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Quantitative measurements of changes in DNA tertiary structures induced by physical and chemical agents Hung, Yip-Chan Jacyln 1988

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Q u a n t i t a t i v e Measurements Of Changes I n DNA T e r t i a r y S t r u c t u r e s Induced By P h y s i c a l And Chemical Agents by Yip-Chan J a c l y n Hung B.Sc., U n i v e r s i t y o f P r i n c e Edward I s l a n d , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f P h y s i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1988 © Yip-Chan J a c l y n Hung, 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 of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT When Chinese hamster V79 lung fibroblast cells are lysed in solution containing sodium chloride, a non-ionic detergent Triton-X-100, and the DNA intercalating dye propidium iodide, intact loops of DNA can be visualized under the fluorescence microscope as a 'halo' region surrounding the periphery of the nuclear matrix. The size of these halos has a biphasic dependence on the concentration of propidium iodide, suggesting that the DNA is organized into supercoiled loops constrained by attachments to the nuclear matrix. However, the induction of strand breaks in the loops of DNA by ionizing radiation results in loss of supercoiling and a uniform halo size regardless of the concentration of propidium iodide. The goal of this project was to use mathematical procedures and image processing techniques to measure the change in the size of the DNA halo. We suggest that such a quantitative assay will provide a fast and objective means to directly measure the effect of physical and chemical agents on the DNA loops. The image acquisition, processing, and segmentation procedures that are necessary for the development of this quantitative assay are performed with the Fluorescence Image Processing System (FIPS). A gradient-threshold algorithm is implemented to segment the image, which in this case is to separate the DNA halo from the background and nuclear matrix. Damage inflicted by two of the better characterized agents, x-radiation and platinum complexes, are used as a test system for this assay. i i i The results reported here suggest that this assay can detect damage to the DNA loops at a radiation dose of as low as one Gray. The size of the DNA loop is calculated to be about 102/im. The biphasic dependence of the halo size on the concentration of propidium iodide is consistent with published data. Studies on the effect on platinum drugs on DNA loops show a difference in the kinetics of the unwinding and rewinding of the supercoils and the size of the halos at these phases also differ for both cis- and trans-diamminedichloro-platinum(II) treated cells. With our increased knowledge of the genome organization, an objective quantitative assay that can provide some insight to the damage and repair of DNA at the tertiary level of DNA organization should prove to be useful. iv TABLE OF CONTENTS PAGE ABSTRACT I i TABLE OF CONTENTS iv LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS ix INTRODUCTION 1 A. BIOLOGICAL IMPORTANCE OF DNA 1 B. MACROMOLECULAR DNA DAMAGE IN MAMMALIAN CELLS 2 a. DNA as a c r i t i c a l target 2 b. Types of DNA damage 3 c. Techniques to measure and detect DNA damage 8 d. Limitation of current techniques 12 C. ROLE FOR DNA TERTIARY STRUCTURE 14 a. DNA structure and organization 16 b. Experimental techniques to study DNA supercoiling 22 D. APPROACH 24 E. EFFECTS OF IONIZING RADIATION AND PLATINUM DRUGS 25 a. Interaction of ionizing radiation with mammalian cells 25 b. Effect of cis- and fcrans-DDP on mammalian cells 27 MATERIAL AND METHODS 30 A. CELL LINES AND CULTURE TECHNIQUE 30 a. Cell line 30 b. Cell culture conditions 30 B. PROCEDURE FOR FLUORESCENCE-HALO ASSAY 31 V a. Preparation of cell samples 31 b. Fluorescence microscopy 32 C. TREATMENTS 34 a. X-irradiation 34 b. Drug-treatments 34 D. FLUORESCENCE IMAGE PROCESSING SYSTEM (FIPS) 35 a. System configuration 35 b. Image processing and segmentation 35 RESULTS 42 A. SEGMENTATION AND DE-CALIBRATION 42 B. BIOLOGICAL CONDITIONS 43 a. Control 43 b. Effect of x-irradiation 48 c. Effect of cis- and trans - DDP 49 DISCUSSION 53 A. SEGMENTATION 54 B. BIOLOGICAL CONDITIONS 59 a. Radiation 59 b. Platinum drugs 60 SUMMARY 62 APPENDIX 64 A. DESCRIPTION OF FIPS COMPONENTS 64 a. Image acquisition 64 b. Digitization and image processing 64 c. Host computer 65 B. SYSTEM CALIBRATION 65 REFERENCES 69 v i LIST OF TABLES PAGE Table I. Measurements o f o p t i c a l performance o f the charge i n j e c t i o n d e v i c e (CID) s o l i d s t a t e camera 67 v i i LIST OF FIGURES PAGE Figure 1. Schematic representation of the types of DNA damage 4 Figure 2. Schematic diagram of the structure of DNA showing it s double helica l configuration 20 Figure 3. Schematic diagram of the packaging and organization of DNA within a eukaryotic nucleus 21 Figure 4. Fluorescence micrograph of supercoiled loops of DNA as a halo surrounding the nuclear matrix . 26 Figure 5. Possible modes of binding of cis-DDP and trans-DDP to DNA 29 Figure 6. Preparation of microscope slide and DNA halo 33 Figure 7. Schematic presentation of the main module of the Fluorescence Image Processing System 37 Figure 8. Flow chart of the image acquisition, processing, and segmentation procedure 38 Figure 9. Examples of the three histograms generated from a 128 x 128 pixels image 40 Figure 10. Result of the image processing and segmentation procedures on a 128 x 128 pixels image 44 Figure 11. Result of the image processing and segmentation procedures using gradient-histogram algorithm 45 Figure 12. Effect of the concentration of propidium iodide on the appearance of the DNA halo 46 Figure 13. Effect of the concentration of propidium iodide on the size of the DNA halo 47 v i i i Figure 14. Dose-response of x-irradiation on the size of DNA halos 51 Figure 15. Effect of platinum complexes on the DNA halo size 52 Figure 16. The theorectical distribution of the intensity of the DNA halo as a distance function from the nuclear matrix. 57 Figure 17. The intensity distribution of the DNA as a function of the distance from the nuclear matrix 58 i x ACKNOWLEDGEMENTS I wish to thank my supervisor Dr. Branko Palcic for his encouragement, support, and guidance, but especially for being so patient with me in the completion of this thesis. I would like to express my gratitude to Drs. Peggy Olive and Kirsten Skov for giving me their time and help in the course of this work. I am indebted to Bruno Jaggi for countless hours of interesting discussions and for a l l his contributions to this work. In particular, I would like to thank him for his thoroughness, help and guidance. I thank Calum MacAulay and Ingrid Spadinger for sharing their ideas and knowledge with me. I thank Alan Harrison and Brian Pontifex for their contributions in computer programming, and Evan Hobenshield and Steven Poon for their technical support, especially in trying to f i x the camera gain. Last but not least, I thank a l l my colleagues and friends in Cancer Imaging and the Medical Biophysics unit of the B.C. Cancer Research Centre for making the laboratory a pleasant place in which to work. 1 INTRODUCTION A. BIOLOGICAL IMPORTANCE OF DNA Deoxyribonucleic acid (DNA) is a biological macromolecule that carries the vital genetic information required for normal cellular metabolism and reproduction of a l l prokaryotic and eukaryotic cells (Hershey and Chase, 1952; Avery et al., 1944). It is present in a single copy and must be copied precisely when cell divides. Its integrity and stability are essential for normal functioning of proliferating cells. Any modifications in the molecular structure induced by damage to DNA could result in a loss or change of genetic information. In living cells, damage to DNA is repaired rapidly and with high fidelity. DNA repair is a molecular event and is believed to be performed by complex enzymatic mechanisms that are closely associated with DNA replication and transcription processes (Kbrnberg, 1980; Hanawalt et al., 1979). In the absence of repair, or after improper repair, DNA damage can result in the inactivation of cellular proliferating capacity causing cell death, mutagenesis and carcinogenesis leading to aberrant gene expression. In higher organisms (e.g. animals and humans), the consequences can be manifested as genetic diseases and / or cancer. Studies have shown that a number of the cancer-prone human hereditary diseases such as Xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Bloom's syndrome, and hereditary retinoblastoma involve 2 defective or deficient DNA repair mechanisms (Willis and Lindahl, 1987; Hanawalt and Sarasin, 1986; Friedberg, 1985a; Cleaver, 1968). B. MACROMOLECULAR DNA DAMAGE IN MAMMALIAN CELLS A multitude of physical agents in the environment such as heat, ultra-violet light (UV), ionizing radiation, as well as a variety of chemicals both naturally occurring (such as cyasin from cyad plant) and man-made (such as nitrofurantoin) are known to cause direct damage to DNA. In addition, spontaneous damage to DNA can occur during DNA replication by incorporation of a non-complementary base or an unpaired nucleotide causing frameshift errors, loss of bases by depurination and depyrimidination, and by deamination of bases (Friedberg, 1985b; Alberts et al., 1984) a. DNA AS A CRITICAL TARGET A large amount of experimental evidence has strongly implicated DNA as the most sensitive and critical target for physically and chemically induced cell lethality. Studies with electron beams (Zermeno and Cole, 1969) and with alpha-particles of selected penetration energies (Datta et al., 1976) show that the cell nucleus, and more specifically DNA, is the most radiosensitive part of the cell. Many drugs such as bleomycin, a DNA-breaking agent (Ostling and Johanson, 1987; Moore and Little, 1985), mitomycin C, an alkylating agent (Prltsos and Sartorelli, 1986), methotrexate and other folates analogue, which exhibit anti-proliferating activity (Lorico et al., 1988; Chabner, 1982; 3 Goulian et al., 1980), furocoumarins In combination with UVA (Gruenert et al., 1985; Ben-Hur and Song, 1984), cis-diamminedichloro-platinum(II) (cis-DDP), an anti-tumor drug (Harder and Rosenberg, 1970) are known to induce their cytotoxic effects by interacting with DNA, demonstrating that the cell nucleus, in particular DNA, is the important target for cytotoxic compounds. Damage to the other components of the cell such as plasma membrane is usually characterized at a relatively higher dose range, exceeding that causing reproductive cell death. In survival 125 studies with Chinese hamster ovary (CHO) cells using IdUrd 125 incorporated into the DNA and I-labeled concanavalin A attached to the cell membrane, a surviving fraction of 0.5 was observed at 60 disintegration of I per nucleus and a similar survival was observed at about 2 x 10^ disintegrations at the membrane, indicating that cell death results from damage to the nucleus not to the plasma membrane (Warters et al., 1977). b. TYPES OF DNA DAMAGE Several types of damage to the macromolecular structure of DNA can result from physical and chemical DNA damaging agents (Gentner and Paterson, 1984; Tyrnell, 1984; Kohn, 1979). These include base damage and damage to the sugar-phosphate backbone. Damage to the bases can lead to base loss or alterations that cause a mutation; damage to the sugar-phosphate backbone can result in strand breaks, DNA-DNA crosslinks, and DNA-protein crosslinks. Damages to the sugar-phosphate backbone are associated with cytotoxicity and anti-tumor activity. The different types of DNA damage are illustrated schematically in Fig. 1. 4 5 i) Strand Breaks Ionizing radiation (e.g. x-rays) and bleomycin, an anti-cancer drug are known to produce single- and double- strand breaks. Single strand breaks (SSBs) interrupt the linear continuity of one strand of the DNA double helix. Studies have shown that mammalian cells repair SSBs rapidly, therefore these breaks are not implicated in cell killing (Elkind, 1984; Lett et al., 1967). A double strand break (DSB) is a result of two SSBs occurring close together on opposite sites of DNA strands in a single radiation event or as a result of two independent events; DSB occurs less frequently than SSB, about 5% of SSB frequency. Several investigators have suggested that unrepaired or mis-repaired DNA DSBs represent the critical molecular lesion responsible for cell death (Radford, 1985; Bryant, 1985; Blocher and Pohlit, 1982; Frankenberg et al., 1981; Chadwick and Leenhouts, 1973). Most mathematical models postulate that any unrepaired DSBs in DNA of mammalian cells are lethal lesions (Blocher and Pohlit, 1982; Tobias et al., 1980; Dugle et al., 1976; Leenhouts and Chadwick, 1978). However, there is conflicting evidence on whether or not DSBs are repaired in mammalian cells and there are studies with mammalian cells that do not support the hypothesis that unrepaired DNA DSB is a lethal lesion (Koval and Kazmar, 1988; Lehman and Stevens, 1977). i i ) DNA-DNA crosslinks Inter- and intra-strand DNA crosslinks are produced by a variety of bifunctional chemical agents such as mitomycin C, platinum derivatives such as cis- and crans-DDP, busulfan, nitrogen and sulfur mustard, diepoxybutane, and photoactivated psoralens (Friedberg, 1985b; 6 Kohn, 1983). Interstrand crosslinks result from two sequential covalent reactions of the chemicals with DNA. These crosslinks prevent the separation of the DNA strands, thus blocking normal DNA replication and transcription processes and exert cytotoxic and cytogenetic effects. Most of the chemicals that produce such crosslinks have anti-cancer activity, hence many are used extensively in cancer chemotherapy. i i i ) DNA-protein crosslinks With the exception of psoralen, a l l agents which generate DNA-DNA crosslinks in mammalian cells can also produce DNA-protein crosslinks. DNA-protein crosslinks are produced by two sequential addition reactions of these chemicals; one covalent reaction link to protein while the second link to DNA. DNA-protein crosslinks are also formed in human cells exposed to ionizing radiation, uv light, and visible light (Oleinick et al., 1986; Peak and Peak, 1986). Studies with purified DNA using mouse leukemia L1210 cells treated with 1,3-bis(2-chloroethy)-1-nitrosourea (BCNU) and with cis-DDP (Ewig and Kohn, 1977; Zwelling et al., 1979) suggest that DNA-protein crosslinks are formed more rapidly than DNA-DNA interstrand crosslinks. Both types of crosslinks are repaired in mammalian cells, but DNA-protein crosslinks are generally less toxic than DNA-DNA crosslinks. A comparison between cis- and trans- DDP on L1210 cell lines have shown that DNA-interstrand crosslinks are prominent in cis-DDP whereas trans-DDP produces relatively more DNA-protein crosslinks (Zwelling et al., 1979). Cis-DDP are known to be more cytotoxic than trans-DDP and is an effective anti-tumor drug. In cell survival studies, using drug dosages that give similar crosslinks, cis-DDP is observed to be more effective in cell 7 killing than trans-DDP, thus implying that interstrand crosslinks are lethal, but DNA-protein crosslinks generated by trans-DDP are either non-lethal or are repaired before expressing a lethal effect (Kohn, 1979) iv) Base Damage About 80% of the reactions of the hydroxyl radicals formed by ionizing radiation attack thymine base to form a hydroxl radical adduct or an altered base. Ultra-violet radiation can also cause base alteration to DNA by the addition of a water molecule across the 5-6 double bond of a pyrimidine base (thymine or cytosine) to form a pyrimidine hydrate. In the case of cytosine, the pyrimidine hydrates may deaminate and dehydrate to form uracil. UV light also induces the formation of a cyclobutane ring between two adjacent pyrimidines. The resulting pyrimidine dimers are blocks to DNA replication, and hence are lethal lesions to cells. Base damage is repaired by excision (Alberts et al., 1983; Hanawalt et al., 1979). This repair process involves a set of enzymes that carry out the following steps: 1) preincision recognition of damage, 2) incision of the damaged DNA strand near the sites of defect by an endonuclease enzyme, 3) excision of the defective site and localized DNA degradation by an exonuclease enzyme, 4) repair replication to restore the excised region with the original nucleotide sequence by DNA polymerase, which resynthesis this region by inserting nucleotides complimentary to the opposite strand, which then serve as a template. Progression is believed to be 5' to 3' for this enzyme, and finally 5) ligation by DNA ligase to join the repaired region to the parental DNA strand (Hanawalt et al., 1979). 8 c. TECHNIQUES TO MEASURE AND DETECT DNA DAMAGE Currently, many techniques are available to detect and measure DNA damage. Some of these techniques include electrophoresis, alkaline sucrose gradient sedimentation, alkaline gradient elution, alkaline unwinding and hydroxylapatite chromatography, neutral gradient sedimentation, and nucleoid gradient sedimentation. Extensive details of some of these techniques are available in " DNA Repair: A laboratory manual of research procedure " (Friederg and Hanawalt, 1981). The most frequently used methods to study DNA single strand breaks and repair in mammalian cells are alkaline sucrose sedimentation, alkaline elution, and alkaline unwinding followed by hydroxylapatite chromatography. i) Strand breaks Alkaline sucrose sedimentation The procedure for alkaline sucrose sedimentation involves the lysing of cells in alkali on top of a 5-20% sucrose gradient followed by sedimentation in an ultracentrifuge for several hours. The alkali denatures the protein from the DNA and allows the DNA to unwind. Single strands of DNA are then sedimented with minimal hydrodynamic shearing. Some pieces of DNA due to excess strand breaks resulting from treatment by chemical and physical agents sediment at a slower rate, and hence can be detected. Usually, treated and control DNAs are co-sedimented in a single gradient. The sensitivity of detecting these breaks is about one break per 108 daltons (Lett et al.,1967; McGrath and Williams, 1966). 9 Alkaline elution This technique utilize polycarbonate membrane filters to discriminate DNA single-strand sizes in mammalian cells (Kohn and Ewig, 1973). Cells labeled with ^C thymidine are lysed onto a membrane fil t e r and a detergent such as sodium dodecyl sulfate (SDS) at pH 10 is then used to wash most of the cellular protein and RNA through the fi l t e r , leaving the double-stranded DNA intact on the f i l t e r . An alkaline eluting solution, at pH 12 is then pumped slowly through the fi l t e r and the kinetics of the elution of the single-stranded DNA is determined. The rate at which the DNA elutes is a measure of its length (i.e. the distance between single-strand breaks). The resultant DNA is collected and counted for radioactivity. Prior to elution, the lysate on the f i l t e r is treated with proteinase K to remove any effects of DNA-protein crosslinks. The number of strand breaks is usually calibrated against an internal standard of H-labeled control cells x-irradiated at 0°C with a dose of 0.15 or 0.3 Gray (Gy). The sensitivity of the measurement is about one strand break per 10^ daltons of DNA. Alkaline unwinding and hvdroxvlapatite chromatography This technique involves a time-limited treatment of cells in a mild alkaline solution (pH 12) followed by rapid neutralization of the alkaline solution (Ahnstrom and Edvardsson, 1974). Alkaline lysis causes the DNA to undergo denaturation and strand separation (unwinding) from free end points and at sites of single-strand breaks. The number of single-strand DNA transformed by the alkaline treatment is directly proportional to the number of single-strand breaks initiall y present. After neutralization of the alkaline solution, and then sonication to 10 produce small fragments, hydroxylapatite chromatography is applied to separate the single-stranded and double-stranded DNA. The ratio is used as a measure of the number of strand breaks present in DNA. The sensitivity of this technique is similar to that of the alkaline elution method. For the measurements of DNA double strand breaks, the commonly used methods are the neutral sucrose sedimentation assay and the neutral f i l t e r elution technique. An [H+] concentration of pH 10 or less is required to maintain the DNA duplex since the DNA begins to denature at above this pH. Neutral sucrose gradient sedimentation In this method, cells labeled with thymidine are lysed in detergent solution containing sodium dodecyl sulphate (SDS), sarkosyl, sodium deoxycholate, EDTA, Tris, and pronase at pH 9.0 on top of a neutral sucrose gradient and sedimenting the free DNA through the gradients. Gradients are run in an ultracentrifuge. After centrifugation, the gradients are fractionated and samples counted for radioactivity. The relative molecular mass of the double strand DNA is estimated by comparing the mass distribution sedimentation profile of the irradiated sample to a computer simulated profile of the mass spectrum of a control sample (Blocher, 1982). Neutral f i l t e r elution This method has been developed for the measurements of DNA double strand breaks at relatively low doses of radiation. In this assay, pre-mixed H and C labeled cells are suspended in cold phosphate buffer saline (PBS), pulled down by a moderate vacuum through a polycarbonated fil t e r , and are lysed in a solution containing glycine, EDTA, sodium lauryl sulphate, and proteinase K at pH 9.0. The kinetics of the elution of the double stranded DNA is then determined. The DNA eluted is considered double stranded since the pH used in this assay is below the critical pH 11.6, at which the DNA begins to denature. The neutral fi l t e r elution method can detect the number of double strand breaks induced by as l i t t l e as 1 Gy of x-rays dose (Bradley and Kohn, 1979). ii ) Crosslinks and base damage Variations and modifications of the above biophysical and biochemical methods are used to measure DNA-protein crosslinks, DNA-DNA crosslinks and base damage. Crosslinks can be measured using the alkaline elution method on the basis that crosslinks have the effect of reducing the rate of DNA elution. Interstrand DNA-crosslinks induces this effect by increasing the effective DNA strand length, and in the case of DNA-protein crosslinks, the effect is due to the adsorption of the proteins to the fi l t e r . Crosslinks can reduce the apparent SSB frequencies, hence by introducing into the DNA an appropriate known frequency of SSBs, the frequency of crosslinks can then be detected based on the reduction of the rate of DNA elution. This is achieved by exposing cells to 0.3 Gy of x-ray dose, at 0°C prior to the elution procedure (Kohn, et al., 1986). Using this method, sensitivity of one nitrogen mustard crosslink per 10 daltons have been reported. A rapid method for detecting base damage in DNA of mammalian cells has been reported (Bryant et al., 1978). The method involves the 12 application of the alkaline unwinding and hydroxylapatite chromatography assay described above and the used of a specific U.V. endonuclease, present in the crude extracts of Micrococcus luteus. This endonuclease has been shown to break the single strand of DNA adjacent to a pyrimidine dimer, and hence i t can be used to detect dimers in DNA of irradiated mammalian cells. Breaks in the DNA, formed by the action of this endonuclease can then be detected using the alkali unwinding assay and the number of single- and double- strands DNA are separated by hydroxylapatite chromatography. The sensitivity reported for this study 8 2 is one dimer per 10 dalton per J/m of UV dose. d. LIMITATION OF CURRENT TECHNIQUES A survey of the literature shows that there are s t i l l many unresolved difficulties and uncertainty as to the validity of the results presented from these assays. Neither the number of double strand breaks nor the fidelity of their repair are determined with satisfactory accuracy. i) Sensitivity None of the above-listed methods can detect DNA lesions at a sensitivity needed for cell-survival studies in most repair models. In the molecular theory of cell survival, the critical molecular lesion is the DNA double-strand break, one unrepaired double-strand break is considered lethal to the cell (Leenhouts and Chadwick, 1978). At present, the techniques described above do not have the sensitivity nor 13 the reliability needed to detect repair of such a lesion at the dose range of biological significance for mammalian cells. i i ) Alkali-liable sites Most of the methods that are used to detect and measure s ingle-strand breaks in cells require alkaline conditions which can generate alkali-liable sites. It is difficult to differentiate between damage from initiall y existing breaks and breaks that occur from alkali-liable sites. i i i ) Radioactivity Most of the methods also require radioactively labeled cells and hence exclude the studies of lymphocytes, non-dividing cells, and limit in vivo studies. iv) Tertiary structure However, even the sensitive methods that are currently used cannot provide any information concerning the alteration of DNA tertiary structure and its reconstitution during the repair of lesions. This would be useful in many area of interest, which is expanded in the next section. For examples, cells grown in suspension culture as spheroids are known to be more resistant to killing by ionizing radiation than cells grown as a monolayer (Durand and Sutherland, 1972). Using alkaline sucrose gradient as well as the more sensitive alkaline elution and hydroxylapatite chromatography assay, no differences was observed in the amount of radiation-induced DNA single-strand breakage and repair between spheroids and monolayers (Durand and Olive, 1979). However, in 14 an earlier study, using similar assays, repair of single strand breaks in spheroids was faster than in monolayers (Koerner et al., 1978). Using the alkaline-unwinding assay, a difference between the rate of monolayer and spheroid unwinding was observed under specific conditions of salt and pH (Olive and Durand, 1986). One possible explanation for these conflicting results is that changes in DNA packaging (tertiary structure) occur when cells are grown as spheroids and these changes account for the difference in radiation sensitivity. However, the assays used in these studies do not measured the damage and / or repair to the tertiary structure of DNA. Damage to DNA, whether in the form of damage to individual bases, single- and double-strand breaks, or crosslinking, can also result in the changes of the tertiary structure in DNA. Repair of damaged DNA is considered as incomplete unless the tertiary structure to DNA and the packaging of DNA at the higher level of organization is correctly restored to i t s original configuration. C. ROLE FOR DNA TERTIARY STRUCTURE In mammalian cell s , the tertiary structure of DNA may play an important role in the distribution and repair of damage induced by chemicals and radiation. There is evidence for intragenomic heterogeneity in the distribution of DNA damage and repair efficiency over the mammalian genome (Bohr et al., 1988). A number of studies have suggested that the repair of damage occurs preferentially in the transcriptionally active genes (Oleinick et al., 1986; Mellon et al., 15 1986; Bohr et al., 1985). Since active genes are transcribed at the nuclear matrix (Ciejek et al., 1983; Robinson et al., 1983; Cook et al., 1982), this suggests a possible role for the DNA loops attachments sites at the nuclear matrix in the repair process. Studies have indicated that the repair process for UV- and chemically-damaged cells is non-random and is localized in the proximity of the DNA-nuclear matrix attachment sites (Mullenders et al., 1987; McCready and Cook, 1984; Harless et al., 1983). Evidence has been presented that repair in the human XP-C cells occur preferentially in DNA associated with the nuclear matrix (Mullenders et al., 1986). However, at high doses of uv light (30 Jm ), evidence for a random distribution of repair events in the DNA loops has been reported for Syrian hamster fibroblasts, human XP-D cells, and HeLa cells. But at o low and biologically relevant doses (5 Jm" ) at least part of the excision repair process is initiated at the nuclear matrix (Mullenders et al., 1987). In addition, DNA regions containing transcriptionally active sequences and newly replicated DNA, which is associated with the nuclear matrix, are found to be more susceptible to damage by ionizing radiation (Chiu and Olenick, 1982; Chiu et al., 1982; Walters and Childers, 1982). However, there is general agreement that ionizing radiation induces a random distribution of lesions on the DNA, but the distribution of chromosomal aberrations resulting from this radiation is non-random and does not always correspond to the areas where the greatest amount of damage occurs. The tight packaging and organization of the DNA in the nucleus may render damage sites at regions of the chromatin organization inaccessible to the DNA repair enzymes. 16 Studies have also indicated that nuclear matrix associated DNA may be the most radiosensitive DNA of mammalian nucleus (Chiu et al., 1986), suggesting that the structural organization and packaging of DNA in the nucleus may play an important role in determining cellular response to damage. There are also suggestions that alteration in the packaging or the changes in DNA conformation is related to differentiation, carcinogenesis, and radioresistance (Olive et al., 1986; Lipetz et al., 1982a; Hartwig et al., 1981). A majority of the studies of DNA damage have emphasized repair enzymes and their roles at the primary and secondary levels of DNA structures. It is important to note that damage to DNA may also affect the tertiary structure and organization of DNA. The changes in the packaging and organization of DNA must be returned to its original configuration as any alteration in this structure is crucial. DNA supercoiling is an essential feature in the structural and functional organization of eukaryotic DNA. Hence, these important aspects of DNA tertiary structures should merit further investigation. a. DNA STRUCTURE AND ORGANIZATION IN MAMMALIAN CELLS The DNA of eukaryotic (mammalian) cells is present in the nucleus as a number of discrete fragments called chromosomes. In human cells, each chromosome contains about 100,000 kilobase pair (kbp) of DNA. If a l l the DNA were stretched out to the extended linear double stranded form, i t would be about 1.74m long. This is too long to f i t into a nucleus that is typically about 5um in diameter. Therefore, to package this large amount of DNA into the nucleus of a eukaryotic cell, the DNA must then be highly organized into several structural levels. Recent 17 studies have indicated that DNA are organized into supercoiled loops or domains, and these repeating loops are constrained by attachment to a supporting skeletal structure of the nucleus termed the nuclear matrix, cage or scaffold. The primary structure of DNA is the nucleotide base pairs sequences which in part exist in the form of the double helical configuration as proposed by Watson and Crick (Watson and Crick, 1953). The double helix consist of two polynucleotide chains that run in parallel but opposite direction with a right-handed twist. Each polynucleotide has a backbone in which a phosphate group alternates with a deoxyribose sugar to form a covalently linked chain. One of the four bases, adenine (A), guanine (G), thymine (T), or cytosine (C) are attached to the sugar ring of each nucleotide. A specific hydrogen-bonding arrangement, known as the Watson-Crick pairing occurs between the bases. Guanine forms hydrogen bonds with cytosine, while adenine always pairs with thymine. The geometry of the duplex is such that sequential bases are 0.34»ym apart and are related by a 36° rotation about the helical axis; thus there are 10 bp per turn of DNA with a pitch height of 3.4»;m. The DNA has a diameter of about 2.0r/m. A schematic of the DNA double helical configuration is illustrated in fig. 2. The tertiary structure of DNA involves the association of DNA molecules with histone and non-histone proteins to form the chromatin. Histones are basic proteins with a molecular weight range of 11000 to 21000. There are five types of histone, HI, H2A, H2B, H3, and H4. Chromatin consists of about 146 bp to 166 bp of the double helical DNA coiled around an octomer of histone proteins to form a 10»;m 18 structure called nucleosome (McChee and Felsenfied, 1980; Olin and Olin, 1974) . The octomer core consists of two molecules each of H2A, H2B, H3, and H4. Cores are connected by linker region where DNA enters or exist the core particle. Histone HI is located on the linker-DNA region. The distance between two nucleosomes called the "repeated length" varies in length but is approximately 60bp long. The coiling of two turns of the double helix around the nucleosome core reduces the length of 166 bp of DNA from 60»jm into coil of 5.5ijm (Romberg, 1977, 1974). The strings of nucleosomes are then packed into an array of 20 to 30»/m chromatin fibers by a solenoidal mechanism. Each solenoid consists of a further coiling of the coiled nucleosomes, with six to nine nucleosomes per turn (McGhee et al., 1983; Finch and Klug, 1976). However, a zig-zag helical ribbon model (Woodstock et al., 1984), a rope-like "fibersome" model (Nicolini, 1983; Cavazza, 1983), and a superbead model (Weintraub, 1984) have also been proposed for the packaging of the nucleosomes into chromatin fiber. In the interphase nuclei and metaphase chromosomes the fibers appears to be folded into structure termed loops (Cook and Brazell, 1975) or domains (Paulson and Laemmeli, 1977). Each loop contains about 30kbp to 100 kbp of DNA (Nelkin et al., 1982) which is anchored to a supporting structure of the nucleus that has been termed the nuclear matrix or cage for interphase cells and the nuclear scaffold for metaphase cells. (Pienta and Coffey, 1984; Adolph et al., 1977; Berezney and Coffey, 1975; Cook and Brazell, 1975). The nuclear matrix is an ubiquitous, insoluble, structural framework composed of about 10% of the total nuclear proteins. It consists of residual elements of the pore-complex and lamina, nucleolus, 19 and an intranuclear network of the ribonucleoprotein particles attached to a fibrous protein mesh of cytoskeletal actins and keratins. This network provides the basic shape and structure of the nucleus (Nelson et al., 1986). The organization of DNA in supercoiled loops is non-random. These loops are constrained by attachment to the nuclear matrix or scaffold through specific DNA sequences and sites (Mirkovitch et al., 1984; Nelkin et al., 1982; Cook and Brazell, 1980). It is reported that the nuclear matrix and sites of the attachments of the DNA loops are involved in various nuclear functions. Actively transcribed genes are enriched on the nuclear matrix (Ciejek et al., 1983; Robinson et al., 1983; Cook et al., 1982). RNA is reported to be synthesized at the nuclear cage (McCready et al., 1982; Jackson et al., 1981). In addition, DNA replication is also associated with the nuclear matrix and the attachment sites (McCready et al., 1982, 1980; Hunt and Vogelstein, 1981; Mattern and Painter, 1979). During replication, the DNA loops may be "reeled" through fixed matrix-associated replication complexes (Pardoll et al. , 1980; Vogelstein et al., 1980). Each loop of DNA appears to function as an individual replicon, and the sizes of the loops are reported to be approximately the size of replicons for different cells (Buongiorno-Nardelli et al., 1982, Nelkin et al., 1981). Repair process and cellular radiosensitivity have been implicated at the sites of attachments of the DNA loops to the nuclear matrix (McCready and Cook, 1984; Mattern, 1984; Chiu et al., 1986). A schematic representation of the packaging and organization of DNA within a nucleus is shown in fig. 3. 20 Figure 2. Schematic diagram of the structure of DNA showing i t s double h e l i c a l configuration (Alberts et al., 1983). F O U R B A S E S A S B A S E P A I R S O F D N A m.nor groom m J | 0, g r o o v. 21 22 b. EXPERIMENTAL TECHNIQUES TO STUDY DNA SUPERCOILING The formulation of the domain or loop model for DNA organization is based mainly on the experimental methods of nucleoid gradient sedimentation (Cook and Brazell, 1975), fluorescence-halo technique (Roti Roti and Wright, 1987; Vogelstein et al., 1980), and electron microscopy (McCready et al., 1979). i) Nucleoid sedimentation The principle of this method consists of the gentle lysis of cells in the presence of a non-ionic detergent such as Triton-X-100 and high salt to release nucleoids, followed by neutral gradient centrifugation (Cook and Brazell, 1975). Nucleoids retain many of the morphological features of a nucleus, containing most of the nuclear ribonucleic acid (RNA) and DNA. The DNA of a nucleoid is supercoiled into loops which are attached at their bases to the nuclear matrix. A l l nucleoids sediment as one aggregate, and not as individual particles. The rate is dependent on the number of strand breaks. A single-strand break in a loop will release the supercoiling of the DNA which will result in a decrease in density and thus a decrease in the sedimentation rate of the nucleoid. By adding fluorescent dye, such as ethidium bromide in the lysis solution and illumination with long UV light, the position of the fluorescent DNA band can be observed directly in the gradient. Hence the gradient need not be fractionated. Single-hit kinetics are used to calculate the number of strand breaks that must be introduced to relax the DNA supercoiling. The difference between the number of strand breaks required to completely relax the supercoiling of a treated and untreated control sample is the measure of the number of pre-existing 23 breaks in the treated sample. Hence, this technique does not quantitate the absolute numbers of DNA strand breaks. The nucleoid sedimentation technique gives a measure of the compactness of the packaging of higher DNA organization. This method, though sensitive, has a very large number of artifacts and moreover, the measurements are dependent on the mass of the total particle, i.e., both the DNA loops and the residual nuclear matrix. i i ) Fluorescence-Halo technique Cells growing on monolayers or in suspension are gently lysed at neutral pH in a solution containing a non-ionic detergent and high salt concentrations, including an intercalating dye like propidium iodide or ethidium bromide (Roti Roti et al., 1987; Vogelstein et al., 1980). After relaxation of the DNA as a result of the intercalating dye, intact loops of DNA can be visualized under the fluorescence microscope as a 'halo' surrounding the nuclear matrix. Increasing concentration of the intercalating dye causes the negatively supercoiled DNA to unwind until a configuration without supercoiling is reached. Further increase in the concentration of the intercalating dye after this relaxation point cause the DNA loops to rewind positively until they are fully compact. Under the fluorescence microscope this can be visualized as a change in the halo size. Radiation-induced damage to the DNA loops will release the supercoiling of that loop and precludes these unwinding and rewinding. The ability to unwind or rewind is inhibited in a dose-dependent manner. If cells are exposed to drugs that bind selectively to DNA and cause changes in the DNA tertiary structure, the rate at which the DNA 24 unwinds and rewinds at a certain concentration of the intercalating dye may be inhibited or reduced. Any damage induced either by radiation or drug will result in a change in the size of the DNA halo. Measuring the resulting changes in the diameter of the DNA halo is a measure of the damage to DNA induced by such agents. The illustration on fig: 4 is a color print of a typical fluorescent DNA halo surrounding the nuclear matrix. D. APPROACH The fluorescence-halo techniques and the nucleoid gradient sedimentation assays have been used in the study of repair and damage induced by UV radiation, ionizing radiation, and chemicals on a variety of cell types (Jaberaboansari et al., 1988; Mullenders et al., 1987; Roti Roti and Wright, 1987; van Rensberg et al., 1987, 1985; Mattern, 1984; Harless et al., 1983; Lipetz et al., 1982b; Weniger, 1982; Cook et al., 1978; Cook and Brazell, 1976). These studies, in particular the fluorescence-halo assay uses manual methods to measure the corresponding changes in size of the DNA halo. This is subjective, inaccurate, and labour intensive. One way to overcome these problems is to apply image processing techniques from image cytometry using mathematical algorithms to determine the halo size. Quantitative analysis using procedures employing different algorithms not only saves a large amount of time and effort, but also provides objective measurements. This project was undertaken to provide a quantitiative and objective measurement of the change in size of the DNA halos. To 25 accomplish this, a Fluorescence Image Processing system (FIPS) that has been developed for low-light level imaging is employed and mathematical procedures and image processing will be performed on the image (Jaggi et al., 1988). For an objective image analysis, the crucial task is to segment the image, which in this case is tb separate the DNA halo from the background and the nuclear matrix: Segmentation by thresholding is implemented for the measurement of the size of the halo under different biological conditions. The effect of ionizing radiation and platinum drugs on Chinese hamster lung V79 fibroblasts are studied using this quantitative assay. E. EFFECTS OF IONIZING RADIATION AND PLATINUM DRUGS IN MAMMALIAN CELLS a. INTERACTION OF IONIZING RADIATION WITH MAMMALIAN CELLS Ionizing radiation is a form of energy; and for energy to have an effect on biological molecules, i t must first be absorbed. On passing through biological material, ionizing radiation does not transfer its energy uniformly, but instead deposits i t as discrete packets called 'spurs'. It is relatively non-selective with respect to the energy deposition. The action of ionizing radiation on biological molecules can be: (i) direct action, in which the energy is absorbed directly by the target molecule; (ii) indirect action, in which the majority of the energy-loss events occur in water to form reactive species called free radicals. These free radical species are the hydroxyl radical, the aqueous electron, and the hydrogen radical. These radicals are highly reactive and diffuse rapidly to give rise to indirect damage. 26 F i g u r e 4. Fluorescence micrograph o f the s u p e r c o i l e d loops o f DNA as a h a l o surrounding the p e r i p h e r y o f the n u c l e a r m a t r i x , prepared from Chinese hamster V79 c e l l s . 27 Both direct and indirect damage to DNA can result in base damage and damage to the sugar phosphate backbone, such as single-strand breaks, double-strand breaks, DNA-DNA crosslinks and DNA-protein crosslinks.. b. EFFECT OF CIS- AND TRANS-DD? ON MAMMALIAN CELLS The discovery that cis-DDP exhibits anti-tumor activity in a wide spectrum of cancer cells (Rosenberg et a l . , 1969), while its trans isomer is inactive has stimulated research into understanding the mode of action and selectivity of cis-DDP (Plooy and Lohman, 1980) and the search for alternative platinum complexes. Both isomers have been shown to bind to DNA, and studies have implicated that DNA is the cellular target for the drug action (Roberts and Thomson, 1979), although details are s t i l l not completely established. For examples, the anti-tumor activity of cis-DDP was proposed to result from direct covalent binding to DNA, which then manifests selective inhibition of DNA synthesis (Harder and Rosenberg, 1970). Both cis- and trans-DDP bind preferentially to the guanine and cytosine regions of DNA by either monofunctional base binding or formation of inter-strand crosslinks (Roberts and Friedlos, 1981; Pera et al., 1981). However, cis-DDP also forms intra-strand crosslinks to adjacent guanines and this mode of binding that is exclusive to cis-DDP has been implicated for cis-DDP anti-tumor activity (Fichtinger-Schepman et al., 1985; Lippard et al., 1983; Cohen et al., 1980). There is s t i l l a disagreement as to whether the inter-strand crosslink or the intra-strand DNA crosslink is the critical lesion. There is a correlation 28 between cis-DDP cytotoxicity and the formation of inter-strand DNA crosslinks (Zwelling et al., 1981; Laurent et al., 1981). In addition to the DNA-DNA crosslinks, both cis- and trans-DDP also form covalent crosslinks between the DNA and its associated proteins (Hnilica et al., 1985; Filipski et al., 1983; Zwelling et al., 1979; Lippard and Hoeshele, 1979). A schematic representation of the different binding modes of cis- and trans-DDP is shown in fig. 5. Studies on SV40 DNA and PM-2 DNA show a difference in the effect of cis-DDP and trans-DDP on the DNA tertiary structure (Scovell and Kroos, 1982; Mong et al., 1980; Cohen et al., 1979). Gel electrophoretic studies on these closed circular DNAs show that cis-DDP shortens the double helix, and the unwinding and rewinding of DNA supercoiling is faster and more effective than those induced by trans-DDP. However, using the nucleoid sedimentation technique, cis-DDP shows l i t t l e i f any effect on the sedimentation behavior of mammalian nucleoids (Weniger, 1982). These studies cited have yet to resolve the question as to why cis-DDP exhibits anti-tumor activity. Both cis- and trans-DDP bind covalently to DNA, inducing a distortion in the DNA structure. Perhaps subsequent changes in the degree of supercoiling at the higher level of DNA organization may determine the differential cytotoxic activities of cis-DDP in mammalian cells. 30 MATERIALS AND METHODS A. CELL LINES AND CULTURE TECHNIQUE a. CELL LINE The cells used in a l l of the studies in this project were Chinese Hamster V79 lung fibroblasts. The culture was originally obtained from Dr. Warren Sinclair at Argonne National Laboratories as line V79-171b. This cell line grows well as a monolayer culture on tissue culture plastic - both in flasks and in petri-dishes. It has a high plating efficiency and a rapid population doubling time of approximately 12 hours. b. CELL CULTURE CONDITIONS The cells were routinely grown as monolayer culture in 25-cm polystyrene tissue culture flasks (Falcon) in Eagle's F-15 minimum essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco). Cell cultures were incubated at 37°C in a 5% C02-humidified atmosphere environment. Cells were maintained in exponential growth and the cell's doubling time was about 12 hours. The cells were routinely subcultured when grown to confluence, about twice per week. Subculturing were usually achieved by trypsinization. The cells were first washed with about 3ml of 0.1% trypsin solution (Gibco) and then exposed to about 2ml of the trypsin for about 6 minutes at 37°C. Trypsin action was neutralized by the addition of fresh medium, which 31 was then pipette vigorously to obtain single cells. Cells were then plated in new tissue culture flask. B. PROCEDURE FOR FLUORESCENCE-HALO ASSAY a. PREPARATION OF CELL SAMPLES Samples of cells for each experiment were removed from the growth medium by centrifugation at 600rpm, at 4°C (Model RC-3, Sorvall) for 6 minutes and resuspended in 10ml of cold Spinner's salt solution (0.8mM MgS04; 9mM anhydrous NaH2P04; 5mM KC1; 0.1M NaCl; 26mM NaHC03, and 5.6mM D-glucose; at pH 7.4). The cell suspension was vortex for 5 seconds to disperse the cells uniformly, and the cell density was determined using a coulter counter (Model Zg, Coulter). The cell suspension in spinner's salt solution were centrifugated again at 1800rpm, at 4°C for 5 minutes. Thereafter, the supernatant was removed and the cells resuspended in cold Spinner's salt at a cell concentration of about 1 x 10"* to 3 x 10^ cells/ml. The cell suspension was kept on ice until lysis procedure. For lysis, an aliquot of about 50 to 100^ *1 of the cell sample was spread over the enclosed area of the prepared microscope slide. To obtain better optical clarity, each microscope slide is soaked in 1% acid alcohol, rinsed in distilled water, washed in 95% alcohol and dried with a cheese cloth. Prior to the lysis procedure, the pre-cleaned microscope slides are rinsed with poly-1-lysine solution; this will decrease the movement of the negatively charged DNA. An equal volume of the lysis-dye buffer containing 2.0M NaCl; lOmM EDTA; 2mM Tris [pH 8.0]; 0.5% Triton-X-100; and the desired concentration of propidium iodide was 32 added to the cell sample on the microscope slide. A glass cover slip (12-545, Fisher Scientific) was then placed over the top of the microscope slide. This was then left in the dark at room temperature (placing the slide inside a box is convenient) for about 5 to 8 minutes to ensure the completion of lysis. An illustration of the preparation of DNA halo on the microscope slide is shown in fig. 6. b. FLUORESCENCE MICROSCOPY The DNA halos were viewed through a Zeiss III RS epi-fluorescence microscope (Model D-7082, Zeiss) under excitation with green light (Band pass 546/7»/m excitation filter, a FT 580fjm beam splitter, and a long pass 590»jm barrier fi l t e r ) , using a HBO/100W mercury lamp equipped with a stabilized power supply. A 25x NEOFLUOR objective with a numerical aperture of 0.60 was used. The fluorescing DNA halos were moved into the centre of the field, focused and images were captured using an image intensified charge injection device (CID) solid state camera, which is mounted on the microscope image receiver. The CID solid state camera is part of the FIPS system. Detailed information of this camera and measurements of its optical performances are described in the appendix. Fluorescence observations and image acquisition were made within the first 60 seconds after illumination. This was necessary because on continuous illumination, a rapidly expanding halo will develop around the original limits of the DNA halo-nuclear matrix boundary. This effect may be caused by the photoactivation and / or photo-bleaching of the propidium iodide, resulting in the breaking of the DNA loops. 33 Figure 6. Preparation of microscope slide and DNA halo. About 50 -100/il of cell suspension at 1 - 3 x 10"* cells/ml in cold Spinner's salt solution are pipette into a poly-l-lysine coated pre-cleaned microscope slide. Equal volume of the dye-lysis buffer solution are added to the slide. A cover glass is placed over this solution. To obtain better optical clarity, the microscope slides are first soaked in IX acid alcohol, rinse in distilled water, and finally soak in 95% alcohol. The slides are then dried with a cheese cloth. B c o v e r g l a s s m i c r o s c o p e s l i d e •vacuum g r e a s e t e f l o n s p a c e r l y s i s c e l l s b u f f e r s u s p e n s i o n 34 C. TREATMENTS a. X-IRRADIATION Cell suspensions of about 1 x 10^ to 3 x 10"* cells/ml in cold Spinner's salt solution were placed in 20ml plastic tubes (Falcon) and were kept on ice during the course of irradiation. Irradiation was conducted using a Phillips RT 250 x-ray unit, operating at 250KVp, at 15mA, and calibrated at 7.58 Gy/minute. The dose rate was calibrated using the ferrous sulphate dosimetry technique. The dose for each experiment was controlled by adjusting the time of exposure. To prevent cellular repair of DNA damage, the cells were lysed immediately following irradiation. b. DRUG TREATMENTS Cis-DDP and trans-DDP (mol. wt. 300.05; Sigma Chemicals) were dissolved in fresh media and gently stirred at 37°C for an hour before used. Solutions (100/iM) were freshly prepared and used immediately for each experiment. o Cells were seeded in 25-cm polystyrene tissue culture flask and allowed to attach to the flask for 48 hours at 37°C, and fed with fresh medium 24 hour prior to drugs treatment. Cells were treated with 50/iM concentration of the drug solution at 37°C for an hour. Drug was then removed and a single cell suspension was prepared using conventional trypsinization. 35 D. FLUORESCENCE IMAGE PROCESSING SYSTEM (FIPS) The image acquisition, processing, and segmentation procedures that were necessary for the development of a quantitative method for measuring the size of the DNA halo was performed using the FIPS system. FIPS is part of the modular Cell Analyzer and Imaging System developed at the British Columbia Cancer Research Centre (Palcic and Jaggi, 1988; Jaggi and Palcic, 1986). The Cell Analyzer is an optical scanner for the recognition, feature extraction, and observation of live cells. FIPS can be operated as a self standing fluorescence imaging system. It was designed and developed specifically for quantitative microscopy in low-light level fluorescence imaging (Jaggi et al., 1988). a. SYSTEM CONFIGURATION The basic components of the system consists of an image acquisition module linked to an image processing system for real-time digital signal processing, which is controlled by an IBM PC-AT host computer. The main modules of the system is shown schematically in fig. 7. Detailed information on certain aspects of the system is described in the Appendix. An interactive modular program (Halopro) was written in-house in the C-programming language. This program allows the user to acquire images of DNA halos and to build up an imaging processing and segmentation routines for the measuring of the sizes of the DNA halos. b. IMAGE PROCESSING AND SEGMENTATION The steps of the algorithm of the image acquisition and processing program are represented by the flow chart in fig. 8. In the first step, a 512 x 512 x 8 pixels image at about 10" lux is continuously scanned using the image intensified charge injection device (CID) camera set at a fixed gain. From this image, a window of 128 x 128 pixels containing a DNA halo surrounding the nuclear matrix is selected, acquired and stored for image processing. The images stored on the disk of the IBM PC-AT can be recalled, processed, and quantified at any time. For the removal of background shading, bright and dark spots caused by the optics of the image acquisition system and for the reduction of random noise which is particularly important in low light level imaging, the acquired images are de-calibrated. For the de-calibration procedure, dark images taken with the camera shutter closed and bright images taken with the camera shutter opened with just background light were captured and averaged for every experiment. Each newly read-in image is corrected by the formula: I r a w(x,y) - I d a r k(x,y) W a i ^ - y ) = k  Ibright<x>y> - W k ^ - y ) where I(j e c al(x,y) * s t n e de-calibrated image, I r a w(x,y) is the newly read-in image acquired from the device, Ibright(x'50 is the averaged bright image, ld a rk( x>y) * s t* i e averaged dark image, and k is a constant used to linearize the scale. A 3 x 3 median rank filtering is then performed on the de-calibrated image. For this filtering, the image field is scanned by a 3 x 3-pixel window. The output of the window is the median gray level value of the nine pixels in the window. This filtering removes small intensity discontinuities due to random photometric noise. This filtering will not result in any significant loss of resolution. 37 Figure 7. Schematic presentation of the main modules of the Fluorescence Image Processing System. IMAGE ACQUISITION IMAGE PROCESSING IMAGE INTENSIFIED C O CAMERA JT FLUORESCENCE MICROSCOPE 7^. MERCURY LIGHT SOURCE A/O T T ILUT.MUX 3L 512"512"12 FRAME MEMORY I BUFFER/CONTROL 1 U PJD/A T RGB MONITOR 80286/87 HOST COMPUTER 38 Figure 8. Flow chart of the image acquisition, processing, and segmentation procedures using the Florescence Image Processing System for the measurements of the DNA halo size. A C Q U I R E I M A G E D E - C A L I B R A T I O N I J , , - k I r a w < x . y ) " r dark(x ,y) 1 dec(x ,y) ' 'br ight(x,y) * 1 dark(x,y) D I G I T A L F I L T E R I N G - rank 3 x 3 m e d i a n f i l ter <• S E G M E N T A T I O N - g rad ien t -h is togram - es t ima t i on o f th resho ld I D I S P L A Y - con tou r over lay - constrast enhancement - pseduco iou r - bit p lane m a n u a l th resho ld select ion F E A T U R E E X T R A C T I O N - rat io (nuc lear m a t r i x / D N A halo d iameter ) 39 In the next step, segmentation by thresholding is performed on the median filtered image. For automated segmentation of DNA halo from the background and nuclear matrix, thresholds selection using the Three Histogram Analysis Method (MacAulay and Palcic, 1987) is implemented. In this method, three histograms are generated; Histograms H^(i), Histogram ^ ( i ) , and Histogram HjCi). Histogram^(i) and Histogran^Ci) are gradient weighted histogram. These are normal histograms except that pixels with gradient magnitudes larger than a specified cutoff value, c, are not included in the histograms. The difference between histograms H^(i) and ^ ( i ) is that the value of the gradient cutoff value, c, is twice as large for histogram ^ ( i ) than that of H-^(i). Hence, ^ ( i ) includes more of the edge pixels than does H^(i). The third histogram H 3(i) is a normalized gradient histogram; i.e, for each pixel intensity i , the gradient value is calculated and the average gradient for a l l pixels with this intensity is determined. To remove small irregularities in these histograms, a 1x3 median fil t e r and a 1x3 mean fil t e r are applied. Subsequently, a valley finding algorithm is used on histogram H-^(i) to locate the threshold for the boundary between the background and the DNA halo, and the boundary between the DNA halo and the nuclear matrix. Similarly, this valley finding algorithm is applied to histogram ^ ( i ) . A peak finding algorithm is used to find the equivalent thresholds in H ^ i ) . Typical examples of these histograms are shown on fig. 9. These threshold values are averaged to give the mean background and DNA halo boundary, and the mean DNA halo and nuclear matrix boundary which are then used to segment the image. 40 Figure 9. Examples of the three histogram generated from a 128 x 128 pixels image of a DNA halo surrounding the nuclear matrix. Histogram H^i), Histogram H 2(i) are gradient weighted histogram, and Histogram ^ ( i ) is a normalized gradient histogram. —1 L 255 0 Gray-lev els 41 The selected threshold values for the boundary between the background and the DNA halo as well as the boundary between the DNA halo and nuclear matrix are contour overlaid over the original raw image and the de-calibrated image to check for accuracy. If segmentation fails, manual threshold selection is performed. If the process image passes the visual verification, the ratio of the diameter of the nuclear matrix and DNA halo is calculated and stored in an appropriate f i l e for further analysis. 42 RESULTS The change in the tertiary structures of the DNA of x-irradiated and cis- and trans-DDP treated V79 cells were studied using the fluorescence DNA halo assay. The resulting changes in size of the DNA halos under these biological conditions were measured using the image processing and segmentation techniques described. A. SEGMENTATION AND DE-CALIBRATION The result of the image processing and segmentation procedures on a typical 128 x 128 pixels image is demonstrated in fig. 10. Segmentation is achieved by thresholding the non-background pixels into DNA halo and nuclear matrix regions. The DNA halo size is described by the ratio of the diameter of the nuclear matrix region to the diameter of the DNA halo region. Hence, for a l l the data presented here, a minimum value for the ratio implies a maximum halo size. Each segmented image was checked for accuracy. This was achieved by generating a contour (the selected threshold values) over the original and de-calibrated images and displaying on the RGB monitor for visual verification. Typically, the segmentation procedure failed (the segmented image was not in agreement with the visual check on the RGB monitor) with the raw (original) or median filtered images. It was observed that with the implementation of the de-calibration procedure, the noise level of the digital image can be reduced by at least a factor 43 of five. Furthermore, de-calibration improved the automated selection of the thresholds for the segmentation of the non-background pixels into DNA halo and nuclear matrix regions. Illustrated in fig. 11 are the results of using the three histogram analysis for automated thresholding, performed on a raw image, the median-filtered image, and the de-calibrated image. B . B I O L O G I C A L C O N D I T I O N S A. CONTROL Chinese Hamster V79 lung fibroblast cells lysed in a non-ionic detergent (Triton-X-100) with 2M NaCl and treated with the intercalating fluorescent dye propidium iodide were characterized by a uniformly fluorescing residual nuclear matrix surrounded by a less intensely fluorescing DNA halo. The size of this fluorescing DNA halos varied with the propidium iodide concentration. This effect of propidium iodide concentration on the appearance of the DNA halo is illustrated in fig. 12. At low concentrations of propidium iodide (< 1.5/ig/ml), the DNA halo was indistinguishable from the intact nucleus. With increasing concentrations of the dye (2-5/ig/ml) , a large, well defined halo appeared surrounding the nuclear matrix. The maximum halo was seen at a propidium iodide concentration of 5/ig/ml. At higher concentration of propidium iodide, the halo size decreased until i t was diminished at concentration of propidium iodide greater than or equal to 40/ig/ml. 44 F i g u r e 10. The r e s u l t o f the image p r o c e s s i n g and s e g m e n t a t i o n p r o c e d u r e s on a 128 x 128 p i x e l s image. From top l e f t c l o c k w i s e , the raw image, d e - c a l i b r a t e d image, segmented image, and m e d i a n - f i l t e r e d d e - c a l i b r a t e d image. 45 Figure 11. Results of the image processing and segmentation procedures on a typical 128 x 128 pixels image using the gradient-threshold algorithm on a raw median-filtered image (top panel) and de-calibrated median-filtered image (bottom panel). 46 F i g u r e 12. The e f f e c t o f the c o n c e n t r a t i o n o f p r o p i d i u m i o d i d e on the appearance o f the DNA h a l o . Photograph i s from the RGB m o n i t o r . 47 Figure 13. The effect of the concentration of propidium iodide on the size of the DNA halos. Minimum ratio value implies maximum halo size. The error bars reflect the standard error of mean of 5-8 independent experiments. C6 10 15 20 25 30 35 40 Propidium Iodide (pg/ml) 48 Characteristically, measurements of the sizes of the DNA halo at increasing concentration of propidium iodide resulted in a biphasic curve, as shown in fig. 13. The ratio of the nuclear matrix diameter to the DNA halo was plotted as a function of the concentration of propidium iodide. Each data point represents the average of 50 experimental values. Data from 5 - 8 independent experiments was combined to give this biphasic curve for the average size of the DNA halo versus the concentration of propidium iodide. Since the cells used in this study were in log-phase growth (asynchronous), the changes in nucleolar morphology (de la Torre and Navarrete, 1974; Erlandson and de Harven, 1971; ) were also observed in nucleoids. For example, the individual chromosomes of the mitotic cells remain identifiable and form aggregates; nucleoli disappear during mitosis and reappear at telophase; small and isolated nucleoli aggregated and enlarged during G^ ; and S-phase nucleoids were small and contain prominent nucleoli. However, the constraints that maintain the basic DNA conformation remained stable in the presence of 2M NaCl and the non-ionic detergent Triton-X-100. Despite these changes, the DNA halo size was constant throughout the cell cycle. Hence, there were twice as many DNA-nuclear matrix attachment sites in G2 as compared to G^ , since the DNA content was doubled (Warren and Cook, 1979). b. EFFECT OF X-IRRADIATION To study the relationship between the radiation dose and DNA halo size, V79 cells were exposed to increasing doses of x-irradiation (0 -20 Grays), and then lysed at a fixed concentration of propidium iodide (50j*g/ml). The dose-respond curve for the ratio of the nuclear matrix 49 diameter to DNA halo diameter versus x-ray dose is shown in fig. 14. Again, each data point is the average of 50 experimental values and data from 5-8 experiments were combined to give the dose response curve. As shown in fig. 14, the loss of the ability of DNA supercoils to rewind was dependent upon the radiation dose up to about 15 Gray. Increasing the radiation dose increased the DNA halo size; small doses had a marked effect while larger doses had a progressively smaller effect. The results indicate that the loss of DNA supercoils was considerable in the first 4 Grays. Damage to DNA can be detected at radiation doses of probably 0.1 Gray or less. From the curve relating the x-ray dose to the increase in DNA halo size, the DNA loop size can be quantitated by applying target theory (Lea, 1969). Assuming there are 8xl0" 1 2g of DNA per V79 cell (Ross and Mel, 1972) and a value of 33ev per single-strand break for V79 cell (Ahnstrom and Edvardsson, 1974), then there are about 1513 single-strand breaks per cell per Gray. If one Gray of X-radiation relaxes the supercoils configuration and there is one single-strand break per loop, then there are about 1513 loops nicked per nucleoid.. If each V79 cell contains 30.9cm DNA per cell (Romberg, 1980), then there are about 204/jm DNA per loop per cell. Hence, the minimum distance of the DNA loop from the nuclear matrix is about 102/im as indicated by the results presented in fig. 14. c. EFFECT OF CIS- AND TRANS-DDP Cis- and trans- DDP were added to V79 cells and the change in the DNA conformation was examined using the halo assay. Indeed, there was a difference in unwinding and rewinding ability as a function of propidium 50 iodide concentration. The result of this effect was observed as a difference in halo size as shown in fig. 15. Both cis- and trans- DDP treated cells exhibited maximum halo size at 5/ig/ml of propidium iodide concentration. However, at this concentration of propidium iodide, the size of the cis-DDP treated DNA halo was smaller than the untreated and trans-DDP treated cells. There was no difference in maximum halo size between trans-DDP treated and untreated cells. The supercoils of both cis- and trans- DDP treated cells are completely rewound at a propidium concentration of 40/ig/ml or more, which was similar to the untreated cells. But, for cis-DDP treated cells, the size of the DNA halos at the unwinding, relaxation, and rewinding phases are smaller compared to trans-DDP treated cells. Moreover, for cis-DDP treated cells, the rate of supercoils rewinding was faster. 51 Figure 14. Dose-response of x-irradiation on the size of DNA halos. Minimum value for the ratio implies a maximum halo size. The error bars reflect standard error of mean of 5 - 8 independent experiments. Cells are treated with 50>g/ml of propidium iodide. Dose (Gray) 52 F i g u r e 15. E f f e c t o f p l a t i n u m complexes on the DNA h a l o s i z e . A) shows the e f f e c t o f trans-DDP on the DNA h a l o , B) shows the e f f e c t o f cis-DDP on the DNA h a l o , C) shows the c o n t r o l , and D) i s a r e p r e s e n t a t i o n o f the e f f e c t o f these t h r e e s e t s o f c o n d i t i o n s on the DNA h a l o s i z e . 0.6 . 0 .5 -0.4 03 10 15 20 25 30 0 5 10 15 20 25 30 D) # control , A ' • trans—OOP/' A c is -DDP / j r" / / * / -• IT"" 1 1 1 1 0 5 10 15 20 25 30 Propidium iodide (vg/nd) 0 5 10 15 2 0 2 5 30 Propidium Iodide (^g/mi) 53 DISCUSSION The intent of this project was to measure changes in halo diameter objectively and quantitatively. A survey of the literature shows that there are very few assays available for the qualitative and quantitative measurements of the changes in the DNA tertiary structures resulting from damage inflicted by various physical and chemical agents. Currently, the nucleoid sedimentation technique and variations of this technique is perhaps the most sensitive and commonly used method to study damage to the tertiary structure of DNA and the role of this conformation in the replication, transcription, and repair processes of DNA. However, criticism of the use of nucleoids stem largely from the method of isolation and from possible artifacts. Moreover, the sedimentation of the nucleoid, which measures the superhelical density of DNA is dependent on the DNA loop extension as well as the total mass of the nucleoid, i.e. both the DNA and the residual nuclear matrix. In view of these limitations, the fluorescence halo assay (Roti Roti and Wright, 1985; Vogelstein et al., 1985) which visualizes DNA loops as a fluorescent 'halo' surrounding a nuclear matrix has been adapted and used as an experimental system for this project. This assay allows the visualization of the changes of the DNA loops as a change in the size of the DNA halo. The advantages of this assay are that i t is possible to measure damage to Individual cells and to obtain data from a moderate number of cells; in this project about 10^ cells were used per experiment. Moreover, i t is not necessary to use radiolabeled DNA. 54 Since the method by Roti Roti and Wright do not required cells to grow on cover slips and allows for the observation of single cells, i t was chosen as the method to use in this project. However, the manual method of measuring the size of the DNA halo used by these workers is subjective. This project attempts to quantify the change in size of the DNA halos objectively and quatitatively. This is achieved by employing mathematical algorithms to separate the DNA halo from background and the nuclear matrix. Damage inflicted by two of the better characterized agents, x-radiation and platinum complexes were used as a test system for this quantitative assay. A. SEGMENTATION From the result presented, the Three Histogram Analysis method only gives a moderately reliable segmentation of the background and the DNA halo as well as the DNA halo and nuclear matrix, in particular the boundary between background and DNA halo (about 60% of the time correct). A mathematical model is proposed to provide a description of the theoretical fluorescence intensity distribution of the DNA halo as a distance function from the nuclear matrix. This model proposed that i t is inappropriate to define a threshold for background and DNA halo boundary because the theoretical intensity distribution of the DNA halo as a function of distance from the nuclear matrix can be approximately an exponential distribution. Therefore, there is no sharp edge or defined boundary between the DNA halo and the background; thus, the selection of a defined boundary would be inappropriate. However, the 55 theoretical expression for the exponential decay of the DNA halo as a function of distance involves parameters which are related to the DNA halo size. Hence, fitting the curve to the observed experimental data should yield the relative DNA halo size under the given experimental conditions. The model assumes the following: i . a l l the protruding DNA loops have the same size (length of DNA loops); i i . the spatial orientation over the surface of the nuclear matrix of these DNA loops are random; i i i . the staining of the intercalating dye propidium iodide is uniform on a l l DNA loops; iv. the number of DNA loops extended out beyond the surface of the nuclear matrix is the same for a l l nucleoids of the same cell type; v. no spatial interaction exists among the DNA loops for a particular nucleoid; v i . the DNA loops are uniformly distributed around the nuclear matrix; Based on assumptions (i), ( i i ) , and ( i i i ) , for a loop exiting at r^, the loop density or the probability of finding a loop at any point r will be proportional to the Gaussian function:--(|r 2|)/*a 2 1 e where a' is related to the length of the loop. 56 Also based on assumptions (iv), (v), and (vi), the density of the loop starting and ending on the nuclear matrix is:-p(r) - p<5(r) where S(r) is the Dirac Delta function. From these equations, we can describe the intensity distribution of the DNA halo as a function of the distance r 2 by this :-This function, representing the theoretical intensity distribution of the DNA halo is plotted, as shown in fig. 16. Unfortunately, i t is not trivial to measure the experimental DNA halo distance from the nuclear matrix because the pixels from the image sensor are rectangular. Counting the number of pixels in the x and y direction cannot give exact distances. Square pixels are needed, but transformation of rectangle pixels to square pixels can be easily performed using another image processor (MATROX MVP-AT, Dorval, Quebec). However, this imaging board is at present not implemented in the FIPS system. Illustrated in fig. 17 is an example of a 256 x 256 pixels image of the DNA halo that is acquired using the CID camera but processed by the MATROX imaging board. The intensity distribution of this image, from the centre of the nuclear matrix to the background level has been plotted and is illustrated in fig. 17. 57 Figure 16. The theoretical distribution of the intensity of the DNA halo as a function of the distance from the nuclear matrix. 0 10 20 30 40 D i s t a n c e 58 Figure 17. The intensity distribution of the DNA halo as a function of the distance from the nuclear matrix of the 256 x 256 pixels image below (in bit-plane). This image is acquired by the CID camera but processed and averaged 20 times (in real time) using the MATROX board. 59 B. BIOLOGICAL CONDITIONS The results described in fig. 14 shows that supercoiled loops can be relaxed and visualized as halos of DNA surrounding the nuclear matrix. The size of the DNA halo is indeed dependent on the concentration of the intercalating dye and a biphasic dependence have been demonstrated. As propidium iodide intercalates into the DNA, the DNA loops unwind and negative supercoils are relaxed (at the relaxation point, the halo size is maximum). Higher concentration of the propidium iodide dye will overwind the DNA, supercoiling i t in the positive direction. The concentration of propidium iodide producing the minimum and maximum halo size and the size variation of the halos are similar to previously reported sedimentation behavior of nucleoids and the direct measurements of halos diameters using the fluorescence halo assay. This demonstration of supercoiling indeed indicates that DNA is organized into loops and constraints at its base at the nuclear matrix. The dependence on the intercalating dye concentration would not be expected i f the halos were made up of linear strands that are attached to the nuclear matrix at one end of the DNA. a. RADIATION From the data presented in the dose-response curve in fig. 14 , i t is observed that one gray of x-radiation can indeed relax many of the DNA supercoils. Using sedimentation technique (Cook and Brazell, 1975), i t was shown that introduction of a single strand break in one loop of the HeLa nucleoids relaxes the supercoiling in that loop but not in the adjacent loops. 60 By applying single-hit target theory (Lea, 1969) to the dose-response curve, an approximate estimation of the sizes of the supercoiled loops has been made. The minimum DNA loop diameter is calculated to be about 102/un. Other investigators, using data from the sedimentation technique, estimated the size of the HeLa nucleoid loops to be about 2.2 x 105 base pairs long (about 75/xm) (Cook and Brazell, Q 1978), and large mouse thymocytes to be about 5 x 10 dalton (about 250/im) (Filippovich et al., 1982). The minimum size of the DNA loops determined using this quantitative assay is within the range of the size of a replicon, which is determined to be about 5 - 150/im (Nelkin et al., 1982). Cook (1974) has hypothesized that a DNA loop corresponds to a replicon and Vogelstein et al. (1980) suggest that DNA replication begins at the point where a DNA loop is attached to the nuclear matrix and then continues through the entire loop. A relationship between DNA loop length and replication size have been reported (Buongiorno-Nardelli et al., 1982). Using the quantitative method, radiation damage at one Gy and less can be detected. b. PLATINUM DRUGS From the results presented in fig. 15, the platinum complexes indeed have no effect on the superhelical density of DNA. This agrees with studies using the nucleoid sedimentation technique (Weniger, 1983). Both cis- and trans-DDP exhibit complete relaxation of supercoils (maximum halo size) at 5/ig/ml of propidium iodide dye as in the case of the untreated cells However, there is a difference in the kinetics of the unwinding and rewinding of the supercoiled loops. This can be 61 attributed to the different mode of binding of the platinum drugs to DNA which in turn may alter the conformation of the supercoiled DNA. Alternatively, propidium iodide may intercalate selectively at certain regions of the DNA duplex, which can then enhance or inhibit the binding of the platinum complexes at these regions; or the presence of platinum complexes may alter the intercalation of the propidium iodide with the DNA duplex. 62 SUMMARY The essential features of the assay are rapid, objective, and quantitative measurements of changes in DNA tertiary structures. Using, this assay, i t is possible to measure damage to individual cells and obtaining data from a moderate number of non radioactively labeled cells (10^ cells). Although the segmentation procedure does not give a reliable segmentation of the DNA halo and nuclear matrix (about 60% correct segmentation), the implementation of the mathematical model may provide a better measurement of the size of these DNA halos. The experiments involving the x-irradiation of cells have provided some information on the length of DNA constrained at the nuclear matrix which agrees with other estimates.. This assay can detect damage in DNA at radiation dose of 1 Gy or less. The repair of this damage can also be studied using this assay. Studies on the effect of platinum drugs on DNA supercoiling suggest that the mode of binding of the drugs to DNA has an effect on DNA supercoling changes. Perhaps this change of DNA conformation could explain the different cytoxicity and anti-tumor activities of these drugs. Further studies of the repair mechanisms of the crosslinks and the effects of ionizing radiation under hypoxia and aerobic conditions in combination with drugs treatment can be investigated using this assay. With our increased knowledge of chromatin structure and genome organization, research to correlate the biological events of DNA damage and repair with various biological end points should also be focused at 63 this level of DNA organization. An assay that can provide some insight to the damage at this level of DNA organization and repair of the damage sites should prove to be useful. 64 APPENDIX A . DESCRIPTION OF FIPS COMPONENTS a. IMAGE ACQUISITION Fluorescent images from the microscope are acquired through a proximity focused channel intensifier tube fiber-optically coupled to a solid state charge injection device (CID) imaging array of 480x380 picture elements (pixels) image sensor (Model F-4562; Electro-optical Product Division, ITT). The camera has a manual gain control featuring a maximum spatial resolution of 18 line pair/mm, a digitization resolution of 8 bits giving discrimination between 256 gray levels and a photo-cathode spectral response of approximately 60% for the range of 400»7m-800»)m. The scanning rate or integration time of the sensor is 33ms or 30 frames/second. The important characteristic of this image sensor is its minimum threshold sensitivity of 10'^lux, which allows the detection of fluorescence at very low light levels. b. DIGITIZATION AND IMAGE PROCESSING The image processing and digitization of the incoming analog image of the sensor is performed in real time by a Digital Image Processor (Model FG-100-AT; Imaging Technology Inc.). The operation and installation of the imaging board is described in detail in the Imaging Technology Inc. manual. 65 For digitization, an analog-digital converter (ADC), converts the incoming analog video signal containing image data to digital data at a sampling rate of 10MHz, producing digital values or pixels from 0 to 255, with an 8-bit resolution. These pixels are then transferred in real-time to a 512 x 512 array of 12-bit pixel frame memory for storage or simultaneous display on an external high resolution RGB analog monitor. Since the ADC is digitized to 8-bit, there is s t i l l 4-bit plane of frame memory that can be used for graphic overlay processing. A key feature of the imaging board is its multiplexer and feedback/input look-up table. Its feedback modes allow real-time processing of the incoming or stored image under the control of the host computer program. It's look-up table provides pseudo-color capability that allows the image from a single channel to be displayed in color, with an arbitrary programmed color assigned to each of the original gray levels. c. HOST COMPUTER The image acquisition and processing is controlled by an 80286/87 based IBM PC-AT. The host computer is responsible for communication between the modules and for various aspects of timing and control of automated functions. It has a 30Mbyte disk drive, a monochrome graphics display and a dot-matrix printer. B. SYSTEM CALIBRATION The optical performances of the charge injection device (CID) image intensifier of FIPS were measured. All measurements were taken 66 with a background light intensity set at 75% of the device saturation; the camera manual gain was set at 50%. The dynamic range, the photo-response non-uniformity (PRNU), the modulation transfer function (MTF), aspect ratio and linearity of the CID sensor were measured. The results are summarized in Table I. i) Dynamic range: noise and standard deviation To specify the dynamic range of the device, the standard deviation and the noise (in gray levels) of the background intensity of "raw" and "average" images were determined. Noise in an imaging system can be defined generally as any intensity variation in the image that is not part of the object. It limits the amount of information that can be extracted from an image and defines the lower limit of the dynamic range of the camera and, therefore, determines the sensitivity of the camera. The noise (in gray levels) was measured by looking at the fluctuation of a line scan of the image set at the background intensity level. The standard deviation was determined from the gray level histogram, which is a representation of the function between the gray level and the number of pixels in the image that have that gray level. i i ) Photo-response non-uniformitv The photo-response non-uniformity (PRNU) of the device was determined by using the formula:-PRNU (%) - [ ( I m a x / I m e a n ) -1 ] * 100% where I m a x is the maximum intensity gray level value and I m e a n is the mean gray level value of a bright field image. 67 TABLE I. Measurements of optical performance of the charge-injection device (CID) solid state camera. MTF 0.25 PRNU raw 16% @75% f u l l range average 9.5% Standard deviation raw 5.3 @75% f u l l range average 3.5 Noise raw 20 (gray-levels) average 12 Aspect ratio 1.24:1 Linearity 4.6 68 i i i ) Modulation transfer function The modulation transfer function (MTF), which specifies the spatial and photometric resolution of the imaging device, was determined at limiting resolution of 300 lines/mm and 600 lines/mm. The MTF was calculated from the formula: M T F - (w - w / + w where I m a x is the maximum intensity gray level value and I m ^ n is the minimum intensity gray level value. iv) Aspect ratio The aspect ratio is a measure of the device pixel dimension. An image of the Zeiss micrometer was taken with the camera at 0° and another taken with the camera turned 90°. 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