o n of r e l a t i v e angle f o r untreated c e l l s 101 3.15 Two-dimensional random walk model of untreated c e l l s 102 3.16 Forecast analysis for untreated c e l l s : d i s t r i b u t i o n 104 of displacement 3.17 Forecast analysis for untreated c e l l s : d i s t r i b u t i o n 105 of r e l a t i v e angle 3.18 Forecast analysis f o r untreated c e l l s : increasing 106,107 forecast length 3.19 C e l l area vs. scan number f o r untreated c e l l s 110 3.20 C e l l brightness vs. scan number f o r untreated 111 i n d i v i d u a l c e l l s 3.21 C e l l c i r c u l a r i t y vs. scan number f o r untreated 112 i n d i v i d u a l c e l l s 3.22 C e l l brightness f o r a d i v i d i n g c e l l 113 3.23 Average c e l l morphology features f o r untreated c e l l s 115 3.24 Combined m o t i l i t y and morphology information from an 116 untreated c e l l s 3.25 Walk patterns of i r r a d i a t e d 3T3 c e l l s 118 3.26 Displacement analysis f o r i r r a d i a t e d c e l l s 119 X Figure T i t l e Page 3.27 C e l l area vs. scan number for i r r a d i a t e d i n d i v i d u a l c e l l s 121 3.28 C e l l brightness vs. scan number for i r r a d i a t e d 122 i n d i v i d u a l c e l l s 3.29 C e l l c i r c u l a r i t y vs. scan number f o r i r r a d i a t e d 123 i n d i v i d u a l c e l l s 3.30 Average c e l l speed following i r r a d i a t i o n 125 3.31 D i s t r i b u t i o n of c e l l speed following i r r a d i a t i o n 126 3.32 The change i n average c e l l speed as a function of 127 x-ray dose 3.33 Two-dimensional random walk model of i r r a d i a t e d 128 c e l l s : 2 Gy 3.34 Two-dimensional random walk model of i r r a d i a t e d 129 c e l l s : 8 Gy 3.35 Forecast analysis f o r i r r a d i a t e d c e l l s : d i s t r i b u t i o n 130 of displacement 3.36 Forecast analysis for i r r a d i a t e d c e l l s : increasing 131 forecast length 8 Gy 3.37 Forecast analysis for i r r a d i a t e d c e l l s : increasing 132 forecast length 2 Gy 3.38 Forecast analysis for i r r a d i a t e d c e l l s : d i s t r i b u t i o n 133 of r e l a t i v e angle 8 Gy 3.39 Forecast analysis for i r r a d i a t e d c e l l s : d i s t r i b u t i o n 134 of r e l a t i v e angle 2 Gy 3.40 Average c e l l morphology features for i r r a d i a t e d 136 c e l l s : 8 Gy 3.41 Average c e l l morphology features for i r r a d i a t e d 137 c e l l s : 2 Gy 3.42 Micrograph of control and i r r a d i a t e d c e l l s stained 139-141 with Coomassie blue 4.1 Time response of c e l l movement following i r r a d i a t i o n 146 4.2 Average c e l l speed vs. time following t r y p s i n i z a t i o n , 151 i r r a d i a t i o n and r e p l a t i n g 4.3 Schematic diagram of mechanism of x-ray induced damage 153 x i Figure T i t l e Page 6.1 Absorption vs. i r r a d i a t i o n time for Fricke dosimetry 170 6.2 Percentage of c e l l growth for glass and p l a s t i c 172 x i i ABBREVIATIONS BSA - bovine serum albumin CCD - charge-coupled device CHO - Chinese Hamster ovary DDHgO - doubly d i s t i l l e d water DMEM - Dulbecco's modified Eagle medium DMIPS - Dynamic Microscope Image Processing Scanner DNA - deoxyribo n u c l e i c a c i d DSP - d i g i t a l s i g n a l processor FACS - Fluorescence Activated C e l l Sorter FBS - f e t a l bovine serum Gy - gray-unit of r a d i a t i o n dose (Joule/kilogram) IF - intermediate filaments 1 - l i t r e : ml - m i l l i l i t r e MPM - multiported memory MT - microtubule MTOC - microtubule organizing centre NIH - National I n s t i t u t e of Health OMDR - o p t i c a l memory disk recorder PBS - phosphate buffered s a l i n e 2D - two-dimensional: 3D - three-dimensional urn. - micron UV - u l t r a v i o l e t [ ] - concentration of a chemical DEFINITION OF TERMS actomyosin - the combination of a c t i n and myosin proteins i n a network autoregressive process - a discre t e d i g i t a l process i n which the measured output at time t, z f c, i s r e l a t e d to the output at e a r l i e r times. Thus z t " a i z t - l + a 2 zt-2 + ••• + a n z t . n + s t where |a^|<l and s f c represents a stochastic v a r i a b l e . bit-map - a representation of an image, i n which the i n d i v i d u a l p i x e l s of the o r i g i n a l image have been converted to the values of eit h e r 0 or 1. B l a i r and B l i s s f a c t o r - a global shape parameter defined as A <2*CJ r da> / where A i s the c e l l area, and r i s the distance between the area element da and the c e l l centre. The in t e g r a t i o n i s performed over the c e l l area. The d e f i n i t i o n can be extended to discre t e images ( i . e . - p i x e l s ) . chain code - a compact d e s c r i p t i o n of the border of an object i n an image frame, i n which the s t a r t i n g p i x e l i s given along with the sequence of incremental steps needed to follow the object edge. chemotaxis - increased propensity of a c e l l to locomote toward (or away from) a substance. convexity analysis - an image processing routine i n which the border of an object i s tracked and tested f o r segments i n which the border i s convex, that i s , the object has a protrusion. cytogel - the cytoplasmic substance which i s i n a g e l - l i k e state. f i l o p o d i a - a general name for c e l l u l a r protrusions of various types and si z e s . GO phase, GI phase, G2 phase, M phase, S phase - the designations of c e r t a i n f unctional periods during the l i f e c y c e l of a c e l l , extending from one mitosis to the next. The sequence of progression f o r normal c e l l s following the completion of mitosis i s : G0/G1 phase, S phase, G2 phase, M phase. g e l a t i o n - the conversion of monomers to a cr o s s l i n k e d network of polymers. xiv l a m e l l a , l a m e l l l p o d i a - a f l a t sheet of cytoplasm that extends from a portion of the main body of a c e l l , usually associated with the leading edge. p i n o c y t o s i s - the process of active cellular intake of extracellular f l u i d and membrane material from the c e l l periphery to the cellular interior, via pinocytotic vessicles pinched off from the c e l l membrane. p s u e d o p o d - a thick cytoplasmic extension, chacteristically occurring in certain cells upon c e l l stimulation, and usually associated with the leading edge of the c e l l . r u f f l e - an extension of cytoplasm and membrane that has failed to attach to the substrate and subsequently folds back onto the main surface of the c e l l . It appears dark under phase contrast microscopy, and is usally associated with an active lamella. s o l a t i o n - the conversion of a crosslinked network of polymers (a gel) into monomers. s t a r g r a p h - an image processing description of an object in which the object has decomposed into the main body and a set of extensions from the main body. 1 1. INTRODUCTION 1.1 Preliminaries The second h a l f of the 20th century has seen an enormous expansion i n man's use of i o n i z i n g r a d i a t i o n f o r a v a r i e t y of therapeutic, diagnostic, i n d u s t r i a l and other purposes. There has been a corresponding increase i n concern f o r the e f f e c t s of i o n i z i n g r a d i a t i o n on l i v i n g organisms. A large part of society's i n t e r e s t has focused on the p a r t i c u l a r i n t e r a c t i o n of r a d i a t i o n with l i v i n g organisms which r e s u l t s i n l e t h a l i t y , carcinogenesis, and genetic mutations. I t i s generally believed that these endpoints are manifestations of r a d i a t i o n damage to the c e l l u l a r genetic material, the DNA. These s p e c i f i c e f f e c t s on the organism have been studied i n d e t a i l i n c e l l u l a r systems i n which c e l l i n a c t i v a t i o n , transformation, and mutation are the corresponding endpoints. I t i s often s a i d that the DNA i s the " c r i t i c a l target" f o r i o n i z i n g r a d i a t i o n (1). However, the deposition of energy by an i o n i z i n g ray i s a stochastic process, and thus the en t i r e c e l l u l a r f a b r i c i s i n d i s c r i m i n a t e l y damaged. Lesions to b i o l o g i c a l macromolecules other than DNA must also be processed by the c e l l and may a f f e c t c e l l behaviour and function, i n the short or long term. Radiation induced changes i n other aspects of c e l l behaviour have not been as extensively studied, i n part because there i s a lack of s e n s i t i v e , quantitative assays to measure c e l l behaviour; and also because the ro l e of c e l l behaviour i n c e l l u l a r physiology and m u l t i c e l l u l a r processes i s not well understood. Various reports have suggested that r a d i a t i o n causes a l t e r a t i o n s i n at l e a s t one aspect of c e l l behaviour, c e l l m o t i l i t y (2-5). This i s of concern to r a d i o b i o l o g i s t s because r a d i a t i o n induced changes i n c e l l m o t i l i t y may have important consequences i n embryogenesis, immune response, and metastasis. I t 2 was the intent of t h i s thesis project to examine the e f f e c t of x-rays on c e l l m o t i l i t y i n more d e t a i l . 1.2 C e l l M o t i l i t y - General Many eukaryotic c e l l s have the a b i l i t y to locomote. The process of i n t e r e s t here i s the attachment of a c e l l to a substrate and the active crawling along the substrate, which d i f f e r s from the swimming ac t i o n of sperm or b a c t e r i a . 1.2.1 Examples of c e l l m o t i l i t y i n biology The m o t i l i t y of i n d i v i d u a l c e l l s within a complex m u l t i c e l l u l a r organism i s important i n a number of normal and pathological processes. Embryogenesis provides many s t r i k i n g examples of c e l l m o t i l i t y (6-8). In the process of gastrulation, the primary mesenchyme c e l l s migrate from the vegetal pole to various locations within the b l a s t o c o e l . In higher organisms the d i f f e r e n t i a t i o n of the neural crest involves myriad processes of c e l l m o t i l i t y . C e l l s from the neural crest migrate and eventually form a v a r i e t y of t i s s u e s , including the v i s c e r a l skeleton, the dermis of the face, the walls of the a r t e r i e s , and various connective tissues (9,10). The immune system depends upon the migratory c a p a b i l i t i e s of the myeloid and lymphoid c e l l s . These c e l l s are among the most motile i n the body and are constantly moving and responding to movement s t i m u l i . An immunological stimulus r e s u l t s i n the dir e c t e d aggregation of lymphocytes and macrophages i n the stimulus area (10,11). These c e l l s , unlike most normal adult c e l l s , ate able to migrate across i n t e r - t i s s u a l boundaries. The process of tumour metastasis i s r e l a t e d to the a b i l i t y of cancerous c e l l s to free themselves from movement i n h i b i t i n g signals within an organism. Studies with i n v i t r o and i n vivo systems have i d e n t i f i e d evidence for m o t i l i t y i n the onset of metastasis (12-13b). In the bulk of human tumours, 3 mainly represented by carcinomas, cancer c e l l m o t i l i t y appears only very l a t e i n the process of tumour progression, and the ea r l y stages of invasion may proceed without m o t i l i t y (13,13a,13b). This i s i n contrast to melanomas and hematopoietic malignancy, i n which i n d i v i d u a l cancer c e l l m o t i l i t y appears quite frequently (13,13a,13b). In most normal tissue, the c e l l s are prevented from moving by movement i n h i b i t i n g s i g n a l s . These signals include c e l l - t o - c e l l contact (contact i n h i b i t i o n ) (14), c e l l - c e l l juntions, the absence of growth factors (15,16), and c e r t a i n components of the e x t r a c e l l u l a r matrix (17). Abercrombie went so fa r as to say that the motile c e l l "needs movement f o r growth" (18), thus the same forces that hold a c e l l i n p o s i t i o n may also regulate the c e l l p r o l i f e r a t i o n . For u n i c e l l u l a r metazoan organisms, c e l l locomotion i s necessary f o r pursuing an optimal environment and avoiding a detrimental one. Very l i k e l y , the same set of m o t i l i t y s t i m u l i and mechanisms developed by u n i c e l l u l a r organisms are u t i l i z e d i n some form by sing l e c e l l s i n m u l t i c e l l u l a r organisms. 1.2.2 In v i t r o model systems of c e l l m o t i l i t y The process of c e l l m o t i l i t y i s also observed i n tissue culture. In many instances, the environment suitable f o r c e l l p r o l i f e r a t i o n i s also s u i t a b l e f o r c e l l migration. C e l l s require a su i t a b l e substrate: glass, t i s s u e culture p l a s t i c s , or i n v i t r o e x t r a c e l l u l a r matrix systems enable c e l l s to adhere and migrate. Similar to i n vivo, d i f f e r e n t types of c e l l s move i n tissue culture at d i f f e r e n t rates. Leukocytes, smooth muscle c e l l s , f i b r o b l a s t s and others are observed to move i n tiss u e culture (10,19). Leukocytes are among the most active c e l l i n v i t r o , and move approximately lOx more r a p i d l y than f i b r o b l a s t s (10). 4 Certain processes of i n vivo c e l l m o t i l i t y can be simulated i n v i t r o . The d i r e c t e d elongation of a neural c e l l axon can be promoted by appropriate growth factors and e x t r a c e l l u l a r matrix components (19,20). A process of i n v i t r o wound repair i s possible by producing a cut i n a confluent monolayer of c e l l s i n tiss u e culture following which the c e l l s migrate and p r o l i f e r a t e i n the depleted area (21,22). The process of contact i n h i b i t i o n i s also observed i n v i t r o (14), i n which the area of i n t e r c e l l u l a r contact ceases to r u f f l e or dis p l a y a c t i v i t y . In a ddition to the c e l l s ' a b i l i t y to move i n tissue c u l t u r e , cytoplasmic fragments also r e t a i n some motile properties (23). I f c e l l s that are attached to the substratum are exposed to mechanical shear, the c e l l body w i l l be torn away and cytoplasmic fragments w i l l remain adherent to the substrate. These fragments r e t a i n t h e i r r u f f l i n g and c o n t r a c t i l e c a p a b i l i t i e s , but are unable to execute directed, coordinated m o t i l i t y (23). There are several advantages f o r performing studies on c e l l m o t i l i t y i n v i t r o . One i s the obvious ease of observation, and ease of performing other measurements, not attainable with i n vivo systems. There i s also the ease of manipulation, such as a l t e r i n g substrate, medium, temperature, and c e l l type. Furthermore, one can, i n a sense, di s s e c t the m o t i l i t y process with the use of ( s p e c i f i c ) drugs. Although c e l l m o t i l i t y i n v i t r o has been up u n t i l now described as motion on a 2-dimensional surface, systems also e x i s t to look at i n v i t r o c e l l movement i n 3-dimensional hydrated gels (24,25). 1.3 How C e l l s Move 1.3.1 Basic m o t i l i t y functions Many types of c e l l s are able to locomote and there i s a correspondingly large d i v e r s i t y i n the locomotive phenotype expressed by d i f f e r e n t c e l l types and by s i m i l a r c e l l s under d i f f e r e n t conditions. F i b r o b l a s t s , amoeboids, 5 granulocytes, lymphocytes, macrophages, keratocytes, smooth muscle c e l l s a l l assume somewhat d i f f e r e n t c h a r a c t e r i s t i c morphologies and m o t i l i t y patterns (19,10). In addition, axon growth i n nerve c e l l s displays many c h a r a c t e r i s t i c s s i m i l a r to c e l l m o t i l i t y , and the growth cone can be considered as a motile apparatus s i m i l a r to a c e l l (19,26). Despite the wide d i v e r s i t y of locomotive c h a r a c t e r i s t i c s , there are c e r t a i n morphological features common to a l l of the above mentioned motile c e l l s , and these are believed to perform the e s s e n t i a l functions of c e l l m o t i l i t y . A s i m p l i f i e d d e s c r i p t i o n of the locomotive phenotype includes the following morphological features: 1) An active leading edge i s morphologically d i s t i n c t i n most motile c e l l s . This may take the form of a broad f l a t t e n e d lamellipodium, as i n f i b r o b l a s t s , keratocytes, etc. or a more narrow, thickened psuedopodia as i n amoeboids and leukocytes; however i t seems i n a l l cases to provide a s i m i l a r function. 2) The c e l l also has means to adhere to the substrate. D i f f e r e n t microscopy techniques have revealed several types of adhesion regions (27,28). These regions form underneath the leading edge of the c e l l , and are often modified as they pass to the rear of the c e l l . 3) F i n a l l y , a l l motile c e l l s seem to have c o n t r a c t i l e properties, which are involved i n force generation during locomotion (29,19). These common morphological features, as well as pos s i b l y others not i d e n t i f i e d here, must provide the three functions required f o r c e l l m o t i l i t y (19): 1) Adhesion of the c e l l to the substrate. The adhesion strength must be c o n t r o l l a b l e i n order to achieve net displacement. 2) Force to extend the cytoplasm onto a new region of the substrate. This process must occur i n p r e f e r e n t i a l regions of the c e l l periphery i n order to maintain a d i r e c t i o n of movement. This provides the basis f o r c e l l p o l a r i t y . 3) Force to move the bulk cytoplasm. In addi t i o n to extending the c e l l periphery onto new substrate, the bulk of the c e l l must move r e l a t i v e to the substrate. 6 1.3.2 Structure and function of cytoplasm Recent techniques i n biochemistry, biophysics, and c e l l biology have revealed a great deal about the structure and function of the cytoplasm. What was once treated as a bag of chemicals i s now recognized as a h i g h l y complex, structured v i s c o - e l a s t i c substance capable of f i n e l y regulated mechanochemical and physicochemical behaviour. The cytoplasm of f i b r o b l a s t s i s permeated by a set of filaments known as the cytoskeleton. The cytoskeleton i s composed of 3 separate systems; the microfilament s y s t e m , the microtubule (MT) s y s t e m , a n d t h e i n t e r m e d i a t e filament system. Each system has d i f f e r e n t properties and thus d i f f e r e n t r o l e s i n c e l l function. The microfilament system (10,30) consists of polymers of a c t i n molecules, plus many a c t i n - i n t e r a c t i n g proteins such as myosin, tropomyosin, g e l s o l i n , p r o f i l i n , etc. The a c t i n filaments (F-actin) can e x i s t e i t h e r as t i g h t l y bundled arrays known as stress f i b e r s , or as a f i n e meshwork of s i n g l e filaments (a g e l ) , or a combination of the two. This component of the cytoskeleton provides the c o n t r a c t i l e force-generating functions necessary f o r m o t i l i t y . The MT (10) system consists of polymers of the p r o t e i n tubulin, plus, again, many t u b u l i n - i n t e r a c t i n g proteins. The system i s usually centered about a MT aster, l i k e the c e n t r i o l e i n interphase c e l l s , and t h i s structure acts as a nucleating s i t e f o r t u b u l i n polymerization. During interphase the c e n t r i o l e i s located near the nucleus and Golgi apparatus i n the c e n t r a l region of the c e l l . The p o s i t i o n of the c e n t r i o l e , and hence the MT array, seems important i n c o n t r o l l i n g the s p a t i a l organization of c e l l s . The intermediate filament (IF) system (31-33,10) i s composed of h i g h l y ordered h e l i c a l polymers of c e r t a i n intermediate f i b e r proteins which vary according to c e l l type. Keratin, f o r example, i s the primary IF p r o t e i n i n e p i t h e l i a l c e l l s (33), while vimentin i s associated with mesenchymal c e l l s such as f i b r o b l a s t s (33). This filamentous structure seems much les s dynamic than the 7 other two and i t s r o l e i n c e l l m o t i l i t y appears to be l i m i t e d . The IF system may act as a s t r u c t u r a l s c a f f o l d . A vast amount of information has developed on the biochemical and b i o p h y s i c a l properties of each c y t o s k e l e t a l component. A few of the more relevant aspects of the microfilament and microtubule systems w i l l be outlined here. The microfilament system contains a c t i n and numerous other proteins that can be grouped into 5 categories according to t h e i r f u n c t i o n a l i n t e r a c t i o n with a c t i n (34). The microfilament system also contains the proteins necessary for actomyosin based contraction, ( i . e . myosin), with modifications from the s t r i a t e d muscle system. In the t e s t tube and i n c e l l s , i_2 many of the p r o t e i n interactions and enzymatic rates are s e n s i t i v e to the Ca +2 concentration, and an increase i n [Ca ] can cause both s o l a t i o n of an actomyosin gel, and contraction (34,29). A c t i n filament assembly i t s e l f i s a complex process, involving the condensation of a c t i n monomers (G-actin) from the soluble pool. The process involves at l e a s t 2 steps (35). F i r s t a slow step i n which small a c t i n oligomers are formed (nucleation), then the rapid elongation to form long a c t i n polymers. The process of polymerization/depolymerization i s an important force generating mechanism i n at l e a s t one dynamic system, the sperm c e l l (36) , and i t quite l i k e l y also plays a r o l e i n c e l l m o t i l i t y . The microtubule system i s a network of polymeric f i b e r s of t u b u l i n with a set of t u b u l i n i n t e r a c t i n g proteins. The i n t e r a c t i n g proteins include dynein and k i n e s i n , both of which may act i n the active d i r e c t i o n a l transport of material along a microtubule (37,37a). The microtubule system i s s e n s i t i v e + 2 to Ca , temperature (cold), and several drugs, which act to depolymerize the microtubules. Disruption of the system seems to prevent d i r e c t e d or coordinated c e l l m o t i l i t y (38,39). The MT system i s disrupted and reassembled i n an ordered fashion around the process of mitosis. Certain c e l l u l a r 8 structures, such as c i l i a and f l a g e l l a , contain h i g h l y organized arrays of microtubules that are more stable than t h e i r c y t o s k e l e t a l counterparts (10). In the cyt o s o l , the microtubule system i s i n a dynamic equilibrium with the soluble t u b u l i n f r a c t i o n . This i s evident from studies which have demonstrated "treadmilling" (40,41). 1.3.3 C e l l membrane The c e l l membrane also plays a key ro l e i n the process of c e l l m o t i l i t y . The cytoplasmic membrane of eukaryotic c e l l s i s a dynamic 2-D f l u i d structure c o m p o s e d of a b i l a y e r of l i p i d molecules. N u m e r o u s p r o t e i n s a r e d i s s o l v e d i n the membrane which act as ion channels, ion pumps, receptors, s i t e s f o r c e l l adhesion, and anchor s i t e s f o r cy t o s k e l e t a l components, among other functions. The b i l a y e r i s asymmetrical due to the preferred o r i e n t a t i o n of the many membrane proteins, and the d i s t r i b u t i o n of the l i p i d molecules themselves. The membrane i s important i n at l e a s t 3 processes r e l a t e d to c e l l m o t i l i t y : 1) adhesion to the substrate; 2) control of solutes and osmotic pressure that regulates the cytoskeleton; and 3) determination of c e l l p o l a r i t y through the preferred o r i e n t a t i o n of membrane re c y c l i n g . C e l l adhesion i s a very complex subject i n i t s own r i g h t . Recently the molecular architecture of the c e l l adhesion s i t e s has begun to be elucidated (42,43), and the ro l e of c e l l adhesion i n the con t r o l of c e l l behaviour has begun to be appreciated (44,45). I t appears that e x t r a c e l l u l a r matrix proteins, such as f i b r o n e c t i n , must be present on the surface of p l a s t i c and glass tissue culture vessels to f a c i l i t a t e f i b r o b l a s t locomotion (44-47). The adhesion plaques which anchor the motile c e l l to the substratum are dynamic structures that form at the leading edge and are dismantled at the t r a i l i n g edge of the c e l l (27). In a c t i v e l y motile f i b r o b l a s t c e l l s , the adhesions are broad, unspecialized and transient, while i n stationary f i b r o b l a s t s , the adhesion structures are stable and well-defined, often forming at the terminus 9 of a stress f i b e r (48). These structures, c a l l e d f o c a l adhesions, have been well characterized morphologically (42,43). The c e l l membrane(s) i s a s e l e c t i v e l y permeable osmotic b a r r i e r that maintains differences between the cytoplasmic and extra-cytoplasmic milieu. Free Ca , which was previously mentioned to have an important r o l e i n c o n t r o l l i n g the state of gelation of the cytogel as well as the c o n t r a c t i l e actomyosin machinery, i s maintained at very low l e v e l s i n the cytoplasm 7 J.2 (approximately 10 M) (49). A large Ca chemical p o t e n t i a l gradient exists across the plasma membrane, and across organelle membranes such as the mitochondria. Concentration differences of Na +, K +, CI" and other solutes are also maintained: these regulate the osmotic and e l e c t r i c a l forces on the c e l l . The c e l l membrane houses the enzymatic machinery that dynamically controls the concentration of these solutes. Gated channels and active pumps r e s i d i n g i n the membrane act to regulate l o c a l concentrations of solutes. A l e s s obvious r o l e of the c e l l membrane i n locomotion i s the determination of c e l l p o l a r i z a t i o n and the subsequent d i r e c t i o n of locomotion. The c e l l o r i e n t a t i o n appears to be due to the preferred i n s e r t i o n of new membrane mass at the leading edge of the c e l l , and the concomittant r e t r i e v a l of membrane from the middle and rear of the c e l l (19,50). This i s accomplished by the o r i e n t a t i o n of the microtubule organizing center (MTOC), and hence the microtubule system, toward a p a r t i c u l a r region of the c e l l (51,52). Stimulation of chemotaxis i n f i b r o b l a s t and endothelial c e l l s r e s u l t s i n the perinuclear MTOC being oriented i n the d i r e c t i o n of migration (19). The current view i s that the p o s i t i o n of the MTOC d i r e c t s the transport of v e s i c l e s to a p a r t i c u l a r region of the c e l l periphery, and the region of membrane i n s e r t i o n i s the preferred l o c a t i o n f o r the leading edge. 10 1.3.4 Phenomenological d e s c r i p t i o n A b r i e f phenomenological d e s c r i p t i o n of c e l l morphology and c e l l physiology relevant to the process of c e l l m o t i l i t y w i l l be given here (see Figure 1.1). I t i s generally believed that the leading edge of the c e l l , i n the form of a lamellipodia f o r f i b r o b l a s t s , i s the key cytoplasmic region i n c e l l locomotion, the "locomotive organelle" (19,14). The forward extension of the lamellipodium appears to generate tension i n the d i s t a l regions of the c e l l , which r e s u l t s i n t h i s p o r t i o n being p u l l e d toward the lamella (19,53). The leading edge i s an area of rapid a c t i v i t y , evidenced by the continuous formation of r u f f l e s and microspikes. The lamellae and microspikes extend out onto new regions of substrate. Some of the processes adhere to the substrate, while others are c e n t r i p e t u a l l y swept over the c e l l surface toward the rear of the c e l l (54). Once attached, the lamella consolidates, and tension i n the c e l l propels the cytoplasmic bulk forward, often leaving a long r e t r a c t i o n t a i l which may suddenly break i t s adhesions and snap into the c e l l . When the r e t r a c t i o n f i b e r i s p u l l e d into the c e l l , a period of ra p i d lamellae extension often follows (55-57). Measurements have been performed on the c y t o s k e l e t a l state i n various regions of the c e l l during locomotion. The c e l l periphery, p a r t i c u l a r l y the leading edge, consists of a f i n e meshwork of a c t i n filaments rather than the more organized a c t i n cables i n the main c e l l body (10,58,59). Intermediate filaments and microtubules do not generally extend into the leading edge (19). I t i s believed that the leading edge i s the s i t e of t r a n s i t i o n s from the gel state to the solated state of the a c t i n filament system (60,61), and that t h i s provides the force for protrusion of the cytoplasm. The leading edge i s also the s i t e of membrane i n s e r t i o n . V e s i c l e s are direct e d from the Golgi apparatus and rough endoplasmic reticulum, and p r e f e r e n t i a l l y i n s e r t near the leading edge (19,50). The v e s i c l e contents are 11 lamellipodia Figure 1.1: Schematic diagram of a motile f i b r o b l a s t . Active lamella i s at front of c e l l , r e t r a c t i o n t a i l behind (from Alberts, B., Bray, D., Lewis, J., Raff, M. , Roberts, K. , and Watson, J.D., Molecular BJOIOEV of the C e l l . Garland Publishing, N.Y., 1983). 12 deposited i n the e x t r a c e l l u l a r milieu, and the proteins and l i p i d s are inser t e d into the c e l l membrane. This process has been proposed to be a key feature i n providing the c e l l o r i e n t a t i o n , as well as po s s i b l y other motile functions. I t i s not known how the c e l l i s able to d i r e c t membrane t r a f f i c toward a c e r t a i n cytoplasmic region, nor the fun c t i o n a l consequences of t h i s . Some measurements have also been made on the i o n i c environment and membrane p o t e n t i a l i n regions of the c e l l during locomotion (62). These studies are s t i l l i n an early stage. 1.3.5 Biophysical model of c e l l m o t i l i t y The process of c e l l m o t i l i t y , and p a r t i c u l a r l y i t s global c e l l u l a r c o n t r o l , have r e s i s t e d a quantitative theory, yet some physicochemical models e x i s t that provide hypotheses concerning c e r t a i n steps i n the o v e r a l l process. These models are almost c e r t a i n l y not the ultimate quantitative d e s c r i p t i o n , yet they provide a conceptual framework f or the process, and provoke more pointed questions and experiments. One such model w i l l be described here. The model proposed by Oster and Odell (63,64), and l a t e r r e f i n e d by Oster and Perelson (65,66), describes the ac t i o n of the leading edge of the c e l l . The forward extension of the lamella, and the subsequent tension exerted by the r e s t of the c e l l to p u l l toward the leading edge, are explained i n terms of the c o n t r a c t i l e - g e l properties of the actin-myosin meshwork. Within a gel, the cross-linked polymers which constitute the gel are supported by the surrounding f l u i d , i n th i s case water (67). At steady state, the osmotic forces i n the system are balanced, and the f l u i d i s at r e s t . I f a l o c a l region of the gel i s disrupted, i n the form of breaking the cr o s s - l i n k s between filaments or depolymerization of the filaments themselves, the osmotic forces come into e f f e c t (67). F l u i d may eit h e r rush into the disrupted region to produce expansion, or flow out of the region to give contraction. The d i r e c t i o n of the flow depends upon whether the osmolarity of the filamentous 13 molecule i s larger i n the polymerized or depolymerized form. This has yet to be determined e x p l i c i t l y f o r cytogel. The actomyosin cytogel has a property that i s unique amongst commonly studied gels i n that i t can use the energy stored i n ATP to a c t i v e l y contract. The Oster/Odell/Perelson model proposes that a cycle of events goes on i n the cytogel (64), i n i t i a t e d at or by the leading lamella. F i r s t a l o c a l _i_2 increase i n the Ca concentration i n the leading edge causes the breakdown of the a c t i n meshwork and the gel solates. The s o l a t i o n of the gel induces an o s m o t i c f o r c e , a n d f l u i d f r o m t h e r e m a i n d e r o f t h e c e l l f l o w s i n t o t h e l e a d i n g edge causing i t to swell onto new substrate. The increase i n f l u i d , plus • 2 active pumping mechanisms, reduces the [Ca ] i n the leading edge and the gel 4-2 then repolymerizes into a f i n e meshwork. Concomittant with the Ca -induced s o l a t i o n , the actomyosin c o n t r a c t i l e machinery i s also activated, however the k i n e t i c s of t h i s a c t i v a t i o n are slower than the s o l a t i o n . Thus as the gel re-anneals, the c o n t r a c t i l e machinery takes over and a c t i v e l y exerts tension on the remainder of the c e l l , p u l l i n g the c e l l forward. The cycle of s o l a t i o n , expansion, gelation, and contraction i s then poised to repeat. This model was formulated i n terms of a set of non-linear p a r t i a l d i f f e r e n t i a l equations. The equations were based upon the measured physicochemical properties of the actomyosin cytogel i n response to [Ca ] and i n response to mechanical s t r e s s . Several assumptions were made regarding the source and nature of the Ca f l u x . The set of equations were solved numerically, and the r e s u l t was a measurement of the l i n e a r extent of the model lamella corresponding to the cycle of actomyosin s o l a t i o n , expansion, gelation, and contraction. 14 1.3.6 Control of motility There are many important issues that are not addressed by the b i o p h y s i c a l models of c e l l m o t i l i t y : p a r t i c u l a r l y notable are the control of i 2 c e l l adhesion, and the mechanism of generating a l o c a l i z e d increase i n Ca This r e f l e c t s our lack of d e t a i l e d understanding about the c o n t r o l mechanisms of c e l l m o t i l i t y . In order to achieve a global c o n t r o l , the c e l l must r e v e r s i b l y s p e c i a l i z e c e r t a i n regions of the cytoplasm and c e l l membrane that w i l l perform the s p e c i f i c tasks i n c e l l m o t i l i t y . At the l i g h t microscope l e v e l , a f i b r o b l a s t contains several morphologically d i s t i n c t regions, such as the main c e l l body, the leading lamella, f i l o p o d i a , r e t r a c t i o n f i b e r and others. Inherent i n c e l l morphology are c e l l u l a r regions of d i f f e r e n t s p a t i a l scales; f or example, the lamella may contain smaller f i l o p o d i a and microspikes. I t i s the net i n t e r a c t i o n of these c e l l u l a r regions with each other, with the global c e l l shape, and with i n t r i n s i c morphology determinants ( i . e . c e l l type or p o s i t i o n of the c e n t r i o l e ) , that produces the global c e l l movement. The control mechanisms, at l e a s t i n f i b r o b l a s t s , do not seem to be omnipotent; t h i s i s r e f l e c t e d i n the apparently incomplete c e l l c o n t r o l i n which various c e l l regions compete f o r prominence. An i n d i v i d u a l motile f i b r o b l a s t displays a v a r i e t y of movement patterns and shapes, and tends to move i n a complex, undulatory manner. 1.4 General Aspects of the Interaction of Ionizing Radiation with  Living Matter A few general comments w i l l be made regarding the i n t e r a c t i o n of r a d i a t i o n with b i o l o g i c a l material. As x-rays or other types of r a d i a t i o n traverse b i o l o g i c a l material, energy i s transferred from the r a d i a t i o n f i e l d to the material. For x-rays, t h i s transpires because the electromagnetic 15 f i e l d i n t e r a c t s with the electrons of the material. I f the i n t e r a c t i o n event transfers s u f f i c i e n t energy, the electron i s stripp e d away from i t s host atom or molecule to produce an i o n i z a t i o n , i . e . - a free e l e c t r o n (e~) and a p o s i t i v e l y charged molecule, hence the term " i o n i z i n g r a d i a t i o n " . This free e l e c t r o n i t s e l f has k i n e t i c energy, and as i t moves through the material i t dis s i p a t e s i t s energy i n a serie s of electromagnetic in t e r a c t i o n s with other e l e c t r o n s / n u c l e i . The free electrons represent a l t e r a t i o n s to the chemical bonds of the material. The net r e s u l t i s one or more i o n i z a t i o n events, and a heating of the material. In most b i o l o g i c a l material, t h i s process occurs i n aqueous media. This has several implications f o r r a d i a t i o n biology. One i s that the free electrons may become solvated and e x i s t as long l i v e d species along with such free r a d i c a l s as H' and OH-. Another i s that damage to the chemical bonds of the c e l l u l a r f a b r i c may occur e i t h e r by d i r e c t i n t e r a c t i o n with the i o n i z i n g p a r t i c l e , or by i n d i r e c t i n t e r a c t i o n v i a a reactive r a d i c a l or electron created i n the s o l u t i o n and d i f f u s i n g to the bond. The phy s i c a l deposition of energy and the subsequent a l t e r a t i o n of chemical bonds i s a very rapid process, generally complete within 10 seconds. This process i s stochastic within the c e l l volume. I t i s the subsequent processing of the damage by the chemical and biochemical molecules of the c e l l that determines the ultimate consequences of the i n i t i a l damage. These processes occur on a much longer time scale than the damaging events (Figure 1.2). Biochemical reactions take place on the order of milliseconds, while c e l l u l a r and whole body e f f e c t s may take days, months or even years to manifest. For the measurement of r a d i a t i o n damage to b i o l o g i c a l material, an assay system i s employed which measures one (or more) endpoints. Referring now s p e c i f i c a l l y to i r r a d i a t i o n of tissue culture c e l l s , an endpoint such as c e l l 16 TIME SEQUENCE OF RADIOBIOLOGICAL EVENTS IO I8 IO rl6 IO IO io IO"8 sec y  initial electronic energy transfer -k RADIATION PHYSICS decay into vibrational,rotational states and heat IO xl4 IO' 2 -IO I.0 C8 IO I0 4sec primary ion and free radicals Long lived macro molecular CHEMISTRY Radical-radical v lesion radical molecule interaction IO"' IO" 1 sec 10 Ihr 10 Iday IO6 sec -I r-macromolecular relaxation and recognition enzymatic repair Cell division BIOCHEMISTRY AND CELL BIOLOGY 10' 10 8 . 1 0 \d2 sec genetic recombination and integration V y -gene expression,carcinogenesis,late effects evolutionary impact GENETICS AND EVOLUTIONARY PROCESSES FIGURE 1.2: Time sequence of events following i r r a d i a t i o n of l i v i n g organisms. (From Tobias, C.A., Blakely, E.A., Ngo, F.Q.H., and Yang, T.C.H., The Repair - Misrepair model of c e l l s u r v i v a l . In Radiation Biology i n Cancer Research, ed. R. Meyn & H. Withers, Raven Press, N.Y., 195-230, 1979). 17 s u r v i v a l i s often measured (68,69). This measures the c e l l s ' a b i l i t y to p r o l i f e r a t e and form a colony following r a d i a t i o n treatment. The phrase "DNA i s the c r i t i c a l target" i s often used (70,1), which has an operational d e f i n i t i o n as follows: of the indiscriminate damage to the c e l l u l a r f a b r i c , the damage to the DNA i s the most c r u c i a l f o r determining whether or not a c e l l i s capable of proceeding to form a colony. Other c e l l u l a r endpoints may measure the r a d i a t i o n damage to other parts of the c e l l . This point i s being stressed here because i t i s f e l t that radiation-induced perturbations to c e l l m o t i l i t y may r e f l e c t damage to b i o l o g i c a l molecules other than DNA. This perturbation may have s i g n i f i c a n t consequences i n c r i t i c a l tissues such as the developing fetus or a malignant tumour. 1.5 Radiation as a Perturbation of C e l l M o t i l i t y and C e l l Morphology. 1.5.1 In vivo A v a r i e t y of investigations have reported that low doses of i r r a d i a t i o n a l t e r the m o t i l i t y of c e l l s . In vivo studies have looked p r i m a r i l y at perturbations to the very motile and r a d i a t i o n s e n s i t i v e lymphocyte c e l l s . One approach has used r a d i o l a b e l l e d lymphocyte c e l l s from mice, i r r a d i a t e d them e i t h e r i n s i t u , or i n v i t r o , and r e i n j e c t e d the c e l l s into a host (5,71,72). Untreated c e l l s accumulated i n the lymph nodes and other organs within 4 hours of i n j e c t i o n , while x-ray doses as low as 1.0 Gy produced a large decrease i n the number of l a b e l l e d c e l l s that accumulated i n the lymph nodes (5,72). C e l l l e v e l s i n the l i v e r and other organs were not perturbed by i r r a d i a t i o n . This e f f e c t was a t t r i b u t e d to a l t e r a t i o n of the glycoprotein antigenic markers on the i r r a d i a t e d lymphocytes (71), although damage to other "mechanisms r e l a t e d to c e l l m o t i l i t y " was also suggested. The e f f e c t of u l t r a v i o l e t r a d i a t i o n on the movement and c o n t r a c t i l e properties of the t i p of fungus has been studied. During fungal hyphae t i p 18 growth, ac t i v e c e l l contraction determines the p o s i t i o n i n g of n u c l e i within the t i p . This region was l o c a l l y i r r a d i a t e d with a UV beam to determine the e f f e c t on nucleus p o s i t i o n i n g (73) . The UV treatment induced l o c a l cytoplasmic contraction and affe c t e d the p o s i t i o n of c e l l n u c l e i . This e f f e c t appeared to be mediated by Ca and the a c t i n network (74). 1.5.2 In v i t r o Other studies have looked at the e f f e c t of i r r a d i a t i o n on i n v i t r o c e l l m o t i l i t y . Here again the lymphocyte has been used extensively and c e r t a i n subpopulations appear to be extremely s e n s i t i v e to low doses of r a d i a t i o n . Experiments with normal lymphocytes from human peripheral blood showed a l t e r a t i o n s i n s u r v i v a l and m o t i l i t y a f t e r doses of 0.1 Gy or less (75,3,4). In studies with other c e l l types, Bases and coworkers looked at the e f f e c t of x-rays (and ultrasound) on the m o t i l i t y of mouse f i b r o b l a s t s using the gold dust m o t i l i t y assay (76) (described l a t e r i n Section 1.6) as well as time lapse cinematography (2). They observed that low doses of x-rays a l t e r e d the pattern of c e l l m o t i l i t y , and induced changes i n dynamic c e l l morphology. Tobias and co-workers exposed d i f f e r e n t i a t e d neuroblastoma c e l l s i n v i t r o to low doses of x-rays and heavy ions and observed a r e t r a c t i o n of axonal processes (77). M o t h e r s i l l et a l observed a l t e r a t i o n s i n the m o t i l i t y of primary oesophageal cultures when exposed to x - i r r a d i a t i o n (78). I r r a d i a t i o n has also been reported to a l t e r the morphology of c e l l s . This has been demonstrated i n s t a t i c measurements of morphology, i n fun c t i o n a l assays, and i n assays to look at dynamic morphology. Yau exposed lymphoid c e l l s to high doses of x - i r r a d i a t i o n and, using scanning e l e c t r o n microscopy, found large membranous and morphological deformations (79). Blebs and c e l l protrusions were observed i n a time dependent and dose dependent fashion. At lower doses, Anderson et a l used SEM to show that doses of 0.5 Gy produced s i m i l a r membrane e f f e c t s i n mice thymocytes and B lymphocytes (71). In 19 another report, Anderson et a l used image cytometry techniques to measure the morphology of T and B lymphocytes (80). I t was found that B lymphocytes were very s e n s i t i v e , with changes to the o p t i c a l density of Feulgen-stained c e l l s a f t e r exposure to less than 1.0 Gy (81). Functional assays have revealed damage to the c e l l membrane by low doses. Koteles et a l observed that low doses of x-rays (0.9 to 2.5 Gy) res u l t e d i n the decreased a b i l i t y of human f i b r o b l a s t c e l l s to bind Con A (82), and reported an a l t e r e d surface d i s t r i b u t i o n of Con A and other receptors (83,84). S i m i l a r l y , Facchini et a l (85) and others (86), determined that low dose i r r a d i a t i o n of human lymphocytes r e s u l t s i n a loss of surface membrane immunoglobins and a decrease i n the a b i l i t y of these c e l l s to cap surface immunoglobins. Sato and coworkers (87,88) and other groups (89) have documented that low doses of x-rays a l t e r the electrophoretic mobility of several c e l l types, an assay which measures the fu n c t i o n a l c e l l surface charge due to a c i d i c carbohydrates. Other assays have looked more d i r e c t l y at membrane damage. These assays have t y p i c a l l y involved high doses, since t h e i r inherent s e n s i t i v i t y i s quite low. Properties such as membrane f l u i d i t y , membrane permeability, and membrane transport have been examined (90). In one report using e l e c t r o n spin resonance, the f l u i d i t y of the plasma membrane of lymphocytes was reported to be a l t e r e d by x - i r r a d i a t i o n (71). It has been proposed that the radiation-induced perturbations to c e l l m o t i l i t y and c e l l morphology may have a s i g n i f i c a n t impact on the events during medical uses of x-rays, p a r t i c u l a r l y diagnosis and cancer treatment. The f u n c t i o n a l assays used to study t h i s damage are perhaps the most relevant, yet these are t y p i c a l l y d i f f i c u l t to quantify. The reports of a l t e r e d c e l l m o t i l i t y following x - i r r a d i a t i o n are an example (2): the reports described a 20 s i g n i f i c a n t perturbation to c e l l function, but were unable to provide a dose response or a time response, and a mechanistic explanation was lacking. 1.6 Techniques to Measure C e l l M o t i l i t y and C e l l Morphology A discussion of c e l l m o t i l i t y must mention the techniques employed to measure and describe movement. C e l l m o t i l i t y has been observed since the e a r l i e s t microscopy, and several techniques to measure c e l l movement and dynamic morphology have evolved. In general, these may be divided into two types; d i r e c t assays and i n d i r e c t assays (91). The i n d i r e c t assays t y p i c a l l y u t i l i z e a coarse endpoint f o r c e l l m o t i l i t y and are able to measure many c e l l s . Examples of i n d i r e c t assays include micropore f i l t e r assay, agarose assay, and c o l l o i d a l gold assay. The micropore f i l t e r assay (92) measures chemotaxis by separating two chambers with a micropore f i l t e r . I n i t i a l l y one chamber contains the tes t c e l l s and the other contains the t e s t substance. The apparatus i s incubated f o r several hours, a f t e r which the membrane and c e l l s attached to i t are fi x e d . Microscopic examination then reveals the extent of c e l l migration into the membrane. This technique measures the propensity of c e l l s to migrate toward the t e s t substance. The agarose m o t i l i t y assay (93) u t i l i z e s an agarose f i l m i n which wells have been cut on a p e t r i dish. The wells are f i l l e d with e i t h e r chemoattractant or c e l l s , then the c e l l migration out of the well toward the attra c t a n t i s measured. The c o l l o i d a l gold assay (94) involves spreading a t h i n layer of gold p a r t i c l e s (<1 /xm i n diameter) onto the surface of the p e t r i dish. F i b r o b l a s t or leukocyte c e l l s are then plated onto the gold dust, and as the c e l l s migrate, they ingest the gold p a r t i c l e s , leaving a track of where they have been. The c e l l s are then fixed, and the length or area of the tracks i s measured. 21 In contrast to the i n d i r e c t assays, the direct methods of measuring c e l l m o t i l i t y t y p i c a l l y perform detailed measurements on a small number of c e l l s . Examples of direct m o t i l i t y assays include opto-electronic method, time lapse cinematography, and video image analysis systems. In the opto-electronic method (95), a grid of 32x32 photodiodes was substituted for one ocular of a microscope. The position of the projected image of a c e l l was monitored by e l e c t r o n i c a l l y scanning the sensor array, and c e l l movement was monitored at 11 second intervals. This was the f i r s t step towards automated systems for tracking c e l l s . Time lapse cinematographic records (91,96) of motile c e l l s were developed very early, 1917 to 1919, followed by video recording techniques. This technique was the mainstay of observation of c e l l m o t i l i t y for many years and provided much of the qua l i t a t i v e information. Individual c e l l s are observed i n d e t a i l as they locomote. However, the method suffers i n that a very l i m i t e d number of c e l l s are monitored at any one time. In addition, the analysis of data i s a tedious and time-consuming process, and the production of quantitative, objective data i s d i f f i c u l t . Recent advances i n image processing have led to automated methods for analysis of video records. I n i t i a l l y , interactive systems became available i n which the recorded c e l l images were projected onto a screen, and the experimenter used an interactive program to extract m o t i l i t y (96,97) (and eventually morphology (98-100)) information. A f u l l y automated system was developed by Berns (101) i n which a high magnification video image of a single l i v e c e l l was continuously monitored. Edge detection algorithms were employed to demarcate the c e l l boundary, and the position of the c e l l centre was calculated. The c e l l position was updated as the c e l l moved. A more sophisticated video-based system was very recently developed by Noble and 22 Levine (102) that i s able to monitor several c e l l s i n the same microscope f i e l d . An automated microscope system was also developed i n t h i s laboratory that i s able to concurrently track many motile c e l l s and extract morphological information (103,104). Unlike the system of Noble and Levine, t h i s system u t i l i z e s a computer-driven p r e c i s i o n x-y microscope stage and a l i n e a r array of charge-coupled device (CCD) s o l i d state photodetectors. Together these enable the system to perform high r e s o l u t i o n scans on c e l l s from d i f f e r e n t microscope f i e l d s . This combines some of the advantages of both types of c e l l m o t i l i t y assays, i n that a large number of i n d i v i d u a l motile c e l l s may be examined i n d e t a i l . The system i s described i n more d e t a i l i n Materials and Methods. 1.7 Analysis of Dynamic C e l l Behaviour Behavioral assays i n c e l l biology are complicated by the f a c t that even with a sophisticated measuring apparatus, the data must be analyzed to produce relevant parameters. The recent developments i n automated microscope systems have permitted the concurrent measurement of m o t i l i t y and morphology from i n d i v i d u a l l i v e c e l l s i n tissue culture. The data i s i n the form of a sequence of coordinate positions of the c e l l c e ntroid (the m o t i l i t y data), and a sequence of representations of the c e l l morphology extracted from d i g i t i z e d images (the morphology data). The data i s i n a "frame by frame" format. I t represents a huge amount of information on the m o t i l i t y and morphology from the c e l l s , and various a n a l y t i c a l techniques have a r i s e n to describe the relevant c h a r a c t e r i s t i c s of c e l l m o t i l i t y and c e l l morphology. A few of the approaches w i l l be described below. 23 1.7.1 C e l l m o t i l i t y One approach to describe c e l l m o t i l i t y data, introduced by Boyarski (105), i s based upon a Markov state analysis. The sequence of recorded c e l l displacement vectors were converted to a sequence of Markov states ( f i v e states i n t o t a l ) according to the d i r e c t i o n and magnitude of the displacement. Then the data from many c e l l s over a period of time was pooled and the steady state t r a n s i t i o n p r o b a b i l i t i e s between the various states were calculated. This treatment i s useful to look at the propensity of a population of c e l l s to move toward (or away) from a given (fixed) d i r e c t i o n i n the frame of the tis s u e culture f l a s k , and was applied to the chemotaxis of neutrophils (106). I t s u f f e r s from the drawback that very l i t t l e information i s retained from the actual process of c e l l m o t i l i t y (both chemotactic and non-chemotactic). Another technique for analyzing c e l l m o t i l i t y data i s the two-dimensional random walk model introduced by G a i l and Boone (107). The treatment sampled the recorded positions of c e l l s at d i f f e r e n t time i n t e r v a l s (2.5 hrs and 5.0 hrs) and measured the average mean square displacement as a function of time. The data was f i t to the two-dimensional random walk model with a persistence factor, and values c a l c u l a t e d f o r the augmented d i f f u s i o n c o e f f i c i e n t and the persistence factor. This method has the advantage that the data i s analyzed i n a c l e a r fashion with parameters that are r e l a t e d to average c e l l movement, but i t does not give information on the movement c h a r a c t e r i s t i c s of i n d i v i d u a l c e l l s . I t was f e l t that these a n a l y t i c a l techniques produced parameters that were not f a i t h f u l to the actual process of c e l l locomotion. In the i n t e r e s t of emphasizing the p u l s a t i l e nature of c e l l locomotion, f o r example, a technique was developed i n t h i s laboratory (108) that segments the data into periods of s i m i l a r c h a r a c t e r i s t i c movement. These segments are of v a r i a b l e time scales, but t y p i c a l l y are on the order of 40-80 min. f o r untreated 3T3 24 f i b r o b l a s t c e l l s . The pattern of the c h a r a c t e r i s t i c segments i s analyzed to detect perturbations to the m o t i l i t y of a c e l l population. 1.7.2 C e l l morphology As with the m o t i l i t y data, the information on c e l l morphology must be analyzed to y i e l d appropriate parameters. There are several conceptual approaches for the analysis of the d i g i t i z e d images of c e l l morphology. To date, most reports have used the bit-map data ( i . e . - each p i x e l of the c e l l image i s e i t h e r black or white) to c a l c u l a t e morphological features. Lewandowska et a l (99) analyzed the morphology of motile leukemia c e l l s by recording 16 mm time lapse records, then d i g i t i z i n g them with a TV camera and d i g i t a l converter. The frames were analyzed using a mini-computer, and a b i t -map of the c e l l was generated. From t h i s , the walk pattern of a c e l l was derived, plus numerous morphological features. The morphology was described by a set of global shape features such as area, minimum rectangle, and o r i e n t a t i o n . In addition, more complex global shape features were defined that emphasized a p a r t i c u l a r aspect of c e l l shape: an example i s the B l a i r and B l i s s factor which i s designed to s p e c i f i c a l l y emphasize the extent of c e l l elongation. Vershueren and Van Larebeke (100) used a s i m i l a r experimental apparatus to generate a bit-map of motile c e l l s . A morphological feature was defined that expressed the extent of change of c e l l p o s i t i o n and c e l l shape from one frame to another (Figure 1.3). This provided an excellent means to quantify the dynamic global c e l l morphology, and to d i s t i n g u i s h between quiescent c e l l s , a ctive c e l l s that were non-motile, and a c t i v e l y motile c e l l s . Noble and Levine (102), using the video microscopy system described e a r l i e r , generated bit-map representations of motile leukocytes. They analyzed the c e l l morphology not only with a set of global features, but also by decomposing the c e l l into subregions. The o r i g i n a l decomposition of the 25 area, at t common area before translation after translation FIGURE 1.3: D e f i n i t i o n of dynamic morphology features. a) "Common area" and 2x "motile area". b) Common area before and a f t e r t r a n s l o c a t i o n to match cent r o i d positions (from 100). 26 c e l l used a convexity analysis of the c e l l border, and segmented the c e l l u l a r extensions as d i s t i n c t subregions. Then a star graph d e s c r i p t i o n was produced of the composite c e l l body plus extensions. In t h i s way, the d i s t i n c t pseudopod of motile leukocytes might be i d e n t i f i e d . An approach used by D i l l et a l (109) on motile leukocytes, plus others on stained c e l l s (110), i s to resolve the c e l l shape into a s t i c k figure representation c a l l e d the skeleton. The arms of the skeleton correspond to the various c e l l extensions. This technique was d i r e c t e d at i d e n t i f y i n g the prominent pseudopod of motile leukocytes, and i d e n t i f y i n g the rate of change of the pseudopod (109), 1.8 Thesis Project With t h i s background information, i t i s now possible to state the intent of t h i s thesis more concisely. The e f f e c t s of r a d i a t i o n on c e l l m o t i l i t y were examined i n d e t a i l with the intent of measuring the dose response and time response of relevant c e l l m o t i l i t y parameters. The studies were c a r r i e d out i n t i s s u e culture. Previous studies by other workers used the gold dust m o t i l i t y assay and time lapse microcinematography to study c e l l m o t i l i t y following exposure to x-rays and s i g n i f i c a n t perturbation to c e l l m o t i l i t y was reported following low doses of x-rays. These reports were q u a l i t a t i v e . I n i t i a l l y , these techniques were repeated i n an e f f o r t to reproduce t h e i r findings, and to serve as a control for the assessment of other c e l l m o t i l i t y assays. Then an i n v i t r o c e l l m o t i l i t y assay system was developed that allowed the quantitative measurement of i n d i v i d u a l c e l l m o t i l i t y and dynamic morphology for many c e l l s . Techniques were also developed to convert the data into a meaningful d e s c r i p t i o n of c e l l m o t i l i t y . This system was then employed to study the e f f e c t s of x - i r r a d i a t i o n on the m o t i l i t y of f i b r o b l a s t s i n tissue c u l t u r e . 27 2. MATERIALS AND METHODS 2.1. Cells and Culture 2.1.1 Growth characteristics The c e l l s used i n a l l of these studies were NIH 3T3 c e l l s , a g i f t of Dr. Tracy Yang (Berkeley, CA). These c e l l s were o r i g i n a l l y derived from a mouse embryo (111) as f i b r o b l a s t c e l l s and have since become a very common tissue culture c e l l l i n e . A v a r i e t y of studies on growth con t r o l have been done with these c e l l s (112,113) They are used i n many studies i n v o l v i n g DNA t r a n s f e c t i o n because of t h e i r s u s c e p t i b i l i t y to the SV40 transforming v i r u s and v a r i a t i o n s thereof (114). The c e l l s are also used i n many c e l l m o t i l i t y studies (2,15,94,97), including studies on x-ray induced perturbations to c e l l m o t i l i t y . 2 The c e l l s were ro u t i n e l y grown i n 75 cm p l a s t i c p e t r i dishes (Falcon, Oxnard, CA) i n Dulbecco's modified Eagle medium (DMEM) (Gibco) plus 10% f e t a l bovine serum (FBS) (Gibco). 5 The c e l l s were plated into the p e t r i dish at 5x10 c e l l s / p e t r i and grown o for 2.5 to 3 days i n a humidified incubator at 37 C and 5% C0 2 u n t i l they reached a near confluent monolayer. The c e l l s were then t r y p s i n i z e d with 0.1% t r y p s i n (Gibco), resuspended, counted, and plated again. The c e l l s used for experiments were taken from v i a l s stored i n l i q u i d nitrogen and passaged for not more than 8 weeks. The growth c h a r a c t e r i s t i c s of the c e l l s were examined. The doubling time i n standard conditions was approximately 14 hrs. (Figure 2.1). The c e l l cycle times were measured using a standard technique on the FACS (see below) and found to be G2/M - 2 hrs; S - 5-6 hrs., and Gl - 7 hrs. The doubling time of 3T3 c e l l s measured i n other laboratories i s somewhat longer (19 hrs) (115,116), and the r a t i o s of Gl to S and Gl to G2/M are s i m i l a r to those 28 Time after plating (days) FIGURE 2.1: P r o l i f e r a t i o n rate of 3T3 c e l l s i n various serum concentrations. The doubling time was extracted from the slope of the curve at 2-3 days post p l a t i n g . 2 9 D o u b l i n g T i m e v s T e m p e r a t u r e w 1 3 3 O Q 3 3 . 0 3 5 . 0 37\0 3?7o T e m p e r a t u r e ( ° C ) FIGURE 2.2: Doubling time of 3T3 c e l l s i n DMEM +10% FBS as a function of temperature. 30 22249 • $ v . * 1 FIGURE 2.3: A micrograph of 3T3 c e l l s showing various c e l l morphologies. Photograph taken on Zeiss inverted microscope with 6.3x objective lens and 3.2x pr o j e c t i o n lens. Bar represents 50 nm. Arrow indicate a c t i v e l y migrating c e l l . FIGURE 2.4: A micrograph of 3T3 c e l l s at higher density, showing c e l l - t o - c e l l i n t e r a c t i o n s . Conditions as i n Figure 2.3. Arrow indicates c e l l i n c e l l - t o -c e l l contact. 32 measured here. The growth rate as a function of serum concentration i s shown i n Figure 2.1, and the growth rate as a function of temperature i n Figure 2.2. A micrograph of some i n d i v i d u a l 3T3 c e l l s i s shown i n Figure 2.3. The c e l l s are t y p i c a l of most f i b r o b l a s t s i n that they display a p o l a r i z e d shape, often with an active zone (lame l l i p o d i a ) . A micrograph of c e l l s i n c e l l - t o -c e l l contact i s shown i n Figure 2.4. 2.1.2 Measurement of p r o l i f e r a t i o n rates The rates of c e l l p r o l i f e r a t i o n under various conditions were measured using the following protocol: 4 1) C e l l s were harvested from regular passage, counted, and 5.0x10 2 c e l l s were added to 25 cm Falcon p e t r i dishes along with 4.0 ml of DMEM +10% FBS. o 2) The c e l l s were incubated for 24 hrs. at 37 . 3) A sample count was taken from at l e a s t one dish. 4) The p e t r i dishes were changed to the t e s t condition, while a c o n t r o l set remained i n standard conditions. The t e s t conditions involved a change of media to a new serum concentration, a d d i t i o n of a drug, or incubation at d i f f e r e n t temperatures. 5) At various times, the c e l l s were t r y p s i n i z e d using a standard routine (pour o f f medium, rinse quickly with 3ml of 0.1% t r y p s i n then o pour o f f , incubate for 1 to 1.5 minutes at 37 , rinse and resuspend with 4ml of fresh DMEM+10% FBS), and counted with the Coulter Counter. From t h i s , the average number of c e l l s per dish as a function of time was measured. 6) The doubling rate was estimated from the maximum slope of the p r o l i f e r a t i o n curve. This period was u s u a l l y 2-3 days a f t e r p l a t i n g (see Figure 2.1). 33 2.1.3 C e l l cycle times with Fluorescence Activated C e l l Sorter (FACS) To determine the length of the various phases of the c e l l cycle, the doubling time for exponentially growing c e l l s was determined, then the percentage of the t o t a l c e l l population i n each phase was determined with the FACS. From t h i s information the average duration of G l , S, and G2/M can be cal c u l a t e d . The FACS procedure i s as follows: 6 2 1) 5x10 c e l l s were plated into 75 cm p e t r i dishes and incubated for -24 hrs. The c e l l s were then i n logarithmic growth phase. 2) The c e l l s were t r y p s i n i z e d using a standard technique, centrifuged and r i n s e d with DMEM, then resuspended i n a fixation/DNA s t a i n i n g c o c k t a i l containing propidium iodide. 3) The c e l l sample was analyzed on the flow cytometer (Becton Dickinson 440) and the DNA content per c e l l measured. The d i s t r i b u t i o n f o r exponentially growing c e l l s i s shown i n Figure 2.5.a. As a control, c e l l s t r y p s i n i z e d from a confluent monolayer ( i . e . c e l l s r e s t r a i n e d i n G1/G0) were also analyzed. As expected, the d i s t r i b u t i o n was strongly peaked with G1/G0 DNA content (Figure 2.5.b). 4) The DNA d i s t r i b u t i o n p r o f i l e was analyzed using both a non-linear population f i t t i n g program (written by R. Durand), and by c u t t i n g and weighing the graph paper. Both techniques y i e l d e d r e s u l t s within 5% as to the r e l a t i v e f r a c t i o n of c e l l s i n G l , S, and G2/M, shown i n Figure 2.5.a. 2.2. Gold Dust Assay 3T3 c e l l s , as well as many other f i b r o b l a s t and lymphocyte c e l l types, tend to pick up and ingest small objects that come i n contact with the c e l l . This property has been used as a means of recording the track of motile 3T3 34 a I 1 1 D a t a p l u s c o m p u t e r f i t il G 1 5 0 % ! j i i J i 1 j S 3 6 % G 2 / M 1 4 % 1 , .? K ^ A .i ft*. . A b ) ;r y li •''I II I ! 1 i i i i i •I k FIGURE 2.5: DNA d i s t r i b u t i o n s f o r 3T3 c e l l s : a) exponentially growing and b) plateau phase. Computer f i t to data also given i n (a). The p l o t i s of the f r a c t i o n of c e l l s vs the fluorescence i n t e n s i t y of the DNA s t a i n . The prominent peak on the l e f t represents c e l l s i n G1/G0, the peak on the r i g h t represents c e l l s i n G2/M and i n between are S phase c e l l s . 35 c e l l s . For the assay, a carpet of f i n e p a r t i c l e s of gold onto the surface of a p l a s t i c or glass tissue culture v e s s e l . The c e l l s are then plated onto t h i s surface, and as they attach, spread, and locomote, the c e l l s ingest the gold p a r t i c l e s i n t h e i r reach and leave a clean path where they have been. The path can then be viewed microscopically. The procedure for the gold dust assay i s as follows (94): A) Materials 1) hydrated gold c r y s t a l s (AuCl 4H) to 14mM i n doubly d i s t i l l e d water (D.D.H20). 2) 0.1% formaldehyde i n D.D.H20. 3) 1% s o l u t i o n of bovine serum albumin i n D.D.H20 and f i l t e r e d (0.2 /im syringe f i l t e r ) (BSA s o l u t i o n ) . 4) 3.5% formaldehyde s o l u t i o n i n PBS. 5) 36.5 mM s o l u t i o n of Na 2C0 3 i n D.D.H20. 6) permount. 2 7) coverslips and 25 cm p l a s t i c p e t r i dishes. B) Preparation of coverslips 1) clean c o v e r s l i p s with 70% ethanol. 2) dip coverslips into BSA s o l u t i o n and touch drain on a s t e r i l e kim-wipe. 3) dip c o v e r s l i p immediately into 100% ethanol, and dry near bunsen flame. 4) place 2 drie d coverslips into each p e t r i . C) Gold p a r t i c l e preparation 1) combine 1.8 ml of the 14 mM AuCl 4H s o l u t i o n , 6 ml of 36.5 mM Na 2C0 3 s o l u t i o n , and 11 ml of D.D. H 20 i n a small beaker. 2) heat mixture over bunsen flame u n t i l i t j u s t reaches b o i l i n g point. 36 3) remove heat. 4) quickly add 1.8 ml of 0.1% formaldehyde s o l u t i o n . The formaldehyde reduces the gold and causes i t to p r e c i p i t a t e as small gold p a r t i c l e s . As i t cools, the s o l u t i o n quickly turns from c l e a r to a muddy brown or purple, depending upon the siz e of the gold p a r t i c l e s . Once the reaction i s complete, put 2-4 ml of the s t i l l hot gold dust s o l u t i o n into the p e t r i dishes with c o v e r s l i p s . 5) l e t t h i s s i t f o r 45-90 min. as the gold s e t t l e s . 6) pour o f f gold s o l u t i o n and rinse with DMEM +10% FBS. The actual p l a t i n g of the coverslips may need to be repeated depending on the q u a l i t y and s i z e of the gold p r e c i p i t a t e . Once properly coated, put the cov e r s l i p s into clean p e t r i dishes. Then plate c e l l s into these dishes with regular medium. The c e l l s w i l l attach and locomote. Incubate f or 24 to 48 hrs. D) F i x a t i o n and microscopy When the c e l l s are ready f or viewing: 1) drain medium with pipette. 2) add 3 ml of 3.5% formaldehyde s o l u t i o n . Let s i t for 30 minutes. 3) extract coverslips with forceps. 4) dip successively into PBS, 70% ethanol plus PBS, 100% ethanol. 5) allow c o v e r s l i p s to become almost dry. 6) mount coverslips with Permount. The c o v e r s l i p s , once dried, can be viewed microscopically. The most d e t a i l e d v i s u a l i z a t i o n of the c e l l tracks can be seen with e p i - d a r k f i e l d • il l u m i n a t i o n , t y p i c a l l y with a 2.5 to lOx objective lens. 37 2.3. Cell Analyzer and Cell Tracking 2.3.1 General information A good f r a c t i o n of t h i s project involved the development of a quantitative assay f o r measuring c e l l m o t i l i t y and dynamic c e l l morphology. This was done with the DMIPS C e l l Analyzer, an automated microscope system that was developed i n t h i s laboratory f o r a v a r i e t y of b i o l o g i c a l i n vestigations (117,118). Information on t h i s technique i s described below and i n the Results section. More d e t a i l e d information on c e r t a i n aspects i s found i n the Appendix. The basic components of the automated microscope system are: 1) An inverted microscope - Zeiss 305 or Nikon Diaphot 2) A computer-driven p r e c i s i o n stage, driven by a microprocessor support system (see Appendix 6.2.2). 3) An o p t i c a l sensor - a l i n e a r array of charge-coupled device (CCD) photo detectors with microprocessor support system (see Appendix 6.2.1). 4) A host computer - IBM PC-AT. 5) A focus drive (z-drive) with microprocessor support. 6) A d i g i t a l s i g n a l processor (DSP) - Texas Instruments TMS 32010 (see Appendix 6.2.4). A d d i t i o n a l components are: 1) A video camera - 3-chip CCD. 2) A video image recorder - Panasonic o p t i c a l memory disk recorder (OMDR). 3) A p l e x i g l a s s incubator hood, Nikon hot a i r blower, and thermoresistor feedback (see Appendix 6.2.3). 4) A video monitor. The components are integrated as indicated i n the block diagram (Figure 2.6). The p r e c i s i o n stage i s mounted on the microscope and holds the tissue culture INVERTED MICROSCOPE I T> 3 CHIP CCD Z-DRIVE TISSUE CULTURE VESSEL PRECISION MICROSCOPE X,Y STAGE Z-DRIVE CONTROLLER JOYSTICK RS232 X,Y STAGE CONTROLLER 3ZI IEEE-488 INTERFACE LINEAR SOLID STATE IMAGE SENSOR SIGNAL CONDITIONING, SAMPLED HOLD, A/D DIGITAL SCANNER INTERFACE 8< SCANNER CONTROL DIGITAL SIGNAL PROCESSOR & MULTI-PORTED MEMORY 3 Z COLOUR MONITOR OPTICAL MEMORY DISC RECORDER H PERSONAL COMPUTER SYSTEM FIGURE 2.6: S c h e m a t i c d i a g r a m o f DMIPS C e l l A n a l y z e r w i t h OMDR at t a c h m e n t . 39 f l a s k . The CCD array i s mounted on the camera port of the microscope. Both of these components, as well as the focus motor, are c o n t r o l l e d by the host component v i a t h e i r own microprocessor support systems. The 3 chip CCD video camera i s mounted onto another camera port of the microscope, and functions independently of the c e l l tracking system. 2.3.2 C e l l m o t i l i t y measurements The system was proposed to monitor motile 3T3 f i b r o b l a s t s with the following s p e c i f i c a t i o n s : 1) the c e l l centre be detected with a s p a t i a l p r e c i s i o n of 3 fim. 2) the system monitor c e l l s spread over a 1.5 cm by 1.5 cm area i n a p l a s t i c tissue culture f l a s k . 3) the system monitor up to 100 c e l l s . 4) each of 100 c e l l s be r e v i s i t e d every 10 minutes ( i . e . - le s s than 6 seconds/cell). 5) the system track the selected i n d i v i d u a l c e l l s f o r several hours or more. A general purpose c e l l r e v i s i t i n g program (RSCAN) was developed to meet these s p e c i f i c a t i o n s . For a c e l l tracking experiment, the protocol was as follows: 1) A f l a s k with c e l l s was placed onto the stage of the C e l l Analyzer o and RSCAN i n i t i a t e d . The incubator had been prewarmed to 37 C. A period of approximately 15 minutes was needed f o r the f l a s k to temperature e q u i l i b r a t e i n the stage incubator. 2) The various parameters i n the program were set for the p a r t i c u l a r experiment. These include l i g h t l e v e l , focus, threshold, i n t e r v a l between scans, t o t a l number of scans, and various options f o r data recording. 40 3) A set of c e l l s were selected by the user with the j o y s t i c k control of the x-y stage motors and the z focus motor. These coordinates were stored i n computer memory. 4) The c e l l s were p e r i o d i c a l l y r e v i s i t e d by the system. The stage moved at high speed to the l a s t recorded p o s i t i o n of the c e l l minus 48 nm i n the x - d i r e c t i o n (Figure 2.7). The stage and the sensor then 2 worked to search a 96x96 (im area centered on the previous c e l l p o s i t i o n . This i s done by moving the stage i n 1 /im steps while a segment from the middle of the CCD l i n e a r array was sampled and stored a f t e r each step. The DSP performed an image analysis routine to extract the p o s i t i o n of the centre of the c e l l (the bit-map c e l l c entroid). A f t e r a l l c e l l s were scanned, these new coordinate p o s i t i o n s were appended i n the data f i l e s . 5) I f the c e l l was not detected i n the i n i t i a l search area, then a f t e r the scan through a l l c e l l s was completed, the undetected c e l l was sought using a larger search area and a relaxed detection threshold. I f the c e l l was s t i l l not found, i t was declared as ' l o s t ' and not subsequently tracked. The user may manually override t h i s assignment. 6) Step 4 may be repeated as frequently and for as long as desired. The frequency at which the system could r e v i s i t an i n d i v i d u a l c e l l i s dependent upon the number of c e l l s that the system was concurrently tracking. T y p i c a l l y , the system required approximately 3 seconds per c e l l , so 100 c e l l s could be r e v i s i t e d every 5 minutes. In addition to recording the c e l l centre, there were options to record an image of each or selected c e l l s during the experiment. This may be done by ei t h e r s t o r i n g the 96x96 p i x e l image used by the DSP for data analysis i n a f i l e , or by incorporating the o p t i c a l memory disk recorder (OMDR) into the program (119). The flow chart for the RSCAN program i s shown i n Figure 2.8. 41 Y - d i r e c t i o n C u r r e n t p o s i t i o n of c e l l i P r e v i o u s p o s i t i o n o f c e l l c e n t r e X 9 6 x 9 6 p m s e a r c h a r e a f X-d i r e c t i o n S t a g e i n c r e m e n t s i n 1 p m s t e p s P o r t i o n o f s e n s o r S t a g e m o v e s f r o m p r e v i o u s c e l l FIGURE 2.7: Sche m a t i c d i a g r a m o f c e l l l o c a t i n g r o u t i n e i n RSCAN program. Sensor and m i c r o s c o p e s t a g e a c t i n c o n c e r t t o s c a n a r e a a r o u n d l a s t r e c o r d e d p o s i t i o n o f c e l l c e n t r o i d . 42 CEED i S y i u m I n i t i a l i z a t i o n t S e l e c t c e l l s o r r e e d c e l l p o s i t i o n t i l e R e c o r d i m a g e o n O M O R FIGURE 2.8: Flow c h a r t o f i n t e g r a t e d RSCAN program t o t r a c k c e l l s and/or r e c o r d c e l l images on OMDR. 43 2.3.3 Measuring c e l l morphology In an early version of the RSCAN program, the 96x96 p i x e l image from the scan of the c e l l p o s i t i o n was used only to c a l c u l a t e the c e l l centroid. A modified version of the program used the DSP to perform a more sophisticated analysis of the image i n order to 1) better i d e n t i f y the c e l l centre, 2) i d e n t i f y the correct c e l l i f 2 or more c e l l s were i n the f i e l d , and 3) extract information on of the c e l l morphology. An image of a s p e c i f i a b l e s i z e i s loaded into the multiported memory (MPM) of the DSP from the CCD array v i a d i r e c t memory access. For our purposes, t h i s s i z e i s u s u a l l y 96x96 p i x e l s . At the selected microscope magnification (4.Ox objective and 2. 5x p r o j e c t i o n 2 lenses), each p i x e l represents approximately 1 fim of the image. The flowchart for DSP processing and i t s incorporation into the host program (RSCAN) i s shown i n Figure 2.9. This procedure i s designed to: 1) r a p i d l y and accurately detect a l l c e l l s i n the f i e l d and ignore other objects (for example, deb r i s ) ; 2) determine how many c e l l s are i n the f i e l d , and decide which c e l l corresponds to the c e l l under i n v e s t i g a t i o n (based on si z e correspondence and minimum displacement); 3) use image processing techniques to extract an accurate morphology of that c e l l ; 4) extract some morphological features ( p a r t i c u l a r l y those involving gray l e v e l data) and the c e l l centre; and 5) store a bit-map representation of the c e l l shape for l a t e r v i s u a l i z a t i o n and analysis ( i n a border representation, i . e . , the chain code). This i s done for each c e l l and each scan i n r e a l time while the stage i s moving from one c e l l to the next. Total processing time f o r a 96x96 p i x e l image i s t y p i c a l l y less than 200 msec. The extracted c e l l morphology was q u a n t i f i e d with several shape and brightness parameters. These are shown i n Figure 2.10, and each parameter i s described i n the f i g u r e caption. 44 8 0 2 S 6 / S T ( H a i l ) r—J— , / S c a n c • 11 I m i g t / / i n d l o a d M P M / D S P ( u n c t i o n s M i l • 1 0 n • x 1 C • 1 ( « .1 z ) I W a i t R « l d a n d ft l o r e d i I i C o n t o u r c * M • n d g r o w r • g i o n t 0 2 n d t h r • m o 1 d E x t r a c t f e a t u r e s a n d c h a i n c o d * S t o r a d a t a i n M P M ~ R e t u r n t o 8 0 2 6 6 / 6 7 FIGURE 2.9: Flow chart of routine within RSCAN to detect c e l l s and extract morphology features from the 96x96 p i x e l image. 45 - / \ V I / 1 f I < ( l " I / o Morphology Featu res Centre: (48.38) Area: 759 Perimeter: 27 1 C i r c u l a r i t y : 7.89 Minimum Rectangle : Length: 84 Width: 58 Slope: 6 9 ° L / W: 1.45 FIGURE 2.10: The chain code reconstruction of a 3T3 c e l l , and some calculated morphological features. The centre i s the centroid, and i s denoted by the cross near the middle of the c e l l . The given values represent the x- and y-coordinates i n the o r i g i n a l 96 p i x e l by 96 p i x e l box. The area i s the number of p i x e l s within the c e l l border. The perimeter i s the distance around the c e l l border. The c i r c u l a r i t y i s defined as (Perimeter /4*w*Area) (102), and i s a measure of the deviation of an object from c i r c u l a r i t y . (A c i r c l e has a c i r c u l a r i t y of 1.0 while non-circular shapes have a larger value of c i r c u l a r i t y ) . The minimum rectangle i s c a l c u l a t e d by f i r s t f i n d i n g the longest cord between any two border points. This cord i s c a l l e d the length, i t s slope i s ca l c u l a t e d to give a measure of the c e l l ' s o r i e n t a t i o n . The width i s found by the minimum distance perpendicular to the length necessary to enclose the c e l l . The r a t i o of length/width i s a measure of the c e l l ' s elongation. 46 2.3.4 Choosing RSCAN parameters The RSCAN program i s a f l e x i b l e program for measuring many aspects of dynamic c e l l behaviour. I t has various parameters that were set to optimize measurement of a p a r t i c u l a r type of c e l l , under p a r t i c u l a r conditions, and to measure a c e r t a i n aspect of c e l l behaviour. The s e l e c t i o n of these parameters i s described below. Scanning rate - the most suitable i n t e r v a l f o r r e v i s i t i n g c e l l s depends upon the rate at which the c e l l s migrate, the p r e c i s i o n of the x,y-stage, the magnification of the objective and p r o j e c t i o n lenses, the s i z e of the scanning window (see below), the density of c e l l s i n the p e t r i dish, and the k i n e t i c s of the c e l l u l a r process(es) under i n v e s t i g a t i o n . The selected stage has a p r e c i s i o n of ±1.5 /im (as s p e c i f i e d by the manufacturer and confirmed by measurements - see Section 2.3.5) and t h i s provides a minimum to the measurement of a meaningful displacement. As c e l l s are r e v i s i t e d more frequently, the c e l l has less time to perform a movement and the "chatter" of the stage becomes an increasing f r a c t i o n of the measured c e l l movement. The c e l l morphology parameters, however, are not a f f e c t e d by the stage "chatter". Given the rate of a c t i v i t y of 3T3 c e l l s i n tissue culture conditions, a scanning i n t e r v a l of at l e a s t 5 minutes i s appropriate. On the other hand, the c e l l should be scanned s u f f i c i e n t l y often so that the c e l l cannot migrate away from the previously recorded p o s i t i o n , and also to capture some d e t a i l s of the locomotive procedure i t s e l f . For t h i s , a scanning i n t e r v a l of less than 12 minutes i s s u i t a b l e . Measurements of motile 3T3 c e l l s were generally performed with an i n t e r v a l of 8 or 10 minutes. Magnification - most of the experiments to measure c e l l m o t i l i t y with RSCAN were performed using a 4.Ox objective lens and e i t h e r a 3.2x (Zeiss) or 2.8x (Nikon) p r o j e c t i o n lens. This magnification i s s u i t a b l e to measure the c e l l m o t i l i t y with a p r e c i s i o n that i s compatible with the stage p r e c i s i o n . 47 I t also has a large advantage i n the RSCAN program, i n that as the stage moves i n 1.0 um increments, each element of the sensor detects an image of 1 /im i n both the x and y d i r e c t i o n s (each element i s 13x13 /im i n actual s i z e ) . More p r e c i s e l y , i f an image i s acquired on the Zeiss system, each p i x e l represents 1.00 /im i n the x - d i r e c t i o n and 1.02 /im i n the y - d i r e c t i o n . On the Nikon system, each p i x e l represents 1.00 /im i n the y - d i r e c t i o n (the l i n e a r array i s mounted perpendicular from the Zeiss) and 1.16 /im i n the x - d i r e c t i o n . Thus each p i x e l of the image i s approximately square. I f the magnification i s a l t e r e d from t h i s , and the stage i s s t i l l used to increment the image f i e l d across the sensor, each image p i x e l becomes a rectangle of unequal x and y dimension, and t h i s complicates the image processing. Size of scanning window - at the given magnification, an average 3T3 c e l l covers approx. 400 p i x e l s (20x20 /im) while a larger c e l l may cover 800 p i x e l s and up to 80 p i x e l s i n any one d i r e c t i o n . Thus a frame s i z e of 80x80 p i x e l s i s the minimum needed to capture to the f u l l extent of the c e l l . A larger window must be used to accommodate c e l l movement between scans. A window s i z e of 96x96 p i x e l s was used for most experiments f o r the i n i t i a l search window. This si z e f i t s e a s i l y into the storage space of the TMS. A larger s i z e of 176x176 p i x e l s was used as the search s i z e f or " l o s t " c e l l s . Focus - the o p t i c a l s i g n a l detected by the sensor v a r i e s with focus. The focus drive (z-drive) of the microscope system enables the z-coordinates of each c e l l to be stored i n computer memory, but f i r s t i t i s necessary to s e l e c t the focus l e v e l . The optimal focus as determined by a human eye i s one i n which the sharpest s t r u c t u r a l d e t a i l i s v i s i b l e . At the selected magnification, the human observer can detect changes of approximately ±10 /im i n the v i c i n i t y of optimal focus. In s i g n a l processing terms, the optimal focus translates as the s i g n a l having the larges t content of high ( s p a t i a l ) frequency components i f the s i g n a l were transformed to the frequency domain. 48 A s e r i e s of o p t i c a l signals of a c e l l , taken at various focus l e v e l s , i s displayed i n Figure 2.11. The optimal eye focus s i g n a l i s nearly f l a t , with sharp edges but with very l i t t l e contrast between the background and the c e l l body. As one varies the focus, the s i g n a l e i t h e r becomes br i g h t e r (as the focus l e v e l i s above the c e l l ) or darker (as the focus l e v e l i s below the c e l l ) , and the edges become less sharp. The f a c t that the c e l l becomes bri g h t e r i s due to the lens e f f e c t of the c e l l and i s quite unique to c e l l s vs other objects i n the f l a s k . For RSCAN purposes, the optimal focus l e v e l i s a compromise between a prominent bright (or dark) s i g n a l , and a s i g n a l which gives the sharpest s p a t i a l r e s o l u t i o n . A s i g n a l from a focus l e v e l that i s -50 iim from optimal eye focus seems to o f f e r a good balance. The morphology feature e x t r a c t i o n routine i n RSCAN i s f a i r l y robust to a l t e r a t i o n s of ±40 LITD from t h i s l e v e l (see Figure 2.12). Threshold - the RSCAN program uses two threshold values: the f i r s t i s fo r the i n i t i a l detection of the c e l l , and the second i s the l i m i t of the region growing procedure. The s e l e c t i o n of the f i r s t threshold i s dependent upon the focus l e v e l and the nature of the s i g n a l from the c e l l , while the second depends upon the noise l i m i t a t i o n s of the image. A value of +6 to +10 l e v e l s above background was used for the detection threshold, while a value of +2 was used f o r the region growing threshold. 2.3.5 Program con t r o l experiments Various tests were performed to ensure that the equipment and program performed to s p e c i f i c a t i o n s , and to t e s t program modifications. Some of these are described here. 1) Bead s l i d e s - with c e r t a i n modifications to the program parameters, RSCAN can i d e n t i f y and track small latex beads a f f i x e d to a c o v e r s l i p . 70 /*m latex beads were attached to a glass s l i d e using Crazy Glue or Permount. These s l i d e s were put on the microscope stage, and a set of FIGURE 2.11: A s e r i e s of s i g n a l s from the l i n e a r CCD o p t i c a l detector across an i n d i v i d u a l 3T3 c e l l with a 4.Ox objective lens at various focus settings, a) Z-value of +60 iim from eye focus, b) +20 pm. c) 0 fim, best eye focus, d) -40 Ltm. e) -80 Ltm. f) -140 Ltm. ( p o s i t i v e z-values i n d i c a t e specimen closer to the stage). CO 50 C 1 0 u m FIGURE 2.12: A ser i e s of chain code border representations of a si n g l e 3T3 c e l l taken at various focus settings, a) A z-value of +20 um from the optimal focus (objective lens close r to specimen). b) The focus used for these experiments, c) A z-value of -40 um from optimal focus. 51 i n d i v i d u a l beads were manually located using the RSCAN program. The beads were then p e r i o d i c a l l y r e v i s i t e d . Any detected movement of the beads would indicate problems with the apparatus. The beads were found to be stationary to within ±1.5 fxm which provides the l i m i t of s p a t i a l r e s o l u t i o n measured by the system. 2) CHO c e l l s , and f i x e d 3T3 c e l l s - s i m i l a r experiments tc the above bead te s t were performed with l i v e CHO c e l l s or f i x e d 3T3 c e l l s . Other techniques such as time lapse microcinematography indicate that CHO c e l l s tend to be non-motile, and the fi x e d 3T3 c e l l s are of course also stationary. Tests with RSCAN also show these c e l l s to be stationary, and confirm the pr e c i s i o n of the bead t e s t s . 3) Repeated c e l l tracking - a simple and e f f e c t i v e c o n t r o l that could be r o u t i n e l y performed during an actual experiment was to monitor a given c e l l twice independently. During the manual s e l e c t i o n of a set of c e l l s , the same c e l l could be selected twice (or more). Independent records of the c e l l behaviour were then made, and these were compared for consistency. An example of t h i s i s shown i n Figure 2.13, which shows two records of the c e l l movement of one c e l l . These tests confirmed that the system performed within s p e c i f i c a t i o n s . 4) OMDR and c e l l tracking - the RSCAN program enables the concurrent tracking of c e l l s and independent recording of time lapse images on the OMDR. The time lapse images can be taken from a f i x e d coordinate p o s i t i o n (thus the motile c e l l w i l l migrate i n the f i e l d ) or the images can be taken from the continuously updated p o s i t i o n of the c e l l centre (thus the motile c e l l w i l l appear to be tre a d m i l l i n g ) . Each of these types of time lapse records can be compared to data movement from RSCAN to ensure that the movement recorded by each technique i s i d e n t i c a l . These types of tests were performed and also confirmed program operation. FIGURE 2.13: The same c e l l t r a c k e d t w i c e i n d e p e n d e n t l y w i t h t h e RSCAN program. Shown i s t h e c e l l w a l k p a t t e r n , i n w h i c h each s m a l l c r o s s r e p r e s e n t s t h e c e l l l o c a t i o n o f each s c a n . 53 5) C e l l behaviour a f t e r p l a t i n g - a f i n a l system evaluation can be made on c e l l s behaving i n a well characterized way, i n t h i s case the reattachment of c e l l s following t r y p s i n i z a t i o n and r e p l a t i n g . 3T3 c e l l s were monitored following t r y p s i n i z a t i o n and r e p l a t i n g with the expected r e s u l t s that c e l l s took approximately 6 hrs to r e a t t a i n a flattened, p o l a r i z e d morphology, and approximately 8 hrs to reach a steady state rate of c e l l m o t i l i t y (data not shown). 2.3.6 Display and manipulation of m o t i l i t y data The c e l l movement data from a tracking experiment consists of a set of coordinate positions which represent the sequential l o c a t i o n of the c e l l centre. This data was displayed and manipulated i n a v a r i e t y of ways with the program RDISP. Some of the manipulations were i n f a c t quite involved, thus a d e s c r i p t i o n of c e r t a i n aspects of RDISP i s given below. 1) Walk pattern - the sequential p o s i t i o n s of an i n d i v i d u a l c e l l were displayed i n a walk pattern. 2) Average c e l l speed - the displacements from a l l the c e l l s i n the experiment were combined and divided by the number of c e l l s and the time i n t e r v a l between scans to give the average c e l l speed. This i s p l o t t e d as a function of time, and the options were given to choose which p a r t i c u l a r c e l l s to combine, and which time i n t e r v a l to display. A smoothing f i l t e r was also a v a i l a b l e , of v a r i a b l e lengths, which was a symmetric, equal weight-averaging f i l t e r . 3) D i s t r i b u t i o n of speeds - the c a l c u l a t e d c e l l speeds from a selected set of c e l l s and a selected range of times were pooled to give a frequency d i s t r i b u t i o n . The "bins" used to create the speed d i s t r i b u t i o n were usu a l l y 0.3/xm/min i n width, and 10 i n number, although t h i s could be altered. The average of the resultant d i s t r i b u t i o n was also c a l c u l a t e d . 54 4) D i s t r i b u t i o n of angles - the vector which corresponds to the displacement between two successive c e l l p o s i t i o n s forms an angle within the x-y coordinate system of the microscope stage. In addition, an angle i s formed between the 2 vectors which represent the 2 successive measured c e l l movements. The f i r s t of these i s within the f i x e d frame of reference of the stage and was c a l l e d the absolute angle, while the second was dependent upon the previous c e l l movement and was c a l l e d the r e l a t i v e angle (Figure 2.14). o o Both of these were measured from -180 to +180 or from -u to + 7 r . The r e l a t i v e a n g l e r e f l e c t s t h e d e g r e e o f p e r s i s t e n c e o f c e l l m o t i l i t y f r o m o n e scan to the next, while the absolute angle r e f l e c t s the tendency of the c e l l to move i n a set d i r e c t i o n within the f l a s k . The r e l a t i v e angles from a set of c e l l s and over a segment of time were combined to give a p r o b a b i l i t y d i s t r i b u t i o n . A displacement of less than 2 um was not pooled i n the d i s t r i b u t i o n . The set of c e l l s and the time segment were modified by the o user. A peak i n the d i s t r i b u t i o n near a r e l a t i v e angle of 0 ind i c a t e d c e l l persistence, while random c e l l movements were indicated by a "white noise" or o f l a t d i s t r i b u t i o n . The b i n si z e to create the d i s t r i b u t i o n was 40 and 9 i n number. 5) Random Walk Analysis - the pooled data from many c e l l s can be analyzed i n terms of a 2-dimensional random walk model or 2-dimensional d i f f u s i o n model. The e f f e c t s of persistence can be incorporated by modified c o e f f i c i e n t s f o r d i f f u s i o n , D , and time, t . 6) Displacement Analysis - the short term persistence of i n d i v i d u a l c e l l s was emphasized with the displacement analysis. P l o t t e d as a function of scan number was the net distance from the current c e l l p o s i t i o n to the p o s i t i o n n scans e a r l i e r , where n i s the displacement f i l t e r length (108). 7) Forecast Analysis - another technique to characterize the short term persistence was the forecast analysis. The frequency d i s t r i b u t i o n s of 55 Pn-2 FIGURE 2.14: A d i a g r a m t o i l l u s t r a t e v a r i o u s p a r a m e t e r s i n t h e a n a l y s i s o f c e l l m o t i l i t y . ^ - a b s o l u t e a n g l e , ^ r e l a t i v e a n g l e , d = d i s p l a c e m e n t . 56 the magnitude of the displacement vector, d^ were calculated given the magnitude of the previous displacement, d ^ ( s e e Figure 2.14). The length of the forecast was varied. Also calculated were the dis t r i b u t i o n s for the r e l a t i v e angles <f>\ given the magnitude of the displacement d^ 2.4 Stained Cells 3T3 c e l l s grown i n tissue culture were fix e d and stained to examine aspects of the c e l l morphology. The stains Coomassie blue and and NBD-pha l l a c i d i n were used to study the microfilament component of the cytoskeleton. The two stains gave sim i l a r r e s u l t s , although NBD-phallacidin apparently had a higher s p e c i f i c i t y for the microfilaments than did Coomassie blue. However, NBD phalla c i d i n bleached quite rapidly when illuminated and precluded easy repeated inspection. Thus only the Coomassie blue procedure and results w i l l be described. The procedure for preparing irradiated and control 3T3 c e l l s with Coomassie blue was as follows: 1) Coverslips were cleaned, then dipped into pure alcohol and flame dried. 2 These were then put into 25 cm p e t r i dishes, one coverslip per p e t r i . 2) 3T3 c e l l s were trypsinized from a subconfluent monolayer, resuspended i n fresh media, and counted using a Coulter Counter. 4 3) A t o t a l of 6x10 c e l l s were plated into each p e t r i dish with 4 ml of DMEM+10% FBS. Each coverslip covers approximately 1/4 of the p e t r i 4 area, so each coverslip receives approximately 1.5 x 10 c e l l s . 4) The c e l l s were incubated for 24 hrs to allow attachment and spreading. 5) The c e l l s were taken to the x-ray room, and the treated c e l l s were given the test dose of x-rays, while the control c e l l s were given a mock x-ray exposure of simi l a r duration. The c e l l s were then returned to the incubator. 57 6) At various times following the x-ray treatment (1-6 h r s ) , the c e l l s were stained and f i x e d . The procedure used a formalin s o l u t i o n i n PBS applied to the c e l l s f o r 45 minutes. Then the c e l l s were given a 5 minute a p p l i c a t i o n of Coomassie blue. Following t h i s , the co v e r s l i p s were extracted from the p e t r i dish with forceps, and dipped 3 times into solutions of PBS. The PBS was then washed o f f by dipping the coverslips into doubly d i s t i l l e d water, and allowing a b r i e f drying period on a paper towel. When the coverslips were j u s t dry, they were mounted onto glass s l i d e s with Permount. These were then sealed with c l e a r n a i l varnish. 7) The c e l l s were viewed with b r i g h t f i e l d microscopy with e i t h e r lOx or 25x objective lens magnification. Photographs were taken with a camera mounted onto the camera port, using I l f o r d black and white f i l m . 2.5 Dosimetry The x - i r r a d i a t i o n doses applied to c e l l s attached to p l a s t i c p e t r i dishes were c a l i b r a t e d with Fricke dosimetry. The Fricke technique involves the use of a c i d i c ferrous s u l f a t e s o l u t i o n , and the conversion of ferrous ions (Fe ) to f e r r i c ions (Fe ) Following i r r a d i a t i o n of the s o l u t i o n , the dose i s c a l c u l a t e d by measuring the concentration of f e r r i c ions i n the s o l u t i o n with absorption spectroscopy. The absorbance at 304 nm i s used to c a l c u l a t e the r a d i a t i o n dose. The x - i r r a d i a t i o n dose applied to c e l l s attached to glass c o v e r s l i p s (for c e r t a i n procedures of the gold dust assay and f o r measurement of s t a t i c c e l l morphology) was corrected f o r increased f l u x of low-energy electrons due to the higher atomic number of the glass ( z a v e ~ H f ° r glass vs. z a v e ~ 7 t o 8 fo r c e l l s and tis s u e culture p l a s t i c ) . This was done by using the p r o l i f e r a t i o n of 3T3 c e l l s as a b i o l o g i c a l dosimeter. C e l l s were i r r a d i a t e d 58 while attached to p l a s t i c or glass, and a c o r r e c t i o n f a c t o r of approximately 2 was measured (see Appendix 6.1.4). This f a c t o r was applied to the c a l c u l a t i o n of dose f or those experimental procedures where c e l l s were i r r a d i a t e d while attached to glass c o v e r s l i p s . The x - i r r a d i a t i o n dose applied to c e l l s i n suspension (for the gold dust assay) was measured by ion chamber dosimetry. A Victoreen probe (model #500 electrometer) was inserted into a glass duct that was f i l l e d with 100 ml of DMEM at room temperature and s t i r r e d with a s t i r r i n g rod. The procedure f o r performing Fricke dosimetry i s as follows: A) Solutions 1) Dilute 22.0 ml of H 2S0 4 (pure) i n approximately 0.4 1 of DDH20. 2) Add 0.40 gm of ferrous ammonium s u l f a t e , (NH^^Fe^O^^. 6^0. 3) Add 0.0650 gm of NaCl. 4) F i l l with DDH20 to 1.00 1. 5) S t i r f o r 45 minutes. 6) Store i n 500 ml glass b o t t l e s i n dark area. B) Procedure 1) Various quantities (20,10,5, and 3 ml) of the above s o l u t i o n were added to the experimental tissue culture f l a s k s . 2) The f l a s k s were i r r a d i a t e d at room temperature under i d e n t i c a l conditions as those used f o r i r r a d i a t i n g c e l l s . Control (0 dose) f l a s k s were kept outside the x-ray room for the same length of time. 3) A set of con t r o l and i r r a d i a t e d samples were prepared as l i s t e d i n Appendix 6.1.2. 4) The samples were then analyzed spectrophotometrically using the spectrophotometer i n absorption mode. The samples were exposed to 304 nm near UV l i g h t and the absorbance r e l a t i v e to con t r o l was recorded. 59 5) The absorbance, A, was converted to dose using the equation D(cGy)=2.864*104*A, This equation has been corrected for conversion to absorbed dose i n muscle, and i s for 250kVp x-rays. 6) The graph of absorption vs i r r a d i a t i o n time for the Fricke dosimetry i s shown i n Appendix 6.1.3. 7) Results were checked with Victoreen probe measurements. 60 3. RESULTS 3.1 Gold Dust Assay The motile paths of 3T3 f i b r o b l a s t s i n gold dust show up quite s t r i k i n g l y when the c o v e r s l i p i s viewed with dark f i e l d microscopy. The c e l l s themselves are evident as b r i g h t gold i r r e g u l a r objects at or near the terminus of a track. The c e l l s accumulated the gold p a r t i c l e s i n the perinuclear region, and occasionally the c e l l sloughed o f f small clumps of gold as the perinuclear area became saturated. 3.1.1 Controls Gold dust records of untreated and mock x - i r r a d i a t e d 3T3 c e l l s served as controls (Figure 3.1). Some features i n the tracks are noteworthy: 1) Individual c e l l s often show strong persistence, that i s , a tendency to move for long segments i n a smooth pattern. Note f o r example the l i n e a r track at the top of Figure 3.1.d, and the east-west tracks i n Figure 3.1.b. These cases, while not unusual, are outweighed by dark c l u s t e r s of tracks where one or several c e l l s have migrated within a comparatively small area. Examples of t h i s are seen i n Figures 3.1.c and 3.1.d. The c e l l s within these regions apparently underwent repeated changes of d i r e c t i o n . 2) C e l l s occasionally move i n a zigzag pattern, with many spikes along the track i n d i c a t i n g that the c e l l moved i n an exploratory fashion. Examples of t h i s are seen i n Figures 3.1.a and 3.I.e. 3) A branched track indicates a c e l l d i v i s i o n . Examples are seen i n the upper part of Figure 3.1.c and i n Figure 3.1.a. I t was noted soon a f t e r the development of the gold dust assay that s i s t e r c e l l s frequently d i s p l a y pronounced symmetry i n t h e i r migratory patterns immediately following d i v i s i o n (94). The pedigree tracks often tend to be mirror images of one another (antisymmetric), although i d e n t i c a l patterns were also reported (symmetric). Examples can be seen i n Figure 3.1.a and Figure 3.1.c (upper and FIGURE 3.1: D a r k f i e l d m i c r o g r a p h s o f u n t r e a t e d 3T3 c e l l t r a c k s i n g o l d d u s t . Shown a r e 4 f i e l d s t o i l l u s t r a t e v a r i o u s m o t i l i t y p a t t e r n s . B a r r e p r e s e n t s lOO^m. a) arrows i n d i c a t e b r a n c h e d t r a c k s . b) l e f t a r r o w i n d i c a t e s b r a n c e d t r a c k , r i g h t a r r o w i n d i c a t e s l o n g s t r a i g h t t r a c k , c) arrows i n d i c a t e b r a n c h e d t r a c k s . d) arro w i n d i c a t e s l o n g s t r a i g h t t r a c k . 63 lower). 4) Of p a r t i c u l a r note i s the great v a r i e t y and complexity of the motile tracks. Shown i n Figure 3.1 are a few of many recorded microscope f i e l d s , yet even from these i t i s evident that a simple d e s c r i p t i o n of 3T3 c e l l m o t i l i t y i s not adequate. 3.1.2 C e l l s i r r a d i a t e d before p l a t i n g In the micrographs presented i n Figure 3.2, the c e l l s were i r r a d i a t e d i n suspension to various doses before p l a t i n g them upon the gold dust c o v e r s l i p s . At high doses, the tracks are obviously much perturbed from those of control c e l l s . Figure 3.2. a shows a c e l l i r r a d i a t e d to 6.0 Gy. The c e l l has an abnormal morphology and l e f t an aberrant track. At doses of under 2.0 Gy, most often there i s not an obvious difference between the i r r a d i a t e d c e l l tracks and the control c e l l tracks. This i s evident i n Figures 3.2.b to d (0.6 Gy) and i n Figures 3.2.e to h (1.2 Gy). These figures show a wide v a r i e t y of track patterns, however the v a r i a t i o n u s u a l l y appears to be within the boundaries of the v a r i a t i o n displayed by unperturbed c e l l s . Occasionally, however, tracks are seen that look unlike the tracks of untreated c e l l s . Figure 3.2.h shows the records of c e l l s i r r a d i a t e d to 1.2 Gy. A s i n g l e c e l l i s evident that underwent extensive exploration of the surrounding substrate, yet performed very l i t t l e t r a n s l o c a t i o n . Long filaments of dark extending from the c e n t r a l dark area indicate that the c e l l extended cytoplasmic protrusions into these regions. Another i n t e r e s t i n g example can be seen i n Figure 3.2.b, near the centre of the Figure. This figure shows numerous c e l l s treated to a dose of 0.6 Gy. Of note are the tracks that display long filamentous extensions from the main c e l l track, and the s t r i k i n g zigzag motion of the one c e l l track. Another pattern i s seen i n Figure 3.2.d, which shows a group of c e l l s treated to 0.6 Gy. In the lefthand side of the f i g u r e i s a very prominent wide track that indicates that the c e l l e i t h e r moved as a very broad sheet, or that the c e l l branched a great many extensions and gathered gold 64 FIGURE 3.2: D a r k f i e l d m i c r o g r a p h s o f x - i r r a d i a t e d 3T3 c e l l t r a c k s i n g o l d d u s t . Shown a r e 8 f i e l d s t o i l l u s t r a t e v a r i o u s n ormal and p e r t u r b e d m o t i l i t y p a t t e r n s . a) 6 Gy, b) t o d) 0.6 Gy, e) t o h) 1.2 Gy. d) arro w on l e f t i n d i c a t e s wide t r a c k , arrow on r i g h t i n d i c a t e s l o n g n a r row t r a c k . 68 debris from a large margin around the c e l l . Compare t h i s track to the very t h i n and d i r e c t e d track i n the lower r i g h t of the same f i g u r e . In summary, gold dust tracks of c e l l s i r r a d i a t e d i n suspension p r i o r to p l a t i n g were examined following t e s t doses of 0, 0.6, 1.2 and 6.0 Gy At the high doses there were often obvious differences. In the lower dose regime (<2 Gy) , the tracks often are i n d i s t i n g u i s h a b l e from co n t r o l c e l l s . There are some tracks that appear to be outside of the range of untreated c e l l s , however, i t i s d i f f i c u l t to quantify the time course of the e f f e c t , and the degree of perturbation. 3.1.3 C e l l s i r r a d i a t e d i n the p e t r i dish In an attempt to i s o l a t e the e f f e c t of r a d i a t i o n on the movement of 3T3 c e l l s , a s l i g h t l y d i f f e r e n t protocol of the gold dust assay was introduced. The c e l l s were plated onto the gold dust c o v e r s l i p s and allowed 24 hrs. i n the incubator to attach and locomote. Then the c e l l s were i r r a d i a t e d i n s i t u to the t e s t doses and returned to the incubator f o r a further 24 hrs. The tracks were then analyzed for evidence of s i g n i f i c a n t change i n the track c h a r a c t e r i s t i c s at some point i n the track. As a c o n t r o l , c e l l s were given a mock x-ray treatment. The control c e l l tracks were in d i s t i n g u i s h a b l e from the standard controls described above. Shown i n Figures 3.3.a and b are examples of c e l l s i r r a d i a t e d to 1.2 Gy a f t e r 24 hrs. of incubation. There do not appear to be any s t r i k i n g examples of a l t e r a t i o n s to the track c h a r a c t e r i s t i c s . Another example of the same treatment i s shown i n Figure 3.3.c. Note i n the lower l e f t corner there are 2 c e l l s which may have changed t h e i r movement c h a r a c t e r i s t i c s ; i n both cases a t r a n s i t i o n from a s t r a i g h t smooth track to a c l u s t e r e d e r r a t i c track i s evident. Further examples at s l i g h t l y higher dose are seen i n Figure 3.3.d. Here a small group of c e l l s were i r r a d i a t e d to 2.4 Gy following a 24 hr. 6 9 FIGURE 3.3: D a r k f i e l d m i c r o g r a p h s o f x - i r r a d i a t e d 3T3 c e l l t r a c k s i n gold d u s t . C e l l s were i r r a d i a t e d h a l f w a y t h r o u g h g o l d d u s t a s s a y . Shown a r e 5 f i e l d s t o i l l u s t r a t e m o t i l i t y p a t t e r n s . a-c) 1.2 Gy. d,e) 2.4 Gy. c) and e ) arrows i n d i c a t e s sudden change i n t r a c k c h a r a c t e r i s t i c s . 72 incubation. I t i s not possible to say that there i s a change i n the c e l l movement i n any of the tracks. In Figure 3.3.e, the c e l l to the lower middle of the f i g u r e shows an apparent change i n motile c h a r a c t e r i s t i c s , from a f a i r l y smooth t h i n track to a thick track. This t r a n s i t i o n may have occurred following the x-ray treatment. In summary, following an incubation period, c e l l s were treated with i r r a d i a t i o n (to doses of 1.2 and 2.4 Gy) then allowed further time to locomote. The tracks were analyzed for dramatic changes i n the walk patterns. In many cases, no such changes were observed, yet there are some examples of a l t e r e d walk patterns. There are several complications i n the i n t e r p r e t a t i o n of these r e s u l t s : 1) normal c e l l s also are capable of a l t e r i n g t h e i r motile behaviour, i n response to c e l l cycle changes, c e l l to c e l l contact, etc., and i t i s d i f f i c u l t to c a l c u l a t e the o v e r a l l p r o b a b i l i t y of t h i s occurring i n untreated and i r r a d i a t e d c e l l s . 2) the record of the c e l l locomotion (the tracks i n the gold dust) contains no temporal information, so i t cannot be known i f the a l t e r a t i o n i n the walk pattern occurred following the i r r a d i a t i o n . 3.2 Time Lapse Records Time lapse microcinematographic records were made of untreated and x-i r r a d i a t e d 3T3 c e l l s i n tissue culture f l a s k s . These records are very informative on the motile behaviour of c e l l s i n culture. They provide a f e e l for how a c e l l moves which i s not attainable from the inspection of the gold dust tracks. The c e l l shape i s v i s i b l e during each segment of c e l l movement, thus a great deal i s revealed about the mechanisms of c e l l m o t i l i t y . Temporal information i s also provided and t h i s emphasizes the p u l s a t i l e and undulatory movement of f i b r o b l a s t s . 73 The time lapse images of the c e l l s were recorded on the O p t i c a l Memory Disk Recorder (OMDR). The frames are stored at various locations on three o p t i c a l disks, and w i l l be r e f e r r e d to by disk number and frame number. Certain images have been photographed from the video screen and are displayed here, ei t h e r i n d i v i d u a l l y or i n sequence. Other examples are r e f e r r e d to p a r e n t h e t i c a l l y , and may be viewed by the reader i n the Cancer Imaging Section of the B.C. Cancer Research Centre. 3.2.1 Control c e l l s - uni r r a d i a t e d 3.2.1.a Low magnification and long time span. Records were made of untreated 3T3 c e l l s i n f l a s k s using a 6.3x objective lens and b r i g h t f i e l d microscopy. The RSCAN program was used without the c e l l tracking function. Thus selected microscope f i e l d s were r e v i s i t e d p e r i o d i c a l l y , and a video image recorded from the same coordinate p o s i t i o n . The p r e c i s i o n of the stage produced a smooth record with very l i t t l e "chatter" from one frame to the next. The experiments used 10 selected microscope f i e l d s , each f i e l d containing several c e l l s , and an image of each f i e l d was taken every 5 or 8 minutes. At t h i s magnification, the c e l l shape i s seen i n some d e t a i l . The o v e r a l l morphology i s evident, and some d e t a i l s of the f i n e r scale morphology can be discerned. Noticeable are the leading lamellae, the r e t r a c t i o n f i b e r , the taunt concave periphery of the c e l l , and the c e l l nucleus. Finer scale features, such as d e t a i l s of the lamellae, n u c l e o l i , mitochondria, pinocytotic v e s i c l e s , etc., cannot be discerned. At t h i s scale of morphological descr i p t i o n , the c e l l does not undergo major changes i n morphology from frame to frame i f the images are acquired every 5 to 8 minutes. This i n t e r v a l allows one to make a f a i r l y lengthy record of c e l l behaviour with a minimum number of t o t a l frames: 200 frames 74 per f i e l d at a recording rate of one frame each 8 minutes produces a record of approx. 27 hours of r e a l time. (The time lapse records of unperturbed c e l l s at 6. 3x magnification are located i n the disk space as follows: Disk 1, frames 16,000 to 17,439; disk 2, frames 21,900 to 23,899). From these low power records of untreated c e l l s , several general observations were made regarding the character of 3T3 c e l l m o t i l i t y : 1) C e l l m o t i l i t y consists of events at d i f f e r e n t time scales. This i s evident i f the time lapse records are viewed at d i f f e r e n t speeds. I f the images are displayed at a slow rate, approximately one frame per second, then the frame to frame changes i n c e l l m o t i l i t y and c e l l morphology are evident. The types of morphological changes that are evident from one frame to the next include the extension of a lamella region of the c e l l , the sudden r e t r a c t i o n of the t a i l , the elongation of the c e l l body, and the flow of the bulk cytoplasm toward a p a r t i c u l a r region. These types of processes generally occur without a large scale change i n c e l l morphology. I f the time lapse record i s viewed at a f a s t e r rate, from 5 to 20 frames per second, then m o t i l i t y events that have a time scale of 10 to 100 minutes become evident. One of t h i s type of event i s the cycle which y i e l d s a net c e l l t r anslocation: lamella extension, c e l l elongation, bulk flow of cytoplasm, and r e t r a c t i o n of the t a i l f i b e r (see Figure 3.4). The o v e r a l l dynamic c e l l behaviour i s seen to consist of a s e r i e s of d i r e c t e d movements, punctuated by r e l a t i v e l y stationary periods. These segments generally correspond to large scale changes i n c e l l morphology, such as the change i n angle of o r i e n t a t i o n , or the change from rounded to elongated shape. The time lapse record may also be viewed at a very f a s t speed, 30 or more frames per second. This emphasizes the m o t i l i t y processes that occur on a long term scale, on the order of two hours or longer. From t h i s i t i s FIGURE 3.4: S e r i e s o f images o f m o t i l e 3T3 c e l l s . Images s t o r e d on o p t i c a l memory d i s k r e c o r d e r and p h o t o g r a p h e d from the v i d e o s c r e e n . Note c e l l i n upper m i d d l e o f f i e l d . C e l l had j u s t c o m p l e t e d c y t o k i n e s i s , and t h e n p e r f o r m e d a m o t i l e sequence (as d e s c r i b e d i n t e x t ) t o m i g r a t e t o the r i g h t . Sequence ends as the c e l l t e r m i n a t e d the movement and was i n a n o n - p o l a r i z e d morphology b e f o r e the n e x t movement. 78 evident that the pe r s i s t e n t behaviour that i s seen at smaller scales i s not seen at t h i s temporal scale. Thus i n these ti s s u e culture conditions the c e l l ends up migrating i n a random d i r e c t i o n , and the p u l s a t i l e movements are smoothed into continuous behaviour. However, i t also evident at t h i s viewing rate that c e l l s maintain some c h a r a c t e r i s t i c s over long term periods, and even over successive generations. For example, a c e l l moving very r a p i d l y may continue to do so over the course of several hours, and a f t e r mitosis, both daughter c e l l s may also display t h i s behaviour. Furthermore, i t i s also evident that d i f f e r e n t c e l l s from a supposedly homogeneous population display quite d i f f e r e n t movement c h a r a c t e r i s t i c s , which suggests that c e l l s are not s t r u c t u r a l l y and f u n c t i o n a l l y i d e n t i c a l . The fun c t i o n a l s i g n i f i c a n c e of th i s i s not known. 2) The second general observation from low magnification time lapse records i s that there are several quite d i s t i n c t i v e longer term modes of dynamic c e l l behaviour, each characterized by a c e r t a i n morphology and a pattern of m o t i l i t y . An i n d i v i d u a l c e l l may maintain a p a r t i c u l a r mode of locomotion f o r an extended period of time (several hours or more), and c e l l s are also occasionally observed to switch between modes. As an i n i t i a l d e s c r i p t i o n , three categories of m o t i l i t y and morphology were i d e n t i f i e d . The f i r s t mode i s exhibited by the clas s of c e l l s that are compact, often t h i n and elongated, with small t h i n protrusions. A broad lamella i s not seen. These c e l l s are d i s t i n c t i v e because of ra p i d motion, and often frequent changes i n d i r e c t i o n . An example of t h i s behaviour i s i l l u s t r a t e d i n Figure 3.5. (Examples of t h i s type can be seen on disk #1, frame #16,027. The c e l l i n the middle of the f i e l d has j u s t undergone cytokinesis. Both s i s t e r c e l l s , and p a r t i c u l a r l y the upper c e l l , remain compact f o r a complete d i v i s i o n cycle, and undergo r a p i d c e l l locomotion and rapid changes i n c e l l o r i e n t a t i o n . Another example i s seen on disk #1, frame #16 837. In the centre of the 79 FIGURE 3.5: I l l u s t r a t i o n o f f i r s t mode o f 3T3 c e l l morphology. The c e l l i n the m i d d l e r i g h t o f the f i e l d ( arrow) was l o n g , compact, and s p i n d l y , and d i s p l a y e d r a p i d movement. 80 f i e l d , a c e l l has again j u s t completed cytokinesis, and both c e l l s , i n p a r t i c u l a r the s l i g h t l y smaller c e l l , begin rapid motion while maintaining a compact morphology). The second mode of dynamic c e l l behaviour of untreated 3T3 c e l l s i s displayed by the c e l l s that are very broad, with an extended lamella covering over h a l f of the c e l l margin, and sometimes covering the e n t i r e c e l l margin. These c e l l s appear f a i n t under br i g h t f i e l d microscopy because of the thinness of the broad lamella. The c e l l s move i n a slow p u l s a t i l e fashion, usually moving i n t i g h t c i r c l e s and with a small net tr a n s l o c a t i o n . During t h i s type of locomotion, a r e t r a c t i o n t a i l i s not formed and the c e l l nucleus i s often at the rear of the c e l l . The broad lamella flows to various regions of the c e l l periphery and a defined c e l l o r i e n t a t i o n i s not established. Such a c e l l type i s seen i n Figure 3.6. The t h i r d mode of dynamic c e l l behaviour represents the most common type, and i s i n some ways a compromise between the two above extremes. These c e l l s may also have the largest dispersion of morphology and m o t i l i t y properties, but the common features include a smooth, p u l s a t i l e , undulating m o t i l i t y , the occasional appearance of a r e t r a c t i o n f i b e r at the t r a i l i n g end of the c e l l , a well-defined p o l a r i z e d morphology that accompanies d i r e c t e d locomotion, and a r e l a t i v e l y small leading lamella. As mentioned, most c e l l s f a l l into t h i s broad category. (An example of t h i s type of c e l l i s seen on disk #1, frame #18,880. Of the three c e l l s i n the upper middle of the f i e l d , each d i s p l a y t h i s type of behaviour with the middle of the three being the best example). The f i r s t and second categories each comprise roughly 10% to 20% of observed c e l l s i n the present population of 3T3 c e l l s , with the t h i r d category comprising the remainder ( a l t e r n a t i v e l y , t h i s may be interpreted as i n d i c a t i n g that c e l l s spend roughly 10 to 20% of the time i n the f i r s t two categories). 81 FIGURE 3.6: I l l u s t r a t i o n of second mode of 3T3 c e l l morphology. Note broad, f l a t t e n e d c e l l , with i l l - d e f i n e d lamella and l i t t l e c e l l p o l a r i t y . The c e l l exhibited slow movements. Image recorded as i n Figure 3.4, but with 16x objective lens and phase contrast microscopy. 82 A l l categories of c e l l s are observed to divide. I t i s not known i f the actual p r o l i f e r a t i o n rates of these three categories are i d e n t i c a l . The categories do not appear to be associated with a p a r t i c u l a r phase of the c e l l c ycle. The fun c t i o n a l s i g n i f i c a n c e of these types of behaviour i s not known. 3) As for the t h i r d general observation, some further words are necessary regarding c e l l m o t i l i t y i n r e l a t i o n to the c e l l c ycle. Ohnishi (131) reported that mouse L f i b r o b l a s t s d isplay large changes of morphology and m o t i l i t y as a function of the c e l l cycle, however, t h i s study was q u a l i t a t i v e . O ' Neill et a l (97) reported that mouse 3T3 f i b r o b l a s t s d i d not disp l a y s i g n i f i c a n t v a r i a t i o n i n the average rate of c e l l movement through the c e l l cycle, other than a decrease i n speed as c e l l s round up for mitosis l a t e i n G2, and an increase i n speed following cytokinesis as the s i s t e r c e l l s r a p i d l y migrate away from one another. Their assay was not very s e n s i t i v e and they measured only one parameter, the average c e l l speed. The observations from our own time lapse records are i n basic agreement with the report of O'N e i l l et a l . (97). An increase i n movement following c e l l d i v i s i o n i s observed, which generally l a s t s approximately one to two hours. Otherwise, v a r i a t i o n s i n c e l l m o t i l i t y through the c e l l cycle are subtle. I t should be emphasized that t h i s observation pertains only to low magnification observation of c e l l s . The important observation from the low magnification time lapse records of unperturbed 3T3 c e l l s i s that the c e l l s move i n a smooth p u l s a t i l e fashion. Segments of rapid d i r e c t e d m o t i l i t y are interspersed with segments of morphological reorganization. The o v e r a l l observation i s that 3T3 c e l l m o t i l i t y i s hig h l y complex. The p u l s a t i l e m o t i l i t y i s but one aspect i n the complexity. As discussed above there are also three types of m o t i l i t y and morphology patterns (at l e a s t ) , and c e l l s may p e r s i s t i n one category or oc c a s i o n a l l y switch between 83 categories. This behaviour requires at l e a s t some sort of quantitative d e s c r i p t i o n before a proper assessment can be made as to the perturbation of m o t i l i t y with r a d i a t i o n . 3.2.1.b High magnification and short time scale Time lapse records of 3T3 c e l l s i n tiss u e culture f l a s k s were also made with a higher power magnification. A 16x objective lens with phase contrast microscopy was used, and i n d i v i d u a l selected c e l l s were imaged at various time i n t e r v a l s . This magnification enabled the e n t i r e c e l l to be v i s i b l e i n one microscope f i e l d . The s p a t i a l d e t a i l that was observed was of course much f i n e r than with the low power objective, and the time scale of movements of the f i n e r c e l l u l a r components i s correspondingly smaller. Records were made with frame to frame i n t e r v a l s of 3.0, 2.0, 0.5, and 0.25 minutes. At t h i s l e v e l of s p a t i a l r e s o lution, the peripheral margin of an a c t i v e l y migrating c e l l undergoes rapid movement, and an i n t e r v a l of 0.5 - 0.25 minutes i s appropriate to capture f u l l information. A longer i n t e r v a l tends to miss the smooth flow of morphological events. For the time lapse records where an i n t e r v a l of 0.5 or 0.25 minutes was used, the RSCAN program was modified such that only a sin g l e c e l l was recorded during each experiment, and the microscope stage d i d not move between scans. The r e a l time duration of the recording experiments was usually shorter than with the low power objective because the frame rate was so much higher. T y p i c a l l y , records were made of 2 to 4 hours r e a l time. (These records are stored on disk #2, frames #16,121 to 20,899 (see OMDR frame information booklet f o r f u l l d e t a i l s ) ) . From these high magnification records of untreated 3T3 c e l l s , several observations were made: 1) The s p a t i a l hierarchy of events which characterized the lower power records i s not evident at the higher power. The c e l l behaviour observed 84 at higher power i s p r i m a r i l y the f i r s t temporal l e v e l mentioned e a r l i e r , i . e . - the frame to frame movements. Thus c e l l u l a r persistence and small scale morphology features are most prominent. 2) The lamella of the c e l l i s generally very a c t i v e . Ruffles, microspikes, and other types of c e l l extensions are continuously forming and disappearing. Large p i n o c y t o t i c v e s i c l e s are ingested near the peripheral area(s) of most active r u f f l i n g , and transported back to the perinuclear region (Figure 3.7). The dark granules i n the main c e l l body are constantly moving but are excluded from the lamella. 3) Certain regions of the c e l l periphery form sharp boundaries. These edges tend to appear with a b r i g h t halo when viewed with phase contrast microscopy. They are usually concave and appear to be under tension, due to adhesion and t e n s i l e forces i n the c e l l . These margins generally r e s i s t the appearance of r u f f l i n g i n the same c e l l region. (For example see the sequence on disk #2, frames #19,521 to 19,596. The lower r i g h t margin of t h i s c e l l formed t h i s type of t i g h t boundary and r e s i s t e d the formation of r u f f l e s u n t i l frame #19,596, during which time other regions of the c e l l were a c t i v e l y changing shape. Another example i s seen on disk #2, frames #18.000 to 18,499. The l e f t margin of the c e l l formed a taut edge and r e s i s t e d lamella flow into t h i s margin. These taut edges were modulated according to the tension i n the c e l l , and seem important for the control of c e l l p o l a r i z a t i o n ) . 4) C e l l s often form a long r e t r a c t i o n t a i l . This i s a t h i n filament of cytoplasm that i s f i r m l y anchored to the substrate and remains i n p o s i t i o n as the c e l l migrates forward (Figure 3.7). As the r e t r a c t i o n f i b e r i s stretched, tension i s exerted that may r e c o i l the f i b e r into the c e l l . Once t h i s occurs, the leading lamella undergoes increased r u f f l i n g a c t i v i t y and the c e l l i s propelled forward (55). (An example of t h i s sequence i s recorded on disk #2, frame #20,041 to 20,074). 85 FIGURE 3.7: Image of an active 3T3 c e l l , showing active leading lamella, r u f f l e s , and pinocytosis. Pinocytotic v e s s i c l e s appear as bri g h t c i r c l e s (small arrow) , and move uniformly from the c e l l periphery to the nuclear periphery where they dissolve. Larger arrow indicates long r e t r a c t i o n t a i l . 86 Unperturbed c e l l s show a very complex pattern of morphological changes. There i s some degree of persistence i n the sense that there i s con t i n u i t y of a p a r t i c u l a r s p e c i a l i z e d region of the cytoplasm. The leading lamella tends to remain such. The taunt c u r v i l i n e a r portions of the c e l l periphery r e s i s t formation of lamellae. This translates into a persistence of c e l l m o t i l i t y such that c e l l s move i n segments, with the t r a n s i t i o n s between segments marked by large scale changes i n c e l l morphology. 3.2.2 Irr a d i a t e d c e l l s 3.2.2.a Low magnification and long time span. Records were made of i r r a d i a t e d 3T3 c e l l s using a 6. 3x objective lens and b r i g h t f i e l d microscopy, s i m i l a r to the untreated c e l l s . The images were recorded at 5 and 8 minute i n t e r v a l s . Test doses of 0, 0.75, 1.2, 2.0 and 8.0 Gy were given. There were two types of protocol used, both designed to provide an i n t e r n a l control to compare to the i r r a d i a t e d c e l l s . The f i r s t protocol was as follows: c e l l s were plated into f l a s k s and incubated f o r 20 hrs. A l i n e was i n s c r i b e d across the bottom of the f l a s k and when the f l a s k was i r r a d i a t e d , h a l f the f l a s k was covered by approx. 2 cm. of lead s h i e l d i n g , and the other h a l f was unprotected. A f t e r i r r a d i a t i o n the f l a s k was placed onto the DMIPS stage. A set of 10 microscope f i e l d s was selected to be subsequently recorded, of which the f i r s t 4 f i e l d s were i n the shielded region of the f l a s k , while the remaining f i e l d s were f u l l y exposed to x-rays. (Records of t h i s type can be found stored on disk #1, frame #16,000 to 23,600, which contains the records from several experiments (see OMDR data record fo l d e r f o r f u l l d e t a i l s ) ) . The second protocol required the f l a s k to be i r r a d i a t e d a f t e r a group of c e l l s had been already recorded, then again monitoring these same c e l l s . The c e l l s were plated into the f l a s k and incubated f o r 20 hrs. The f l a s k was then 87 taken to the microscope stage and 10 f i e l d s were manually selected. These f i e l d s were recorded f o r a period of time, the f l a s k was removed from the microscope system and quickly given a t e s t dose of r a d i a t i o n , then the same microscope f i e l d s were subsequently recorded following the treatment. (Data of t h i s type can be found on disk #2, frames #21,900 to 23,900. In t h i s experiment the c e l l s were i r r a d i a t e d a f t e r the 109th scan to a dose of 2.0 Gy). These records were examined f o r perturbations to dynamic c e l l behaviour following i r r a d i a t i o n . The following observations were made: 1) For most c e l l s , at the t e s t doses given (<5 Gy) , a large perturbation to the dynamic c e l l behaviour i s not observed. I f the i r r a d i a t i o n was done i n the middle of the time lapse record, then most c e l l s maintained a s i m i l a r pattern of behaviour a f t e r the treatment. I f the f l a s k contained both shielded and d i r e c t l y i r r a d i a t e d c e l l s , then most i r r a d i a t e d c e l l s d i splay behaviour that i s within the broad l i m i t s of the control c e l l behaviour. For the c e l l s undergoing mitosis during the record, the gross morphological changes that accompany mitosis were s u f f i c i e n t l y large as to obscure any perturbation induced by the r a d i a t i o n . 2) A f r a c t i o n of the c e l l s display an unusual behaviour following i r r a d i a t i o n . S i g n i f i c a n t a l t e r a t i o n s i n the rate of change of c e l l shape, and other parameters, were noted i n some c e l l s . (For example, i n frame #22,701, disk #2, i n the r i g h t middle of the f i e l d , a c e l l was recorded that was quite f l a t t e n e d and well-spread. The c e l l was quiescent u n t i l the i r r a d i a t i o n , and a f t e r treatment the c e l l begins cytoplasmic contractions and undergoes some movement. Another example i s on disk #2, beginning on frame 23,500. A c e l l i n the r i g h t middle of the f i e l d i s again spread and quiescent p r i o r to i r r a d i a t i o n , but following treatment i t becomes quite a c t i v e and undergoes rapid morphological changes). In the dose regime of 0 to 5 Gy i t i s estimated 88 that l e s s than 10% of the i r r a d i a t e d c e l l s displayed s i g n i f i c a n t changes i n c e l l behaviour. To summarize, from the observed cases of behavioral changes that appear to be due to the i r r a d i a t i o n treatment, one feature i s a short term increased i n cytoplasmic a c t i v i t y of quiescent c e l l s . This manifests as i ) more rapid changes i n c e l l morphology, i i ) contractions i n spread c e l l s , and i i i ) as increased a c t i v i t y of c e l l u l a r extensions. These perturbations are most e a s i l y v i s u a l i z e d i n previously quiescent c e l l s , while r a p i d l y moving c e l l s and c e l l s that undergo d i v i s i o n near the time of i r r a d i a t i o n have an inherent wide range of dynamic behaviour and thus i t i s d i f f i c u l t to detect r e l a t i v e l y subtle perturbations with q u a l i t a t i v e assessment of time lapse records. The dose response and time response of t h i s e f f e c t are d i f f i c u l t to assess with t h i s assay. 3.2.2.b High magnification and short time scale Records were made of i r r a d i a t e d 3T3 c e l l s using a 16x objective and phase contrast microscopy. Images were recorded at 3.0 and 0.25 minute i n t e r v a l s . During the shorter i n t e r v a l experiments only a sin g l e c e l l per experiment was monitored and the stage was not engaged. Test doses of 0, 2.0 and 8.0 Gy were applied to c e l l s immediately p r i o r to the s t a r t of recording. 4 The protocol was as follows: c e l l s were plated into f l a s k s at 3 to 5 x 10 c e l l s / f l a s k and incubated f o r approximately 20 hrs. The c e l l s were then i r r a d i a t e d to the t e s t dose, and placed upon the microscope stage i n the pre-warmed stage incubator. Approximately 15 minutes were given f o r the f l a s k to temperature e q u i l i b r a t e , then the t e s t c e l l ( s ) were manually selected and recording begun. Records up to 5 hours of r e a l time were made. (The records are stored on disk #2, frames #19,400 to 20,899 (3 minute i n t e r v a l ) and disk #2, frames #17,500 to 18,499 (0.25 minute i n t e r v a l ) ) . 89 Certain i r r a d i a t e d c e l l s displayed behaviour that seems outside the range of unperturbed c e l l s . Some examples are evident i n the records of c e l l s i r r a d i a t e d to 8 Gy. (For example, the c e l l i n frame #19,403, disk #2, had j u s t been exposed to a large dose of x-rays, 8.0 Gy. From frame #19,403 to #19,458 i t was a c t i v e l y migrating and undergoing r a p i d large scale changes i n c e l l morphology, p a r t i c u l a r l y before frame #19,434. Then the c e l l became rounded and compacted as i f i t was to enter mitosis. I t s m o t i l i t y and most of i t s morphological a c t i v i t y were dramatically reduced. The c e l l remained i n the compact state from scan #19,458 u n t i l the end of the record, a duration of 129 minutes r e a l time. The c e l l ( s ) recorded i n frames #19,803 to 19,899, disk #2, also underwent very active motion and r a p i d large scale changes of c e l l morphology. This i s p a r t i c u l a r l y true i n the e a r l y p o r t i o n of the record, and the rate of a c t i v i t y seems to subside s l i g h t l y toward the end of the record). The c e l l s treated to 2.0 Gy were generally quite active immediately following treatment, but the pattern of a c t i v i t y i s apparently within the range of untreated c e l l behaviour. The c e l l s were somewhat more motile during the e a r l y p o r t i o n of the time lapse recording, and became less active towards the end of the 5 hr recording period. (For example, the c e l l recorded on frames #20,700 to 20,799, disk #2, underwent rapid r u f f l i n g and p i n o c y t o t i c uptake from frames #20,700 to 20,761, then seemed to spread and become les s active. Another example i s found on disk #2, frames #20,500 to 20,599. This c e l l was i n i t i a l l y p o l a r i z e d and r a p i d l y locomoted u n t i l approx. frame #20,580 at which time i t became spread and non-polarized). The c o n t r o l c e l l s f o r these experiments displayed somewhat more conti n u i t y i n terms of maintaining t h e i r motile c h a r a c t e r i s t i c s . (This i s seen upon an examination of the records on disk #2, frames #19,900 to 20,399). 90 3.3 Automated Measurements of M o t i l i t y and Morphology The DMIPS C e l l Analyzer and the RSCAN c e l l tracking program were used to measure the m o t i l i t y of untreated and i r r a d i a t e d 3T3 c e l l s i n f l a s k s . The data were stored i n the form of .TRK and .CHN f i l e s i n computer memory and l a t e r examined with the RDISP and PLOT programs. As i n the cases of the gold dust and time lapse assays, a d e t a i l e d analysis of the movement of untreated c e l l s i s necessary before perturbed c e l l s are examined. 3.3.1 Control c e l l s : M o t i l i t y 3.3.1.a Individual c e l l s The automated microscope system was used to record d e t a i l e d information on the motile performance of i n d i v i d u a l c e l l s i n tissue culture p l a s t i c f l a s k s . The data from each c e l l were examined and/or manipulated i n an e f f o r t to understand the locomotion of 3T3 c e l l s at the i n d i v i d u a l c e l l l e v e l . The most straightforward method to present the data i s i n the form of the walk pattern. Shown i n Figure 3.8 are examples of several walk patterns taken from d i f f e r e n t c o n t r o l experiments. The information i n these patterns correlates with the q u a l i t a t i v e observations of c e l l paths from the time lapse records, but several features are further emphasized. The f i r s t i s the wide v a r i e t y of walk patterns displayed by d i f f e r e n t c e l l s , and indeed, by a single c e l l at d i f f e r e n t times. Certain c e l l s remained within a r e s t r i c t e d area as they exhibited slow movement and/or frequent changes i n d i r e c t i o n . Other c e l l s displayed periods of rapid motion without d i r e c t i o n a l changes, and accumulated large net displacements. C e l l s were observed to switch between these extreme types of behaviour. Another general observation from the walk pattern i s the persistence, or degree of persistence, displayed at d i f f e r e n t times by an i n d i v i d u a l c e l l . When a c e l l was moving i t tended to maintain i t s d i r e c t i o n from one scan to b L 28 fllCRONS c L 28 MICRONS e L 28 MICRONS f i L 28 MICRONS FIGURE 3.8: Examples of walk patterns of untreated 3T3 c e l l s . Each small cross represents the recorded positions of the c e l l centre extracted at 8 minute i n t e r v a l s . The i n i t i a l c e l l p o s i t i o n i s denoted by the '1' i n the centre of the f i e l d . Shown i s the c e l l movement recorded during 70 scans (=560 minutes). 92 the next, which r e s u l t s i n low angles of turn. Conversely a quiescent c e l l also tends to maintain i t s p a r t i c u l a r behaviour. The m o t i l i t y data from an i n d i v i d u a l c e l l may also be analyzed by measuring the magnitude of the displacement from the previous c e l l p o s i t i o n as a function of time. I f the displacement i s modified by the i n t e r v a l between scans, t h i s y i e l d s a p l o t of i n d i v i d u a l c e l l speed vs time. Shown i n Figure 3.9 are two examples of t h i s analysis, from the walk patterns as above. Some ad d i t i o n a l observations on 3T3 c e l l movement were made. F i r s t l y , the maximum recorded c e l l speed was roughly 3.0 microns/minute as measured over an 8 or 10 minute time i n t e r v a l (presumably instantaneous c e l l speeds may be larger but are not maintained for 8 minute i n t e r v a l s ) . Secondly, the large c e l l speeds tend to appear i n c l u s t e r s . Note the burst of high speed movements i n the middle of Figure 3.9.b. These correspond to r a p i d l i n e a r movement i n the walk pattern. A peak of c e l l movement which l a s t s f o r approximately 60 minutes i s often observed. T h i r d l y , the c e l l speed records show a seri e s of peaks and v a l l e y s , and indicates that even for the most r a p i d l y migrating c e l l , the movement process i s undulatory. The 8 to 10 minute sampling i n t e r v a l appears to be s u f f i c i e n t l y r a pid to detect the undulating behaviour. The data from an i n d i v i d u a l c e l l was pooled to give the average speed of that c e l l over a segment of time, or to give the d i s t r i b u t i o n of i t s speeds (Figure 3.10). This analysis showed that amongst a 'homogeneous' c e l l population, i n d i v i d u a l c e l l s showed large differences i n behaviour. For example, the c e l l depicted i n 3.10.a showed an average speed of 0.64 /xm/min, and shows a peak at approximately 1.5 /im/min, while the c e l l i n 3.10.C moved at an average of 0.42 /»m/min and displayed no peak at 1.5 /im/min. This c e l l u l a r inhomogeneity holds true for measurements of at l e a s t up to 12 hours. This observation was also noted by viewing the time lapse records, but with the automated microscope data, a degree of quantitation was pos s i b l e . 9 3 3 . 0 c i 2 . 0 2 . 0 FIGURE 3.9: The c e l l speed vs scan number for untreated 3T3 c e l l s . Shown are two examples corresponding to the walk patterns i n Figure 3.8.b and 3.8.d resp e c t i v e l y . Each point represents the distance moved i n the immediately preceding 8 minute i n t e r v a l . 0 Velocity (um/min) 3 FIGURE 3.10: The d i s t r i b u t i o n o f c e l l speeds f o r u n t r e a t e d 3T3 c e l l s . Shown a r e d i s t r i b u t i o n s from t h e w a l k p a t t e r n s i n F i g u r e 3.8.a-d. The average c e l l s p e e d o f each d i s t r i b u t i o n i s g i v e n i n t h e upper r i g h t o f each p a n e l . 95 The v e l o c i t y vs time p l o t (Figure 3.9) does not contain information on the angle of the c e l l displacement, nor on the persistence. The displacement analysis was designed to emphasize the short term persistence of c e l l movement. D i f f e r e n t lengths of the f i l t e r (section 2.3.6) emphasize d i r e c t i o n a l persistence of d i f f e r e n t time scales. Shown i n Figure 3.11 are several examples of i n d i v i d u a l c e l l movements analyzed with the displacement f i l t e r , derived from the walk patterns above. Several observations regarding normal 3T3 c e l l m o t i l i t y were made from t h i s type of a n a l y s i s : 1) The recorded movement usually resolved into a ser i e s of segments. The segments were compared to the walk patterns and the v e l o c i t y p l o t s and found to be a meaningful r e s o l u t i o n of the data. 2) In the f i r s t approximation, 3 types of m o t i l i t y segments were i d e n t i f i e d . The f i r s t of these i s the d i r e c t e d motile bursts which appear on the displacement analysis graph as a demarcated peak. The second i s the quiescent or d i r e c t i o n a l change behaviour, which appear on the graph as a v a l l e y or trough. The t h i r d i s the nondirected m o t i l i t y which appear as a (high) plateau. 3) C e l l s are able to switch between each segment, and a motile c e l l displays a ser i e s of peaks and troughs rather than a nondescript behaviour. The time length of each segment i s somewhat v a r i a b l e , and the rate of switching between segments i s also not f i x e d . However, bursts of directed movement of duration approximately 60 minutes were also detected with t h i s analysis, and unperturbed c e l l s showed a c h a r a c t e r i s t i c rate of switching between segments. In Figure 3.11, note that the peaks of d i r e c t e d c e l l movement are often of 50-100 minute duration, and are repeated 3-5 times per 500 minute record. 4) Some work was done to examine the c e l l cycle v a r i a t i o n s of m o t i l i t y i n unperturbed c e l l s . For these tests, i n d i v i d u a l c e l l s were tracked for an FIGURE 3.11: The displacement analysis of untreated 3T3 c e l l movement. Shown are the s i x m o t i l i t y records depicted i n Figure 3.8, each analyzed with a displacement f i l t e r of length-5. Compare 3.11.b to 3.9.a and 3.11.d to 3.9.b. CO G» 97 extended period and the point of c e l l d i v i s i o n (cytokinesis) was marked for each c e l l e i t h e r by manual observation or by automated morphology analysis. From t h i s data, and knowing the (constant) duration of the phases of M, G2, and S, the m o t i l i t y was examined throughout the c e l l c ycle. The data (not shown) indicates that c e l l s frequently underwent a set of two or three segments of dire c t e d movement l a t e i n the c e l l cycle (G2 and l a t e S) , immediately p r i o r to rounding up. The data from the other phases of the cycle i s more equivocal and requires further study. 3.3.1.b Population of c e l l s In a d d i t i o n to examining the behaviour of unperturbed i n d i v i d u a l c e l l s , the data from many c e l l s i n one (or more) experiments was combined to produce population averages of various c e l l m o t i l i t y parameters. This approach emphasizes the advantages of the automated system because large numbers of c e l l s were monitored i n a single experiment with r e l a t i v e ease. For the treatment of population data, several techniques were applied to characterize the movement. The co n t r o l behaviour of several parameters w i l l be presented here. The f i r s t parameter analyzed was the average c e l l speed. The average c e l l speed for 3T3 c e l l s measured under these experimental conditions was i n the range 0.4 to 0.7 /im/min (Figure 3.12). The average speed was quite constant throughout the co n t r o l experiments, even i f the c e l l s were removed from the microscope at some point i n the experiment and given a mock x-ray treatment. In addition, the average c e l l speed was f a i r l y constant on a day to day bas i s . However there were changes i n average speed from month to month as the c e l l s were passaged or as new batches of c e l l s were thawed. Quite often the average c e l l speed decreased s l i g h t l y during the course of longer experiments. Generally at l e a s t 8 to 10 hours of observation were needed before t h i s was detected. I t i s not known i f t h i s was due to "natural" Average Cell Speed vs Time 3T3 cells 2.0 c i E 3 . •01.0 0) a . CO Q) o • D • • • 0 • D • D a D . • 0 ° • D n D • • • • D a a D • • u 0 n • • • on ° O Q • • ' 1 ' 1 1 1 i 1 1 1 i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 1 1 i i i 1 1 1 1 1 8 TIME ( M i n ) - > T~T 544 FIGURE 3.12: The average c e l l speed as a function of time for untreated 3T3 c e l l s . Data i s averaged from 37 c e l l s , each measured every 8 minutes f o r a period of approximately 9 hours. Average speed was quite constant throughout experiment at approximately 0.70 ^m/min. CO CO 99 causes such as c e l l p r o l i f e r a t i o n , increased e x t r a c e l l u l a r matrix, or depletion of serum, or whether t h i s was due to perturbation of the c e l l s by the measurement process such as a l t e r a t i o n of the medium by exposure to l i g h t , or non-ideal growth conditions. As mentioned, t h i s e f f e c t was small (<5% of the average c e l l speed a f t e r 8 hours of observation) and d i d not seem to a f f e c t the morphology or p r o l i f e r a t i o n of the c e l l s . The average c e l l speed was s l i g h t l y l e s s i n c a l f serum than i n f e t a l bovine serum, approximately 0.40 vs 0.60 /im/min r e s p e c t i v e l y (data not shown). Another m o t i l i t y parameter for a population of c e l l s i s the d i s t r i b u t i o n of c e l l speed. The c e l l speeds from a selected time segment were pooled for a l l the c e l l s i n the population and a d i s t r i b u t i o n p r o f i l e was made. This was compared at d i f f e r e n t periods i n an experiment. Shown i n Figure 3.13 are the d i s t r i b u t i o n s from a group of untreated 3T3 c e l l s taken at two periods during an experiment (same experiment as shown i n Figure 3.12). The d i s t r i b u t i o n s have s i m i l a r average values; 0.69 /im/min vs. 0.66 /xm/min. A further c h a r a c t e r i z a t i o n of the m o t i l i t y of a population of c e l l s was done by examining the d i s t r i b u t i o n of angles between successive displacement vectors. An example f o r untreated c e l l s i s shown i n Figure 3.14. These data show a peak i n the angle d i s t r i b u t i o n around 0°, and indicate the d i r e c t i o n a l persistence of m o t i l i t y . The peak has a width at half-max of approximately 60°. As described e a r l i e r , a report by G a i l and Boone analyzed the m o t i l i t y of 3T3 c e l l s i n terms of a 2-dimensional random walk. The average square of 2 the displacement, <D >, was p l o t t e d as a function of time to y i e l d the two parameters D and t (see equation i n Figure 3.15). This was repeated i n the present experiments with the a l t e r a t i o n s that a larger number of c e l l s were used, the time i n t e r v a l over which each displacement value was measured was varied, and the parameter values were estimated f o r d i f f e r e n t segments within 1 0 0 Cell speed (Lim/min) FIGURE 3.13: The d i s t r i b u t i o n of c e l l speeds for untreated 3T3 c e l l s . a) data taken from the middle 1/3 of Figure 3.12 (time-170-340 minutes), b) data taken from the end 1/3 of Figure 3.12 (time=340-510 minutes). D i s t r i b u t i o n s show s i m i l a r shape and average. Average speed f o r a) =0.69 ^m/min. b) 0.66 /im/min. —I 1 1 1 h Relative Angle FIGURE 3.14: The d i s t r i b u t i o n o f r e l a t i v e a n g l e s f o r u n t r e a t e d 3T3 c e l l s . D ata a r e from t h e 37 c e l l s d e p i c t e d i n F i g u r e 3.12 a v e r a g e d o v e r the e n t i r e d u r a t i o n . 16012 j * 2 CM CM Q V < D 2 > = 4 D * ( t - f ) D = 5 4 2 urn HI III II III II II 11 Ml III II III II III II III II M i l l II M i l l I TIME (nin) -> 544 FIGURE 3.15: Example o f <D > vs t f o r u n t r e a t e d 3T3 c e l l s . Shown i s the t e c h n i q u e f o r e s t i m a t i n g t h e p a r a m e t e r s D and t . D i s measured from the s l o p e a t l o n g e r t i m e s , and t f r o m t h e x - i n t e r c e p t . D a t a a r e from an e x p e r i m e n t w h i c h m o n i t o r e d 20 c e l l s e v e r y 8 m i n u t e s f o r 60 s c a n s . Each p o i n t r e p r e s e n t s t h e ave r a g e s q u a r e o f t h e n e t d i s p l a c e m e n t t a k e n f r o m the c u r r e n t p o s i t i o n t o the o r i g i n a l p o s i t i o n . Each p o i n t i s t h e r e s u l t a t 24 minute i n t e r v a l s . 103 the same experiment. Shown i n Figure 3.15 i s an example of <D > vs time, along with the estimated values for the augmented d i f f u s i o n c o e f f i c i e n t D and the modified time constant, t . These values agree with those published by G a i l and Boone, although they were found to vary from one experiment to the next. The value for t v a r i e d by 50% between comparable experiments, and the * 9 value for D v a r i e d by 25%. In addition, the p l o t of <D > vs t produced a smooth curve from 0 to 8 hours, and then often displayed kinks at longer times. The curve i s expected to e x h i b i t n o n l i n e a r i t i e s at longer times according to the f u l l t h e o r e t i c a l treatment (107), however not of the type seen i n the data. The explanation f o r t h i s e f f e c t i s not known. A l l of the above techniques have a "snapshot" approach i n that each recorded c e l l displacement i s taken as e s s e n t i a l l y separate from i t s immediate predecessors and successors. As emphasized i n t h i s thesis and elsewhere, the aspect of persistence i s important i n c e l l m o t i l i t y . A technique to characterize the short term continuity of behaviour for a population of c e l l s i s to p l o t the d i s t r i b u t i o n of displacement (and angle) according to the displacement on the previous scan. This type of analysis was c a l l e d forecasting. Examples from untreated 3T3 c e l l s are shown (Figures 3.16 to 3.18), and from these several observations were made: 1) The data show d i s t i n c t l y d i f f e r e n t d i s t r i b u t i o n s of displacement from the d i f f e r e n t bins (Figure 3.16). A c e l l that moved between 0 to 3 microns i n 8 minutes was l i k e l y to move an average of 4.3 microns i n the next 8 minute i n t e r v a l , and had a p r o b a b i l i t y of about 0.10 of moving more than 10 microns. Conversely, a c e l l that moved 12 to 29 microns was poised to move an average of 8.5 microns and had a p r o b a b i l i t y of about 0.40 of moving more than 10 microns during the next 8 minute i n t e r v a l . 2) The d i s t r i b u t i o n s of r e l a t i v e angles were c a l c u l a t e d f o r each of the bins (Figure 3.17). The data show some evidence for differences i n the c o 4— u CO LL .. 20 30 Displacement (um) 6 to 9UM T= 466 a= 6.35 \ . C 1 - a * jn263t3a 37 ce l l s Scans 1 to 69 Forecast L= 1 FIGURE 3.16: The forecast analysis f o r untreated 3T3 c e l l s . Shown i s the same data as displayed i n Figure 3.12. The data has been broken into 5 bins (0-3 um, 3-6 um, 6-9 um, e t c . ) . Shown by the boxes i s the d i s t r i b u t i o n of the next displacement, d^ +^. given that the displacement d^ i s i n the appropriate range. The data show that i f a c e l l moved at a higher speed during the i n t e r v a l , then during the next 8 minute i n t e r v a l i t had an increased p r o b a b i l i t y of again moving at a high speed. Displayed i n the upper r i g h t of _ L each panel i s the range f o r that panel, the number of events that f a l l within O that range to comprise the d i s t r i b u t i o n , and the re s u l t a n t mean. 105 Relative Angle FIGURE 3.17: The forecast analysis of the d i s t r i b u t i o n of r e l a t i v e angles for untreated 3T3 c e l l s . Data as i n Figure 3.12. The d i s t r i b u t i o n of the r e l a t i v e angle for the i ' t h displacement was plo t t e d for d i f f e r e n t sizes of displacement. 106 CO 4 ' 3 ' * ' o N» CO . t (uurl) iu8iuaoB|dS!a FIGURE 3.18: The decay of persistence of untreated 3T3 c e l l s . P lotted i s the average displacement as a function of increasing the forecast length. Open tr i a n g l e s : 12 Ltm to 20 Ltm bin. Closed boxes: 9 fim to 12 Ltm bin. Open boxes: 6 (ii to 9 Ltm bin. Open c i r c l e s : 3 Ltm to 6 Ltm bin. Closed c i r c l e s : 0 pm to 3 Ltm bin. Data taken from same experiment as shown i n Figure 3.16. b) data from only 0-3 Ltm and 12-20 Ltm bins on logaithmic scale. Estimated parameter values: 0-3 Ltm; VA=5.2 Ltm, a=-1.23 Ltm, A=0.0449 /min. 12-20 Ltm; VA=6.5 Ltm, a-2.17 Ltm, A=.0535 /min. 5 7 9 1 1 1 3 Forecast Length 108 p r o f i l e s f o r r a p i d l y migrating vs. slowly migrating c e l l s . This observation i s based upon estimations of the f u l l width at h a l f maximum of the peak i n the d i s t r i b u t i o n of angles. I t should be noted that these estimates are accompanied by large un c e r t a i n t i e s . 3) The decay of the persistence of c e l l movement was estimated by applying increasing forecast lengths. Shown i n Figure 3.18 i s the average c e l l displacement with increasing forecast length. The average c e l l displacement from the d i f f e r e n t bins changes toward the o v e r a l l average rate of movement as the forecast length i s increased. Thus the average displacement from the b i n 0-3 /xm increases from the value of 4.3 /im with increasing forecast length, and the average displacement from the b i n 9-12 /im decreases. The rates of decay of the average displacement were estimated by f i t t i n g the data to the equation V L = V A + a e " A L where L i s the length of the forecast, A i s the i n i t i a l decay rate, i s the asymptotic average displacement of the b i n at large forecast length, and i s the average displacement of the b i n with a forecast length L. The data were f i t by estimating a value for V^, then p l o t t i n g In ( V L - V A) = In a - AL and d e r i v i n g a l i n e a r least=squares f i t for the values of a and A. Shown i n Figure 3.18.b i s the p l o t f or two of the bins depicted i n 3.18.a. Given i n the fi g u r e caption are the c a l c u l a t e d values f o r A and a, along with the estimated value for V^. The curves shown i n the fi g u r e are c l c u l a t e d from the f i t to the data. The important point here i s that the values for the asympotic average displacement, V^, are d i f f e r e n t f o r the d i f f e r e n t bins. This indicates that untreated c e l l s maintain long term persistence of motile behavior, at l e a s t up to a duration of several hours. 109 3.3.2 Control c e l l s : C e l l morphology C e l l morphology i s an important determinant i n numerous c e l l u l a r processes. I t strongly influences c e l l m o t i l i t y and i n return the motile process feeds back to influence the c e l l morphology. The C e l l Analyzer was used to concurrently measure m o t i l i t y and morphology from i n d i v i d u a l c e l l s i n f l a s k s . The program i n the DSP extracted an o u t l i n e of the c e l l shape , and a b i t map representation of the shape along with several shape features were stored i n data f i l e s . These f i l e s were then l a t e r examined with the RDISP and PLOT programs. The c e l l morphology record was examined both on i t s own and i n conjunction with the corresponding record of c e l l m o t i l i t y . Experiments were performed on untreated 3T3 c e l l s to serve as c o n t r o l . 3.3.2.a Individual c e l l s As i n the case of the m o t i l i t y data, the c e l l morphology records were analyzed both at the i n d i v i d u a l c e l l l e v e l , and at the population l e v e l , i n which data from many c e l l s i n a s i n g l e experiment were pooled to derive population averages. At the single c e l l l e v e l , several morphology features were examined, and examples from untreated c e l l s are shown. The c e l l area, brightness, and c i r c u l a r i t y are r e l a t e d global shape features, and each changed on a s i m i l a r time scale (Figures 3.19 to 3.21). Large scale changes occurred on the order of 1 to 2 hrs. A global morphology feature that was stored i n the data f i l e but i s not derivable from the b i t map representation i s the average c e l l brightness. This feature was generally constant f o r an i n d i v i d u a l c e l l during observation, but proved to be useful f o r i d e n t i f y i n g m i t o t i c c e l l s . As a 3T3 c e l l rounded up p r i o r to cytokinesis, the average c e l l brightness r a p i d l y increased. Upon cytokinesis, the s i s t e r c e l l s respread quite r a p i d l y , and the brightness decreased (the brightness was measured f o r one of the s i s t e r c e l l s , a r b i t r a r i l y selected). Thus a c e l l mitosis was i d e n t i f i e d as a very d i s t i n c t FIGURE 3.19: The c e l l area as a function of time f o r 4 i n d i v i d u a l untreated 3T3 c e l l s . Morphology measured every 8 minutes f or 70 scans. Data shown corresponds to c e l l records i n Figure 3.8. 3.19.a-3.8.c. 3.19.b=3.8.d. 3.19.c=3.8.e. 3 .19 . d-3 . 8 . f. C e l l area measured i n // of p i x e l s , where 1 p i x e l - 1 um . 30 3a FIGURE 3.20: The average brightness as a function of time f o r 4 i n d i v i d u a l untreated 3T3 c e l l s . Same c e l l s as depicted i n Figure 3.19. Note change of scale i n (d), i n which y-axis has been enlarged by a factor of approximately 3x. 112 FIGURE 3.21: The c e l l c i r c u l a r i t y as a function of time for 4 individual untreated 3T3 c e l l s . Same c e l l s as depicted i n Figure 3.19. The c i r c u l a r i t y i s defined as C = P 2 4*A This yields a value of 1.00 for a c i r c l e , and a larger value for objects deviating from a c i r c l e . A large value of c i r c u l a r i t y generally indicates an elongated c e l l . 92 (0 CO <D C •*-> sz 0) o CELL FEATURE PLOT 3T3 eel + -15.9 hrs-*-4 v I + + + % * t + f A 0 ' 0 Time (min.) 3969 FIGURE 3.22: The average c e l l brightness as a function of time for a single untreated 3T3 c e l l . The c e l l was scanned every 10 minutes for 400 scans, during which time i t divided 4 times. Each d i v i s i o n was marked by a prominent peak on the f i g u r e . The i n t e r d i v i s i o n time for the f i n a l mitosis i s marked. These data are s i m i l a r to that shown i n Figure 3.20 except the experiment was of much longer duration, and the scale of y-axis i s d i f f e r e n t . 114 peak i n c e l l brightness which l a s t e d approximately 1 hr and corresponded to events i n the area and perimeter records (Figures 3.22, 3.20.d). An example from a long experiment i s shown (Figure 3.22) i n which several c e l l d i v i s i o n s are evident. The time i n t e r v a l between d i v i s i o n s was c a l c u l a t e d and appears on the f i g u r e . 3.3.2.b Populations of c e l l s The morphology data from many c e l l s within an experiment were also combined to give population averages. The average c e l l area, c e l l brightness and c e l l c i r c u l a r i t y were examined for co n t r o l c e l l s (Figure 3.23). In "steady state" culture conditions, the average c e l l area f o r 3T3 c e l l s was measured to be approximately 500-550 p i x e l s using t h i s microscopy set-up, 2 which i s very close to 500-550 /un . Individual c e l l v a r i a t i o n s were from 200 to 900 p i x e l s . The average c e l l brightness was approximately 18. The average c e l l c i r c u l a r i t y was measured to be approximately 4.0, with i n d i v i d u a l c e l l v a r i a t i o n s from 1.3 to 8.0. The feature of c e l l brightness was quite s e n s i t i v e to the focus, and the technique was not s u f f i c i e n t l y developed to compare absolute values of c e l l brightness from one experiment to another. 3.3.2.C M o t i l i t y and morphology The c e l l morphology data was compared to the c e l l m o t i l i t y . Figure 3.24 shows an example of i n d i v i d u a l c e l l behaviour i n which the morphology feature ' c i r c u l a r i t y ' i s displayed along with the displacement f i l t e r analysis graph. Each graph shows a seri e s of peaks and troughs, and i t was observed that rapid d i r e c t e d c e l l movements often correspond to large changes i n c e l l c i r c u l a r i t y . The change i n c i r c u l a r i t y may e i t h e r be an increase (a rounded c e l l undergoing an elongation) or a decrease (an elongated c e l l p u l l i n g forward into a more rounded shape). This i s i n agreement with the r e s u l t s from the time lapse records. 790 CD X Z + CD a> CO CD > < +++, » 1 * \ +++ 358,8 38 567 TiMc(Min) in in CD c w CO CD O) CO CD > < + + +++ + + + + + + + + + + + •H- + + t t t t + + t +++ + + + + + + + ++ ++++ + ++ +++ IB, 8 9.88 367 TiM«<nin) 3 O o CD o> CO CD > < + +++ +++44+ ^ / + + + + + + + +++ * + + +++++ + ++++ + +++ T i n e ( n i n ) 567 FIGURE 3.23: Population average c e l l morphology features as a function time for untreated 3T3 c e l l s . Data from experiment depicted i n Figure 3.1 a) average c e l l area. b) average c e l l brightness. c) average ce c i r c u l a r i t y . C/3 c 3 c £ 0) o CO a CO 116 40 60 80 S c a n n u m b e r 100 20 40 60 80 100 Scan number FIGURE 3.24: Combined m o t i l i t y and morphology information f o r a single untreated 3T3 c e l l . C e l l was scanned every 8 minutes f or 120 scans. a) displacement analysis, with displacement f i l t e r length=5. b) c e l l c i r c u l a r i t y . 117 The d e t a i l e d analysis of the morphology and m o t i l i t y i s a laborious process. Methods have not yet been developed to compare c e l l movement and morphology i n a quantitative way f o r i n d i v i d u a l c e l l s or for populations of c e l l s . 3.3.3 Ir r a d i a t e d c e l l s : M o t i l i t y and morphology Experiments were performed that measured the m o t i l i t y and morphology of 3T3 c e l l s following i r r a d i a t i o n i n tissue culture f l a s k s . The experiments were done eit h e r with a period of c e l l tracking p r i o r to the i r r a d i a t i o n , or without a control period so that c e l l s were tracked immediately post-i r r a d i a t i o n . In the f i r s t case, c e l l s were plated and incubated i n f l a s k s for approximately 14 hours, then a selected group of c e l l s were tracked on the C e l l Analyzer for 1 to 2 hours p r i o r to treatment. Following treatment, the same set of c e l l s were tracked for a subsequent 6 to 8 hours. In the second case, the c e l l s were incubated f o r approximately 14 hours, i r r a d i a t e d to the t e s t dose, and then a set of c e l l s was selected and tracked f o r 6 to 8 hours. In each case, a set of several experiments, both co n t r o l and i r r a d i a t i o n , were performed within the span of less than a week. The r e s u l t s were compared within each set of experiments. 3.3.3.a Individual c e l l s The data was f i r s t analyzed at the i n d i v i d u a l c e l l l e v e l . The walk patterns from i r r a d i a t e d c e l l s were compared with t h e i r c o n t r o l counterparts. Similar to controls, the i r r a d i a t e d walk patterns d i s p l a y a wide v a r i e t y of patterns. The data i n t h i s format i s d i f f i c u l t to quantify, but the walk patterns of c e l l s i r r a d i a t e d to doses up to 8 Gy do not appear s i g n i f i c a n t l y d i f f e r e n t from control (Figure 3.25). At very high doses, c e l l m o t i l i t y i s s u f f i c i e n t l y disrupted as to be evident i n the walk patterns. The displacement analysis was applied to the movements of i r r a d i a t e d c e l l s . As discussed i n the section on control c e l l s (Section 3.3.1.a), the FIGURE 3.25: Walk patterns f o r i r r a d i a t e d 3T3 c e l l s . Each c e l l was scanned every 8 minutes for 70 scans. C e l l s depicted i n a) - d) i r r a d i a t e d to 8 Gy. C e l l depicted i n e) - f) i r r a d i a t e d to 2 Gy. Note scale change i n (b). N e t D i s p l a c e m e n t N e t D i s p l a c e m e n t rt a O M w o M i 13 CI — ' M pa m ce a w <t> • 3 ro r t » * * M l r t (6 i - l len he tm r t 3* a g H * U T3 p j t/3 O 0] <t> 3 3 3 0 r t a> M M p , to 3 » » 01 |-> to v<; U tr 3 in C M c l-t M l o i-t • M ro K • a M -«. 113 p i r t — (t) a r t O 120 relevant feature i n the analysis i s the pattern of c h a r a c t e r i s t i c segments. Shown i n Figure 3.26 are displacement analysis p l o t s of c e l l s i r r a d i a t e d to d i f f e r e n t doses immediately p r i o r to the experiment. I t appears from these p l o t s , and others not shown, that the pattern i n the displacement graph i s a l t e r e d i n some i r r a d i a t e d c e l l s ,for example Figure 3.26.a. The dire c t e d bursts of m o t i l i t y , shown as the peaks of the graph, require the coordinated i n t e r a c t i o n of the e n t i r e c e l l , and w i l l be concentrated upon here. The di r e c t e d bursts of i r r a d i a t e d c e l l s are of shorter duration i n t h i s example and are separated by shorter i n t e r v a l s than non-irradiated c e l l s . This can be qu a n t i f i e d by measuring the average number of peaks i n a 6 hour i n t e r v a l , the duration of the peaks, and the dispersion of these. The global c e l l morphology of i n d i v i d u a l i r r a d i a t e d c e l l s was measured. Shown i n Figures 3.27 to 3.29 are the morphology parameters area, brightness, and c i r c u l a r i t y . The shape parameter ' c i r c u l a r i t y ' was seen to be cor r e l a t e d with the movement of untreated c e l l s , i n p a r t i c u l a r with the displacement analysis pattern. The c i r c u l a r i t y as a function of time f o r several i n d i v i d u a l i r r a d i a t e d c e l l s i s shown i n Figure 3.29 (same c e l l s as displacement a n a l y s i s ) . Again, the c i r c u l a r i t y of i r r a d i a t e d c e l l s i s r e l a t e d to the displacement; periods of rapid d i r e c t e d movement c o r r e l a t e with periods of r a p i d change of c e l l shape. For example the c e l l depicted i n Figure 3.29.a underwent a seri e s of large scale changes i n c i r c u l a r i t y that i s outside the range of untreated c e l l behaviour. 3.3.3.b Populations of c e l l s The c e l l m o t i l i t y and morphology of i r r a d i a t e d c e l l s was analyzed by combining data from many c e l l s i n a given experiment and c a l c u l a t i n g population averages. The average c e l l speed as a function of time i s shown (Figure 3.30) for t e s t doses of 0 Gy, 0.5 Gy, and 1.0 Gy. These experiments tracked 60 c e l l s f o r 2 hours p r i o r to i r r a d i a t i o n , then for another 6 hours FIGURE 3.28: The average c e l l brightness as a function of time for i r r a d i a t e d 3T3 c e l l s . Same c e l l s as depicted i n Figure 3.25. ro ro 14.0a 5*7 • 3t7 B ~ " ~ T I N * ( N I R > l i n t C n i n ) l i n t C i i l f t ) FIGURE 3.29: The c e l l c i r c u l a r i t y as a function of time f o r i r r a d i a t e d 3T3 c e l l s . Same c e l l s as depicted i n Figure 3.25. C i r c u l a r i t y described i n caption to Figure 3.21. 124 p o s t - i r r a d i a t i o n . The time of i r r a d i a t i o n i s marked on the Figures (arrow). The untreated population displayed a r e l a t i v e l y constant average c e l l speed throughout the experiment. The c e l l s i r r a d i a t e d to 1 Gy displayed an increase i n average c e l l speed immediately following treatment, then a gradual decrease. The d i s t r i b u t i o n of speeds from co n t r o l c e l l s and from c e l l s i r r a d i a t e d to 1.0 Gy i s shown (Figure 3.31). At the e a r l i e s t measurable times post-treatment (approximately 2/3 hour), the doses of 0.5 and 1.0 Gy produced a s l i g h t increase i n average c e l l speed. This was not seen at higher doses. The data from several d i f f e r e n t experiments was combined to give a dose response curve for average c e l l speed vs x-ray dose. Shown i n the Figure 3.32 i s the data from an average of 2 experiments per dose, 60 c e l l s per experiment. The data i s shown at times of 50, 100, and 150 minutes a f t e r the treatment. Population averages of other c e l l m o t i l i t y parameters were ca l c u l a t e d for i r r a d i a t e d c e l l s . The 2-dimensional random walk model was applied. In the dose range 0-8 Gy, there were not consistent a l t e r a t i o n s i n the parameters D* or t * (Figure 3.33 and 3.34). The forecast analysis was also applied to i r r a d i a t e d c e l l s . The set of displacement d i s t r i b u t i o n s with varying previous displacement were q u a l i t a t i v e l y s i m i l a r between control and i r r a d i a t e d c e l l s (Figure 3.35). There were some quantitative differences between con t r o l c e l l s and c e l l s i r r a d i a t e d to 8 Gy, p a r t i c u l a r l y i n the large displacement p r o f i l e s . For the b i n 12 to 29 Ltm, the average displacement and standard error of the mean are 7.771.38 Ltm, compared to 8.52±.36 pm f o r controls (Figure 3.16). The values for the 9-12 Ltm b i n are 6.53±.32 Ltm compared to 7.35±.34 Ltm f o r co n t r o l . These differences may r e f l e c t changes to the degree of persistence of the r a p i d l y moving c e l l s . PLOT OF AVERAGE UELOCITY/IHTEIWAL US ELAPSED TIME 3.0 2.0 1.0 control 3 0 0 0 0 0 0 0 0 "T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 T 1 1 1 1 1 . TIME (ain) -> 338 3.0 2.0 1.0 2 < 0.5 Gy b 0 » 0 0 - 1 — i — i — r - - i — i — i — i — i — i — i — i — i — i — i — i — r — i — i — . TIME (ain) -> 325 3.0 2.0 1.0 1.0 Gy C 0 0 0 - 1 — 1 — 1 — I — 1 — 1 — 1 — I — 1 — I — I — I I TIME (ain) -> 385 FIGURE 3.30: The average c e l l speed as a f u n c t i o n o f t i m e f o r i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d a t time i n d i c a t e d by arrow. I n each e x p e r i m e n t , 60 c e l l s were scannned e v e r y 10 m i n u t e s . C e l l s i r r a d i a t e d t o doses i n d i c a t e d i n each p a n e l . The aver a g e c e l l speed i m m e d i a t e l y f o l l o w i n g i r r a d i a t i o n i s a r e s u l t o f removing the f l a s k from the s t a g e , and i s n o t i n d i c a t i v e o f a c t u a l c e l l m o t i l i t y . FIGURE 3.31: The d i s t r i b u t i o n of c e l l speed for untreated i r r a d i a t e d 3T3 c e l l s . Top 3 panels are c o n t r o l , bottom 3 panels i r r a d i a t e d to 1.0 Gy. t^ i s immediately p r i o r to treatment, t 2 i s from 30 to 120 minutes post-treatment and t j i s from 120 to 210 minutes post-treatment. In upper r i g h t of each panel i s the mean c e l l speed ro 127 FIGURE 3.32: The change i n average c e l l speed as a function of x-ray dose. Shown are the averages at 3 time points: closed c i r c l e s - 50 minutes post-i r r a d i a t i o n , open c i r c l e s - 100 minutes p o s t - i r r a d i a t i o n , closed boxes - 150 minutes p o s t - i r r a d i a t i o n . 2 .0 G y 13842 P » 2 FIGURE 3.33: <D% vs time f o r i r r a d i a t e d 3T3 c e l l s . C e l l s i r r a d i a t e d to 2.0 Gy. Values f o r the parameters D and t are shown on graph. Compare to Figure 3.15. ro 00 8 . 0 G y 17641 w. 2 FIGURE 3.34: <D^> vs time f o r i r r a d i a t e d 3T3 c e l l s . C e l l s i r r a d i a t e d to 8 Gy. Values f o r the parameters D and t shown on graph. Compare to Figure 3.15 and 3.33. Displacement (urn) FIGURE 3.35: F o r e c a s t a n a l y s i s f o r i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d t o 8 Gy. Compare t o F i g u r e 3.16. CO o 1 3 1 co m ^ (wH) j u a u j e o B i d S f a FIGURE 3.36: The decay of persistence for i r r a d i a t e d 3T3 c e l l s . C e l l s i r r a d i a t e d to 8 Gy. Plotted i s the average displacement as a function of increasing forecast length (see Figure 3.18). Open t r i a n g l e s - 12-20 um bin. Closed boxes - 9-12 um bin. Open boxes - 6-9 um bin. Open c i r c l e s - 3-6 um bin. Closed c i r c l e s - 0-3 um bin. 132 IS.' O CO (LuH) l U 9 U J 8 0 B | C l S ! a FIGURE 3.37: The decay of persistence f o r i r r a d i a t e d 3T3 c e l l s . C e l l s i r r a d i a t e d to 2 Gy. Open tr i a n g l e s - 12-20 /jm bin. Closed boxes - 9-12 pirn bin. Open boxes - 6-9 Ltm bin. Open c i r c l e s - 3-6 Ltm b i n . Closed c i r c l e s -0-3 um bin. FIGURE 3.38: F o r e c a s t a n a l y s i s o f t h e d i s t r i b u t i o n o f r e l a t i v e a n g l e s f o r i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d t o 8.0 Gy. Compare t o F i g u r e FIGURE 3.39: Forecast analysis of the angles f o r i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d to 2.0 Gy. Compare to Figure 3.17 and Figure 3.38. 135 The decay of persistence i n i r r a d i a t e d c e l l s was further assessed by measuring the average c e l l displacement as a function of forecast length. This technique also enables the examination of the d i f f e r e n t motile subpopulations. Figure 3.36 shows the data from c e l l s i r r a d i a t e d to 8 Gy. Of note i s the f a c t that the curves from the upper 3 displacement p r o f i l e s (namely 6-9 /im, 9-12 /im, and 12-29 /im) a l l converge to the same average displacement of approximately 5.8 /im. The displacement p r o f i l e of 0-3 um appears to converge toward a value of 5.0 um. The e f f e c t of 8 Gy i s apparently to erase the long term persistence of c e l l movement. The e f f e c t of 2 Gy on c e l l persistence i s shown i n Figure 3.37, which shows the average c e l l displacement as a function of forecast length. With t h i s dose, the d i f f e r e n t displacment bins appear to maintain heterogeneity and do not approach a common value f o r average displacement. The two largest displacement p r o f i l e s , 9-12 /im and 12-29 um, quickly converge, but t h i s i s s i m i l a r to control c e l l s (Figure 3.18). The d i s t r i b u t i o n s of angles from the various p r o f i l e s were also examined. Figure 3.38 and 3.39 show the d i s t r i b u t i o n of r e l a t i v e angles for c e l l s i r r a d i a t e d to 8 Gy and 2 Gy, respectively. The c e l l s i r r a d i a t e d to 8 Gy show s i m i l a r behaviour as untreated c e l l s i n the small displacement p r o f i l e s (Figure 3.38.a and b), however i n the large displacement p r o f i l e s , the data do o not show a pronounced peak at 0 (Figure 3.38.d and e). Again, these values have a large uncertainty. Similar data f o r c e l l s i r r a d i a t e d to 2 Gy are shown i n Figure 3.39. The population averages of c e l l morphology of i r r a d i a t e d c e l l s were examined. The values of average c e l l area, c i r c u l a r i t y , and brightness d i d not appear to be a l t e r e d with r a d i a t i o n i n the dose regime 0-8 Gy, seen i n Figures 3.40 and 3.41. 136 FIGURE 3.40: The average c e l l morphology features f o r a population of 37 i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d to 8 Gy. a) average c e l l area, b) average c e l l c i r c u l a r i t y , c) average c e l l brightness. 700 a + + +++ ++ + + + ++ ++++ 350.8 TiMe(Hin) >> « 3 O b O) (0 a> > < + v ++ + V 30 10 in o c k_ m a> Ol CO w a> > < 18.0 367 T i M ( n i n ) c t\ +4 4 4+4 ++ + 4 44444 4 4 4 4 4 4444 444 4444 4 4 4 • 4 44444 U - 4 4 4444 4 H 1+4 44 4 t 4 1 II 1144 4 4 4 TiM«(nin> 367 FIGURE 3.41: The average c e l l morphology features f o r a population o i r r a d i a t e d 3T3 c e l l s . C e l l s were i r r a d i a t e d to 2.0 Gy. a) average c e l l area b) average c e l l c i r c u l a r i t y , c) average c e l l brightness. 138 3.4 Stained Cells In an e f f o r t to detect x-ray induced morphological alterations that may resu l t i n perturbed c e l l m o t i l i t y , 3T3 c e l l s on glass coverslips were fixed and stained, and microscopically examined with lOx, 25x, and 40x objective lenses. The stains that were used have a f f i n i t y for structural proteins, and i n p a r t i c u l a r towards the microfilaments. Photographs shown here are of c e l l s stained with Coomassie blue. A fluorescent s t a i n , NBD-phallacidin, was also used with similar results, but proved more d i f f i c u l t to record photomicrographs. The Coomassie blue stained c e l l s proved more suitable for detailed microscopic examination. The c e l l s were grown on glass coverslips for 24 hrs before being exposed to mock x-ray treatment (controls), or a test x-ray dose. The c e l l s were irra d i a t e d while i n the p e t r i dish i n DMEM+10% FBS. The c e l l s were incubated for 1 hr after the i r r a d i a t i o n , and then fixed and stained. The coverslips were examined microscopically, and photomicrographs made of each test dose. 3.4.1 Control c e l l s The unirradiated 3T3 c e l l s are shown i n Figures 3.42.a and b. The morphology of the c e l l s i s similar to that detected with other techniques i n t h i s study, and several additional features are evident. The nucleus, and p a r t i c u l a r l y the nuclear periphery, tends to be o p t i c a l l y dense as compared with the outer regions of the c e l l . This i s probably due to the high protein and organelle concentration i n the nuclear v i c i n i t y and the r e l a t i v e exclusion of large macromolecular complexes from the c e l l periphery. Also, thi n dark strands can be seen i n some c e l l s , most often i n well-spread, nonpolarized c e l l s . These are l i k e l y the stress fibers seen by many workers i n cultured f i b r o b l a s t s . These are generally absent i n c e l l s with an 'active' morphology. The c e l l periphery i s usually l i g h t l y stained, except for notable dark patches very near the c e l l border on some c e l l s . These areas represent the active 139 ft a ' J r * FIGURE 3.42: 3T3 c e l l s stained with Coomassie blue and photographed with lOx objective lens. Bar i n (a) represents 50 um. a) and b), unir r a d i a t e d c e l l s , c) and d) c e l l s i r r a d i a t e d to 60 Gy. e) and f) c e l l s i r r a d i a t e d to 16 Gy. 142 c e l l margins, and the dark patches are the r u f f l e s which have been shown to contain concentrations of meshwork a c t i n . As an o v e r a l l observation, there are a v a r i e t y of c e l l shapes r e f l e c t i n g the d i f f e r i n g degrees of c e l l a c t i v i t y and m o t i l i t y at the moment of f i x a t i o n . 3.4.2 I r r a d i a t e d c e l l s Examples of 3T3 c e l l s i r r a d i a t e d to high dose (60 Gy) are seen i n Figures 3.42.C and d. These c e l l s are obviously morphologically a l t e r e d from untreated c e l l s . The differences are r e f l e c t e d i n the long, arbourized c e l l extensions, the collapsed c e l l periphery, and the i r r e g u l a r c e l l periphery. However i t should be noted that a small f r a c t i o n of the c e l l s (approx. 5%) are able to maintain a roughly normal morphology at one hour p o s t - i r r a d i a t i o n . These c e l l s are well-spread and nonpolarized, that i s , quiescent c e l l s . Stress f i b e r s are s t i l l observed i n these c e l l s . S t a t i c morphological differences are more d i f f i c u l t to detect i n c e l l s i r r a d i a t e d to low or moderate doses. At more moderate doses (16 Gy), most c e l l s have a global shape that i s within the range of untreated c e l l s (Figures 3.42.e and f ) . Perturbations may be evident i n some c e l l s , f o r example the c e l l periphery may take a minor degree of the i r r e g u l a r i t y seen at high doses. At lower doses t h i s i s seldom seen, and c e l l s are not morphology d i s t i n c t from u n i r r a d i a t e d c e l l s . 143 4. DISCUSSION 4.1 Repeat of Published Experiments This thesis sought to examine the e f f e c t s of x-rays on the process of f i b r o b l a s t m o t i l i t y . To an extent the research was motivated by reports that low doses of x-rays induced dramatic a l t e r a t i o n s i n 3T3 f i b r o b l a s t locomotion i n t i s s u e culture. These studies measured c e l l m o t i l i t y with the gold dust assay or time lapse microcinematography following exposure to low doses (<2 Gy) of x-rays (2,76). I t was reported that c e l l s exhibited a much al t e r e d track pattern i n the gold dust following exposure, i n d i c a t i n g that c e l l s moved i n "a more random" pattern. Similar observations were reported using time lapse cinematographic techniques, and "phrenetic membrane a c t i v i t y " and "tenuous c e l l attachment" were also described. The f r a c t i o n of c e l l s a f f e c t e d was reported to increase with dose, and the a c t i v i t y p e r s i s t e d f o r several generations following treatment. A time lapse cinematographic record, shown at the Radiation Research Meeting (Orlando, F l a . , A p r i l , 1983), suggested a large and s t r i k i n g e f f e c t on 3T3 c e l l m o t i l i t y following low doses of x-rays. This perturbation was suggested to have relevance for the medical use of x-rays, p a r t i c u l a r l y i n b i o l o g i c a l processes where c e l l m o t i l i t y plays a c r i t i c a l r o l e (such as embryogenesis, and tumour metastasis). These r e s u l t s were purely q u a l i t a t i v e and lacked d e t a i l on the time course of the e f f e c t , on the f r a c t i o n of c e l l s a f f e c t e d as a function of dose, and on the dose response of the e f f e c t . In an e f f o r t to properly assess the possible b i o l o g i c a l implications and relevance of x-ray induced perturbation to c e l l m o t i l i t y , we sought to extend the studies and address these issues. The gold dust assay was employed to record c e l l m o t i l i t y following exposure to d i f f e r e n t r a d i a t i o n doses. I t was found that x-ray doses i n the range 0-2 Gy did not induce an obvious perturbation to the gold dust tracks of most c e l l s . 144 Some c e l l s d i d seem to display an a l t e r e d pattern of m o t i l i t y , consistent with the e a r l i e r reports (2,76). S p e c i f i c a l l y there was apparently an increased percentage of "zig-zag" tracks, representing a few per cent of the t o t a l i r r a d i a t e d c e l l tracks. Higher doses (6 Gy) produced some obviously a l t e r e d gold dust tracks, although again not a l l c e l l s were affected. Time lapse cinemicroscopy was also employed to study i r r a d i a t e d c e l l m o t i l i t y . These studies showed that most c e l l s continue to locomote approximately normally following x - i r r a d i a t i o n of up to at l e a s t 2 Gy. Again, i t was noted that a small percentage of c e l l s exhibited a l t e r e d behaviour following i r r a d i a t i o n . An estimate might be that 5% of the c e l l s were noticeably perturbed following 2 Gy of x-rays. The nature of the perturbation was consistent with that seen i n the gold dust studies, that i s , an a l t e r a t i o n to the c o n t r o l of c e l l m o t i l i t y and morphology. The r e s u l t s attained seemed to be i n q u a l i t a t i v e agreement with the previous reports (2,76), however, the magnitude of the response seemed les s dramatic than that reported and documented i n the time lapse record. This emphasized the inherent l i m i t a t i o n s of the assays used for c e l l m o t i l i t y . The gold dust assay d i d not provide a quantitative assessment of the magnitude or pattern of movement, nor d i d i t provide temporal information. The assay also exposes c e l l s to an unnatural and p o t e n t i a l l y perturbative environment (even further removed from the i n vivo s i t u a t i o n than i s an unmarked tis s u e culture p l a s t i c surface). The m o t i l i t y displayed i n time lapse records i s also d i f f i c u l t to quantify, and data analysis becomes a subjective and tedious process. Both techniques can be enhanced with i n t e r a c t i v e d i g i t i z a t i o n and video analysis packages, but these packages are expensive and the extra information gained i s minimal. I t became obvious that a new approach was necessary which provided d e t a i l e d s p a t i a l and temporal information on a large number of motile c e l l s . Such a system required automation. 145 4.2 Design of Automated System f o r C e l l M o t i l i t y An automated microscope system for the measurement of c e l l m o t i l i t y i n tissue culture was developed. The system was designed such that: 1) d e t a i l e d s p a t i a l and temporal information of the m o t i l i t y of i n d i v i d u a l c e l l s was a v a i l a b l e ; 2) a large number (up to 100) of i n d i v i d u a l c e l l s were concurrently monitored; and 3) the c e l l s were exposed to a s t r i n g e n t l y c o n t r o l l e d tissue culture environment. The t h i r d point required that c e l l s be exposed to c o n t r o l l e d temperature, pH, l i g h t i n t e n s i t y and media conditions, as well as being f u l l y recovered from enzymatic d i s s o c i a t i o n ( t r y p s i n ) . We attempted to measure only normal c e l l m o t i l i t y and x-ray induced perturbations to normal c e l l m o t i l i t y . 4.3 Average Rate of C e l l Movement Following I r r a d i a t i o n Once the system was f u l l y characterized, an e a r l y measurement was the change i n average c e l l speed as a function of r a d i a t i o n dose (Figure 3.32). The time course of the e f f e c t can be seen i n Figure 4.1, which shows the change i n average c e l l speed as a function of time f o r c e l l s exposed to 1 Gy. The average speed of 60 c e l l s and was measured from 50 minutes to about 180 minutes post-treatment. The e a r l i e s t time point that was t e c h n i c a l l y f e a s i b l e to measure was at 50 minutes post i r r a d i a t i o n , thus the exact form of the curve from 0 to 50 minutes i s not known. The curve can be interpreted as being a r e s u l t of two competing processes. At e a r l y times, there i s an increase i n the rate of movement, while at longer times the average rate of movement tends to decrease toward control l e v e l s or below. The onset of m o t i l i t y may be due to the early and rapid damage to the c e l l u l a r control of m o t i l i t y , while the l a t t e r decrease i n movement may r e f l e c t c e l l u l a r repair, or the subsequent expression of r a d i a t i o n damage. Further i n t e r p r e t a t i o n c E E "O CD CD a CO 0 O CD > CO CD O) c CO O .050-.025-0- > .025--.050-Dose=1 Gy 50 100 150 200 Time after treatment (min.) FIGURE 4.1: Time response o f c e l l m o t i l i t y f o l l o w i n g i r r a d i a t i o n . C e l l s i r r a d i a t e d i n f l a s k t o 1 Gy. Shown i s change i n average c e l l speed vs time f o r 60 c e l l s . Response b e f o r e 50 minutes c o u l d not be measured, and dashed l i n e s show two p o s s i b l e responses i n the time 0 t o 50 minutes. Cft 147 of t h i s result i s made d i f f i c u l t for several reasons. F i r s t , the parameter of average c e l l speed does not describe the actual process of c e l l m o t i l i t y . Radiation was not thought to act as a stimulant or depressant of m o t i l i t y per se, but rather to disrupt the motile machinery so as to produce changes i n the average rate of movement. The inspection of time lapse records had shown that f i b r o b l a s t movement was undulatory with segments of rapid c e l l movement interspersed with segments of quiescence and reorientation. Further parameters were needed to quantify the process of locomotion, and i t s perturbation. Second, there i s a longer term motile heterogeneity among c e l l s of equal p r o l i f e r a t i v e characteristics. The cytoplasm seems to carry certain morphological characteristics that pass from one generation to another (see 3.2.1.a). A perturbation to the average rate of c e l l movement does not indicate i f the treatment had equal effect on a l l morphology phenotypes. Indeed, such a change would be obscured i f only a f r a c t i o n of the c e l l s were affected and the rest were not. Thus further m o t i l i t y parameters were needed to i d e n t i f y and quantify heterogeneous c e l l populations and heterogeneous radiation response. 4.4 Development of C e l l M o t i l i t y Parameters With these goals i n mind, other c e l l m o t i l i t y parameters i n the l i t e r a t u r e were explored. Two of the more sophisticated techniques were thought to be applicable, the 2-dimensional random walk model (107) and the Markov analysis (105). These were examined i n more d e t a i l (Figure 3.15, and results not shown). However, the combining of data from many c e l l s tends to smear out important c e l l - t o - c e l l variations as well as erasing the pattern of movement of each c e l l . I t was soon realized that i n order to parameterize the undulating f i b r o b l a s t m o t i l i t y , techniques were needed that did not take a "snapshot" view of the m o t i l i t y data. I t i s necessary to quantify and 148 manipulate the recorded movement from each c e l l before combining i t with that from other c e l l s . Two techniques were developed along these l i n e s . One was the displacement analysis i n which the motile record from an i n d i v i d u a l c e l l was modified by the displacement f i l t e r (see section 2.3.6 and 3.3.1.a). This technique emphasizes the segmented nature of the o v e r a l l movement pattern. As w e l l , i t allows a much better c o r r e l a t i o n between the motile record and the morphology record (see Figure 3.25). The other technique was the forecast a n a l y s i s . This method extracts short term behaviour from an i n d i v i d u a l c e l l , and then combines i t with s i m i l a r behaviour from other c e l l s . The short term persistence of c e l l m o t i l i t y was characterized, as well as the longer term heterogeneity of the c e l l population (see Figures 3.16 to 3.18). This technique also enables a set of simulated c e l l m o t i l i t y data to be derived ( v i a a Monte Carlo approach -r e s u l t s not shown), and holds promise for the comparison of c e l l m o t i l i t y data to b i o p h y s i c a l models of c e l l m o t i l i t y (64). 4.5 Further Characterization of X-ray E f f e c t on C e l l M o t i l i t y The a n a l y t i c a l tools to describe c e l l m o t i l i t y were applied to the locomotion of i r r a d i a t e d f i b r o b l a s t s . Examples of the displacement analysis are shown for u n i r r a d i a t e d c e l l s (Figure 3.11), and f o r i r r a d i a t e d c e l l s (Figure 3.27). The analysis emphasized that i n the i r r a d i a t e d c e l l population, c e r t a i n c e l l s display a l t e r e d patterns of c e l l movements. The treatment does not perturb a l l c e l l s i n the population. The forecast analysis for control c e l l s (Fig. 3.16 to 3.18) and i r r a d i a t e d c e l l s ( F i g . 3.35 to 3.39) shed l i g h t on the heterogeneous nature of the x-ray perturbation. The d i s t r i b u t i o n of r e l a t i v e angles for c e l l s i r r a d i a t e d to 8 Gy showed that the d i r e c t i o n a l persistence of the r a p i d l y migrating c e l l s i s l o s t . This suggests 149 that the r a d i a t i o n perturbation i s more evident i n a p a r t i c u l a r segment of m o t i l i t y . Furthermore, the p l o t of average c e l l speed vs increasing forecast length also shows that the short term persistence of r a p i d l y moving c e l l s i s perturbed (evidenced by a more r a p i d i n i t i a l decay), i n a d d i t i o n to the longer term persistence being a l t e r e d (evidenced by the common asymptotic average speed i n Figure 3.36). Thus these doses of x-rays appear to a l t e r the short term and long term behvioural c h a r a c t e r i s t i c s of i n d i v i d u a l c e l l s . In order to measure persistence, data i s needed over an extended period of c e l l movement, thus i t i s d i f f i c u l t to compare the time course of the average speed response (Section 4.3) to the changes i n c e l l u l a r persistence. 4.6 Comparison to Published Reports The r e s u l t s on x-ray e f f e c t s on 3T3 c e l l m o t i l i t y , obtained with the C e l l Analyzer as well as conventional m o t i l i t y assays, are i n accord with some aspects of e a r l i e r reports (2,76). We also observed changes i n the pattern of c e l l m o t i l i t y which apparently r e f l e c t changes i n the c e l l u l a r control mechanisms. Furthermore, the treatment a f f e c t s only a f r a c t i o n of the o v e r a l l population. The current report i s however at odds with e a r l i e r studies (2,76) with respect to the magnitude of the r a d i a t i o n induced perturbation. The large and s t r i k i n g e f f e c t s reported elsewhere were not observed i n our measurements. The nature of the perturbation that we observed i s consistent with that reported, however i t s measurement required d e t a i l e d r e s u l t s near the l i m i t of the s e n s i t i v i t y of the apparatus. The exact cause of the difference between our measurements and those of others i s not known. The process of c e l l m o t i l i t y i s f a i r l y s e n s i t i v e to v a r i a t i o n s i n c e r t a i n external conditions (as well as perhaps to v a r i a t i o n s i n the 3T3 c e l l l i n e s ) . For example, the rate of 3T3 c e l l movement has been o o shown to vary s i g n i f i c a n t l y i n the temperature range 33 to 39 C, thus small 150 v a r i a t i o n s i n temperature may produce anomalous m o t i l i t y r e s u l t s . Further, the i n t e r a c t i o n of these external conditions with the x-ray induced damaged has not been measured. In t h i s thesis, we attempted to measure unperturbed c e l l m o t i l i t y , thus the c e l l s were allowed at l e a s t 14 hrs., and less than 24 hrs., following t r y p s i n i z a t i o n u n t i l beginning x-ray treatment or measuring c e l l movement. Some preliminary data indicates that the process of c e l l reattachment following t r y p s i n i z a t i o n may be perturbed by x-rays (see Figure 4.2). Unfortunately the e a r l i e r reports (2,76) do not provide s u f f i c i e n t procedural d e t a i l to assess these p o s s i b i l i t i e s . The comparison of t h i s work to the larger body of experiments with i r r a d i a t e d lymphocytes i s more d i f f i c u l t . F i r s t l y , the mechanism and pattern of leukocyte m o t i l i t y i s somewhat d i f f e r e n t from f i b r o b l a s t s . Secondly, leukocytes show d i s t i n c t subpopulations with dramatically d i f f e r e n t r a d i a t i o n response, and further, stimulated and non-stimulated leukocytes are d i f f e r e n t i a l l y affected. In addition, the assays used to measure leukocyte m o t i l i t y are generally not compatible with the data reported here. Thus while note should be made as to the extreme s e n s i t i v i t y of leukocyte m o t i l i t y to i r r a d i a t i o n , and to the varying response with d i f f e r e n t metabolic state, i t i s not yet possible to make comparisons between 3T3 c e l l data and leukocyte data. 4.7 Mechanisms Data from other laboratories has suggested that r a d i a t i o n induced changes i n c e l l m o t i l i t y and cytogel contraction are mediated by damage to the c e l l membrane(s). During the course of the current studies, we have hypothesized that the observed perturbations to c e l l m o t i l i t y are due to damage to the permeability of the membrane, eit h e r cytoplasmic or organelle, and the subsequent f l u x of ions into (or from) the c y t o s o l . This i n turn would produce changes i n the polymerization and c o n t r a c t i l e state of the 0.1 5 200 400 600 800 Time after plating (minutes) FIGURE 4.2: Ave r a g e c e l l speed v s ti m e f o l l o w i n g t r y p s i n i z a t i o j i , i r r a d i a t i o n , and r e p l a t i n g . C e l l s i r r a d i a t e d i n s u s p e n s i o n t o 0.5 Gy a t 4 C, t h e n p l a t e d i n t o f l a s k s . C e l l t r a c k i n g begun 3 h r s . a f t e r p l a t i n g . C l o s e d c i r c l e s show c e l l s g i v e n mock x - r a y ( c o n t r o l ) . Open s q u a r e s show i r r a d i a t e d c e l l s . 152 cytoskeleton. The microfilament and microtubule systems are both s e n s i t i v e to i o n i c concentrations. The most obvious candidate to mediate t h i s e f f e c t i s Ca , both because of i t s steep gradient across the plasma membrane (and across organelle membranes such as the sarcoplasmic reticulum) and because of i t s potent regulatory r o l e i n the polymerization and c o n t r a c t i l i t y of the microfilament (and microtubule) system (Figure 4.3). Permeability changes to Ca would r e s u l t i n a f l u x of ions into the cytosol and subsequent a l t e r a t i o n s i n the functional state of the cytoskeleton. Some evidence from _i_2 other investigations i s a v a i l a b l e to support t h i s r o l e of Ca (73,74,120). The r e s u l t s from t h i s thesis are consistent with t h i s hypothesis. The increase i n average c e l l speed was measured at a time of 50 minutes post i r r a d i a t i o n , the e a r l i e s t time t e c h n i c a l l y f e a s i b l e . Although t h i s may be s u f f i c i e n t time f o r a change i n p r o t e i n synthesis or gene expression to be mediated, i t i s an u n l i k e l y p o s s i b i l i t y . Instead, t h i s e f f e c t i s l i k e l y mediated by a non-genomic e f f e c t . Also, i n consideration of the mechanism of normal c e l l m o t i l i t y , i t seems l i k e l y that improperly c o n t r o l l e d fluxes of 4-2 Ca would lead to the type of d i s r u p t i o n observed following i r r a d i a t i o n . As to why only a f r a c t i o n of the i r r a d i a t e d c e l l population expresses the damage, the answer may be i n the nature of 1) the i n t e r a c t i o n of x-rays with l i p i d b i l a y e r s and 2) the mechanism of global c e l l c o n t r o l . F i r s t , experiments with a r t i f i c i a l l i p i d b i l a y e r s have shown that free r a d i c a l formation i n the hydrophobic domain of the b i l a y e r can r e s u l t i n a cascade r e a c t i o n to produce a c l u s t e r of r a d i c a l s (121). The rates for t h i s process i n mammalian c e l l membranes are a complex function of l i p i d composition, membrane pro t e i n concentration, l o c a l curvature of the membrane, etc. and have not been f u l l y measured or understood. Furthermore, i t i s not yet known to what extent the membrane must be damaged before s i g n i f i c a n t l o c a l changes i n c a t i o n permeability occur. I t may be that only a small f r a c t i o n of 154 the i n i t i a l free r a d i c a l s i n the b i l a y e r w i l l produce relevant changes i n permeability. A l t e r n a t i v e l y , the e f f e c t i v e target s i z e may be small, such that only c e r t a i n regions i n the membrane can mediate the e f f e c t . Second, although the global control of dynamic c e l l behaviour i s a poorly understood topic, i t i s recognized that c e l l s d i s p l a y persistence to the extent that quiescent c e l l s tend to remain thus, and motile c e l l s remain motile. The onset of even short term quiescence (a few hours) i s apparently heralded by an accumulation of a c t i n monomers into large stress f i b e r s , which have been shown to be much less l a b i l e to Ca (122). Thus the dynamic cytoskeleton i n a c t i v e l y migrating c e l l s may already be more s e n s i t i v e to Ca . Some evidence for t h i s comes from the morphological examination of stained c e l l s i r r a d i a t e d to moderately high and high doses (Figure 3.42), i n which the only morphologically normal c e l l s were those that were well spread, f l a t , nonpolarized, and presumably quiescent (at l e a s t from other observations these c e l l s were quiescent 100% of the time). Large stress f i b e r s are oc c a s i o n a l l y s t i l l seen i n these c e l l s a f t e r i r r a d i a t i o n . Presumably the other morphological subpopulations were more r a d i o s e n s i t i v e i n terms of morphology. Along these l i n e s , the data from the forecast analysis, i n which the d i s t r i b u t i o n of angles was p l o t t e d for various ranges of c e l l displacement (Figures 3.38 and 3.39), shows that the r a p i d l y moving c e l l s were more perturbed by i r r a d i a t i o n . Also from the forecast analysis, increasing forecast length showed that i r r a d i a t e d c e l l s underwent changes i n long term persistence. Thus, although the r a d i a t i o n e f f e c t may be the most s i g n i f i c a n t i n a c t i v e l y migrating c e l l s , i t i s p r e c i s e l y i n these c e l l s that i t i s most d i f f i c u l t to detect and quantify a perturbation. These c e l l s assume a wide v a r i e t y of shapes and movement patterns, and i n d i v i d u a l c e l l movement has s t i l l not been r i g o r o u s l y characterized. 155 There are several further tests and implications f o r t h i s hypothesis of the mechanism of r a d i a t i o n induced change i n c e l l m o t i l i t y . One t e s t would be _i_2 to measure the ( l o c a l instantaneous) concentration of Ca i n the cytosol or the f l u x of Ca across d i f f e r e n t c e l l u l a r membranes. This might be done • 2 e i t h e r with radioactive Ca , i n which case one would measure the f l u x of the +2 ion between compartments, or with i n t r a c e l l u l a r Ca - s e n s i t i v e fluorescent probes, such as aqueorin, Quin 2, Fura 2, or Indo 1 (123,124). Some experiments have been done to study the f l u x of radioactive solutes (such as _i_ i _i_2 K , Na , Ca , etc.) across the c y t o s o l i c membrane of erythrocytes following i r r a d i a t i o n . The somewhat low s e n s i t i v i t y of the experiments combined with the active transport of these ions has meant that most studies have been done at large doses (> 5 Gy) (122). Also, the s e n s i t i v i t y of the fluorescent probes may not allow detection of l o c a l i z e d changes of Ca i n a small subset of the population. Recent advances i n video microscopy and image processing on images of s i n g l e c e l l s have dramatically improved the s e n s i t i v i t y of t h i s technique (125,126). The hypothesis may be further tested by a more s e n s i t i v e analysis of the structure of the microfilaments, or an analysis of the dynamics of the microfilaments i n l i v e c e l l s following exposure to x-rays. Fluorescent analogue cytochemistry i s a technique for studying the behaviour of a p a r t i c u l a r p r o t e i n i n l i v e c e l l s by introducing an a l i q u o t of functional, f l u o r e s c e n t l y l a b e l l e d p r o t e i n into the c e l l and observing c e l l s m icrofluorometrically (127). Recent experiments have begun to characterize the behaviour of c y t o s k e l e t a l proteins within l i v e c e l l s (128-130), and the technique may enable the study of perturbations to c y t o s k e l e t a l dynamics. 156 4.8 Further Studies This work has begun to elucidate the e f f e c t of x-rays on the process of 3T3 c e l l m o t i l i t y . We have i d e n t i f i e d the complexity of the response, and have developed appropriate measuring instruments and a n a l y t i c a l t o o l s . What was o r i g i n a l l y believed to be a simple task to i d e n t i f y a perturbation to c e l l behaviour, has turned out to be a complex problem. S p e c i f i c a l l y , i t has been r e a l i z e d that 1) there may be d i s t i n c t morphological subpopulations i n a c e l l l i n e of 3T3 c e l l s ; 2) there are d i s t i n c t motile segments during the course of c e l l locomotion; and 3) r a d i a t i o n does not y i e l d a straightforward perturbation, and furthermore, may have d i f f e r e n t i a l e f f e c t s on each of the above. Further studies should thus incorporate the inherent heterogeneity of the r a d i a t i o n response. In order to do t h i s , data must be obtained from a large number of c e l l s i n each of the c e l l subpopulations. The present version of the C e l l Analyzer could r o u t i n e l y monitor up to 100 c e l l s during each experiment (based on a scanning i n t e r v a l of 8 minutes/scan, and a scanning rate of ~5 seconds/cell). A more d e t a i l e d analysis of motile heterogeneity may require an order of magnitude more c e l l s per experiment. This would c a l l for f a s t e r scanning, more e f f i c i e n t s i g n a l analysis, and more storage space. Another important issue i s the i n t e r a c t i o n of r a d i a t i o n damage with other p h y s i c a l and chemical agents - such as temperature and drugs - and the resultant e f f e c t on c e l l m o t i l i t y . This i s of p a r t i c u l a r concern during radiotherapy and the control of metastases. To study t h i s , a m o t i l i t y parameter i s required that 1) provides a meaningful c h a r a c t e r i z a t i o n of the process of c e l l m o t i l i t y (or i t s perturbation), and 2) can be e f f i c i e n t l y and r e l i a b l y measured i n each given experiment. The m o t i l i t y parameters developed i n t h i s thesis may be sui t a b l e f o r t h i s r o l e . From the forecast analyses, the early (and late) decay rates of the average c e l l speed vs forecast length 157 r e f l e c t the nature of the radiation perturbation (Figures 3.18 and 3.36), and are derived from each experiment. Further studies may u t i l i z e these techniques to systematically investigate radiation-drug interactions. This thesis has concentrated on the application of the C e l l Analyzer for the measurement of radiation-induced perturbation to c e l l m o t i l i t y . However, the C e l l Analyzer, and automated microscope systems i n general, have wide applications i n the f i e l d of c e l l biology. Studies on c e l l p r o l i f e r a t i o n , dynamic morphology, d i f f e r e n t i a t i o n , and c e l l function are feasible with such an apparatus. We are currently involved i n several projects to measure p r o l i f e r a t i v e behaviour of tissue culture c e l l s . From a technical point of view the quest w i l l continue for more detailed information on a larger number of c e l l s . The l i v e c e l l morphology analysis i s s t i l l very much i n i t s infancy. Even with the r e l a t i v e l y simple microscopy setup described here ( i . e . bright f i e l d and low magnification) a vast amount of c e l l morphology information can s t i l l be extracted. At present, only selected global shape features are measured, yet the image i s of s u f f i c i e n t quality to calculate relevant grey l e v e l features, to measure edge cha r a c t e r i s t i c s , and to reconstruct the overall c e l l shape from several functional regions. The use of other microscopy setups i n conjunction with the RSCAN program holds great promise. Higher magnification phase-contrast microscopy would enable more detailed measurement of c e l l morphology: accurate measurement of such things as c e l l r u f f l i n g , pinocytosis, and nuclear shape i s feasible. Fluorescent imaging of l i v e c e l l s i s also rapidly becoming a powerful tool for measuring a variety of dynamic c e l l u l a r properties: the position of the microtubule organizing centre (MTOC), the rate of f l u i d uptake, the expression of surface proteins, and the state of the chromatin are examples of what might be measured i n conjunction with c e l l m o t i l i t y and c e l l morphology. 158 The a n a l y t i c a l techniques to describe c e l l m o t i l i t y , the displacement analysis and the forecast analysis, attempt to characterize the actual process of locomotion. Further e f f o r t i s needed to develop these two techniques, and i t i s believed that an e f f i c i e n t synthesis may be achieved by using Kalman f i l t e r procedures from d i g i t a l s i g n a l processing (132). 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For the gold dust studies (section 3.1), the c e l l s were irr a d i a t e d either while i n suspension, or while attached to glass coverslips within a p e t r i dish. For the irr a d i a t i o n s i n suspension, the c e l l s were placed i n a glass i r r a d i a t i o n duct, with magnetic s t i r r i n g system, i n 20 ml of DMEM+10% FBS, with 1x10^ cells/ml. For the irr a d i a t i o n s on glass coverslips, the c e l l s were irr a d i a t e d similar to those on p l a s t i c p e t r i dishes, that i s , at room temperature, from below, and while resting on a 1.0 cm thick sheet of plexiglass. The fixed c e l l studies (section 3.4) were done with c e l l s on glass coverslips, and irradiated as described above. 6.1.2 Control Procedures for Fricke Dosimetry Several control procedures were done to ensure that the absorption value of the Fricke solution represented a true measure of the absorbed dose. The volume of ferrous sulfate solution i n the irradiated f l a s k was varied, from 20 ml to 3 ml. The absorption values for the smaller volumes were consistently s l i g h t l y higher than the large volumes. The absorption value for 5 ml of solution was t y p i c a l l y 2% to 5% higher than the value for 20 ml. The graph i n 169 the following section, and the dose estimates throughout the text, have used the absorption data with 5 ml of s o l u t i o n i n the f l a s k . Another co n t r o l procedure was to confirm that the length of time the s o l u t i o n was i n the p l a s t i c f l a s k s d i d not influence the measured absorption. Thus, various amounts of Fricke s o l u t i o n were l e f t i n the f l a s k s , both control and i r r a d i a t e d , f o r d i f f e r e n t lengths of time. This d i d not a l t e r the absorption value. A further control procedure was to i r r a d i a t e the s o l u t i o n i n both glass vessels and p l a s t i c vessels. For t h i s , 5 ml of s o l u t i o n were added to 20 ml glass t e s t tubes, and i r r a d i a t e d as above. The absorption values were within 5% of the values for f l a s k s . 6.1.3 Absorption and Dose f o r Fricke Dosimetry Figure 6.1 shows the measured absorption versus i r r a d i a t i o n time for the ferrous s u l f a t e dosimetry. The data show a l i n e a r increase up to an i r r a d i a t i o n time of 5 minutes. The data points at 8 and 10 minutes show an upward curvature. This r e s u l t was consistent f o r each of the three separate solutions of ferrous s u l f a t e . The cause i s not known. A l l of the i r r a d i a t i o n s performed i n t h i s project used an i r r a d i a t i o n of 5 minutes or l e s s , so t h i s p ortion of the curve w i l l be used. The dose rate converts to a value of 6.22 Gy/min using the equation i n Section 2.5. 6.1.4 B i o l o g i c a l Dosimetry C e l l s attached to glass and i r r a d i a t e d from below w i l l experience a larger dose than those attached to p l a s t i c . This i s because the larger average atomic number of the glass produces an increased f l u x of low energy electrons. The range of the electrons i n water i s on the order of 100 fim ( i . e . several c e l l diameters), and even a t h i n glass c o v e r s l i p (thickness <0.5 mm) has s u f f i c i e n t material to produce an increase i n e l e c t r o n f l u x . In an .300 171 e f f o r t to measure t h i s increased dose to c e l l s i r r a d i a t e d on glass, a b i o l o g i c a l dosimetry t e s t was performed. The procedure was as follows: 1. several glass coverslips were s t e r i l i z e d and placed one each i n 25 cm2 p l a s t i c tissue culture p e t r i dishes. 2. 3xl0 4 c e l l s / p e t r i were plated with 5 ml of DMEM+10% FBS into the dishes with c o v e r s l i p s and dishes without c o v e r s l i p s . 3. the c e l l s were incubated for 12 to 20 hours. 4. b o t h c o n t r o l c e l l s a n d g l a s s - a t t a c h e d c e l l s w e r e i r r a d i a t e d t o t h e t e s t doses. 5. dishes were returned to incubator, and c e l l s allowed to grow u n t i l the u n i r r a d i a t e d (control) dishes approach confluence (approx. 3 days). 6. c e l l counting procedures were used to determine the number of c e l l s . For the p l a s t i c - a t t a c h e d c e l l s , the procedure i n Section 2.1.2 was followed. For the glass-attached c e l l s , the c o v e r s l i p was c a r e f u l l y transferred from i t s host dish to another dish. Then the c e l l s were t r y p s i n i z e d from the c o v e r s l i p and counted. 7. the percentage of c e l l growth was c a l c u l a t e d by the r a t i o of the number of c e l l s i n the i r r a d i a t e d dish (coverslip) to the number of c e l l s i n the u n i r r a d i a t e d dish ( c o v e r s l i p ) . The r e s u l t s are shown i n Figure 6.2. The r a t i o of dose required to achieve an equal e f f e c t on glass-attached and p l a s t i c - a t t a c h e d c e l l s i s given for several values of % growth. This r a t i o has a value of approximately 2.1 for the % growth of 0.10. A value of 2.0 was used f o r c o r r e c t i n g the dose for Section 3.1.3 and Section 3.4. Several reports i n the l i t e r a t u r e have used b i o l o g i c a l dosimetry to measure the dose to c e l l s on glass vs c e l l s on p l a s t i c . These assays have 172 approx * of cells plated 0.50 1.00 1.50 2.00 Irradiation time (min) FIGURE 6.2: The percentage of growth vs. i r r a d i a t i o n time for c e l l s on glass and p l a s t i c . Open c i r c l e s show data for c e l l s i r r a d i a t e d on p l a s t i c ; closed c i r c l e s f o r c e l l s on glass. The r a t i o between the doses to achieve equal e f f e c t are given for several values of % growth. 173 used c e l l s u r v i v a l (colony forming a b i l i t y ) as the b i o l o g i c a l endpoint. With t h i s technique, a dose r a t i o to achieve i s o e f f e c t i s 1.4 to 1.5 f o r x-rays ( S i n c l a i r , W.K., i n Radiation Dosimetry. Vol. I l l , 2nd E d i t i o n , Editors A t t i x , F.H., Roesch, W.C., and T o c h i l i n , E. , Academic Press, N.Y., 1969, pp. 649-654). The difference between the value quoted i n the l i t e r a t u r e and the value measured here, using a s l i g h t l y d i f f e r e n t endpoint, may be due to several f a c t o r s , including r e l a t i v e b i o l o g i c a l e f f e c t i v e (RBE) differences between the two endpoints. 174 6.2 DETAILS ON DMIPS COMPONENTS 6.2.1 CCD O p t i c a l Sensor The o p t i c a l sensor of the C e l l Analyzer i s a l i n e a r array of charge-coupled device (CCD) elements. The array i s 1728 elements i n length, and each element (pixel) has a r e a l s i z e of 13 nm x 13 (im. Each element acts as a s o l i d state photodetector, and the charge c o l l e c t e d from each p i x e l i s proportional to the l i g h t i n t e n s i t y . The magnified microscope image i s projected onto the array. The CCD element i s a metal-oxide-semiconductor (MOS) structure i n which charge packets are 1) generated (either v i a absorbed photons or input d i f f u s i o n ) , 2) transferred (with a correct sequence of voltages to the electrodes) and 3) output (to produce a voltage). For imaging purposes, the incoming photons are absorbed i n the semiconductor p o r t i o n of the CCD, and generate conduction electrons. These are c o l l e c t e d i n a p o t e n t i a l well at the insulator/semiconductor i n t e r f a c e . The s p e c t r a l response of the semiconductor material (usually doped Si) i s f a i r l y broad i n the v i s i b l e and near i n f r a r e d regions (400-100 nm) and devices with a very high quantum e f f i c i e n c y i n t h i s region have been developed. The CCD device ( F a i r c h i l d ) c u r r e n t l y used i n the C e l l Analyzer has a c o l l e c t i o n time of 3.5 msec and a t o t a l t r a n s f e r time of 3.5 msec, thus the cycle time for the en t i r e array i s 7 msec. At present, the i n t e g r a t i o n time i s constant, although work i n progress i s aimed at c o n t r o l l i n g the i n t e g r a t i o n time. The array i s c o n t r o l l e d by a microprocessor, which clocks and reads the data, and a microprocessor which performs additive and m u l t i p l i c a t i v e corrections to each p i x e l of the array. The device i s c o n t r o l l e d and read by the host IBM-PC-AT v i a a s p e c i a l i z e d camera data bus. 175 6.2.2 Incubator For the l i v e c e l l m o t i l i t y studies with the C e l l Analyzer, a microscope incubator was b u i l t from p l e x i g l a s s which enclosed the microscope and stage. The temperature within the incubator was c o n t r o l l e d with an incubator-blower coupled to a thermoresistor feedback (Nikon). The incubator was designed to allow access to the tiss u e culture f l a s k on the microscope stage v i a a large door, as well as allowing adjustment of the microscope controls (focus, f i l t e r s , e t c . ) . The temperature within the incubator was monitored with mercury thermometers i n the v i c i n i t y of the f l a s k , and during c a l i b r a t i o n tests the temperature of the f l a s k medium was also measured. The a i r o temperature could be maintained at a chosen temperature to within ±0.2 C o within the range 30 to 40 C. the temperature i n the f l a s k could be a l t e r e d by the experimentor at some point during an experiment enabling studies of c e l l m o t i l i t y as a function of temperature. 6.2.3 Microscope Stage The development of the C e l l Analyzer f o r l i v e c e l l tracking depended i n a c r u c i a l way upon the performance and r e l i a b i l i t y of the microscope stage. The stage currently employed (Merzhauser) had a minimum step s i z e of 1.0 /im, a movement range of 10 cm x 10 cm, and maximum speed of 20,000 steps/second (2 cm/sec). The stage was driven by p r e c i s i o n x- and y-stepping motors coupled to a microprocessor support u n i t and operated by the IBM-PC-AT host computer. 6.2.4 D i g i t a l Signal Processor (DSP) The DSP performs analysis on the d i g i t i z e d ( o p t i c a l ) s i g n a l from the CCD array. In the RSCAN c e l l tracking program, the DSP analyzes the 96 p i x e l x 96 p i x e l image of the c e l l i n r e a l time, and extracts information on the p o s i t i o n 176 and morphology of the c e l l . The DSP i s the TMS 32010 (Texas Instruments). I t contains a 64 Kbyte multiported data memory and a 4 Kbyte program memory, mounted on a sing l e plug-in board. The c e l l image i s loaded d i r e c t l y into the DSP data memory v i a d i r e c t memory access (DMA). The DSP i s able to process data i n one portion of memory white data i s being loaded into another. The time required f o r a simple programming step i n the DSP i s 200 nsec (5 MHz cycle time) and r e s u l t s i n a 10 to 100 f o l d decrease i n computation time f o r the 96 x 96 p i x e l image processing compared to the same operation performed i n the host computer. The r e s u l t s of the DSP analysis ( c e l l centre, c e l l area, etc.) are loaded into a t h i r d part of the MPM which i s e a s i l y accessed by the host computer. 

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