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Computer-controlled microscope for the automatic classification of white blood cells. Gabert, Howard Frederick 1973

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A COMPUTER-CONTROLLED MICROSCOPE FOR THE AUTOMATIC CLASSIFICATION OF WHITE BLOOD CELLS  by  Howard Frederick  Gabert  B.A.Sc, The University of B r i t i s h Columbia, 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE  i n the Department of E l e c t r i c a l Engineering We accept this thesis as conforming required standard  to the  THE UNIVERSITY OF BRITISH COLUMBIA  July 1973  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  the Head of my  I t i s understood t h a t copying or  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be written  Department or  permission.  Department of The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada  publication  allowed without  my  ABSTRACT  A microscope was interfaced to a PDP-9 computer i n order to develop techniques suitable f o r on-line c l a s s i f i c a t i o n of white blood cells. The computer visual-input system i s composed of an image d i s sector o p t i c a l l y coupled to a microscope used i n a transmitted l i g h t mode.  The p o s i t i o n of the s l i d e under the microscope and the fine focus  control are controllable from the computer. A technique of auto-focusing was developed to e f f i c i e n t l y focus the microscope under computer control.- This algorithm i s described i n d e t a i l , followed by a discussion of the physical factors that affect the performance of this technique. A method of l o c a t i n g or i s o l a t i n g the leukocytes (white blood c e l l s ) i s described next.  A constant, referred to as the "contrast r a t i o '  i s used to extract the threshold f o r the nuclei of the leukocytes based on the average background i n t e n s i t y . F i n a l l y , contour tracing and curvature function extraction are used as a means of testing the system.  A s p e c i f i c test i s conducted  to obtain a comparison of the system's e f f i c i e n c y as compared to that of "manual" techniques used by a technician. The system described here i s not only suitable for automatic leukocyte c l a s s i f i c a t i o n , but could also be used for many other routine tests requiring the examination of microscopic c e l l s .  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i i  LIST OF ILLUSTRATIONS  .  iv  LIST OF TABLES . •  v  i  ACKNOWLEDGEMENT  v  i  1.  INTRODUCTION 1.1  2.  3.  4.  5.  1  Clinical Count  Procedure f o r the D i f f e r e n t i a l  Leukocyte 3  1.2  C h a r a c t e r i s t i c s of Leukocytes  1.3  System Design O b j e c t i v e s  1.4  Other M i c r o s c o p i c Image P r o c e s s i n g Systems  4 . . . .  THE HARDWARE  6 7 8  2.1  The Computer V i s u a l - I n p u t System  8  2.2  The Image D i s s e c t o r  8  2.3  The M i c r o s c o p e  13  2.4  The Computer I n t e r f a c e  13  2.5  The System R e s o l u t i o n  16  DEVELOPMENT  i  OF THE AUTO-FOCUS ALGORITHM  3.1  Background  3.2  Developing  3.3  Factors A f f e c t i n g  22 22  the A l g o r i t h m  23  the V a r i a n c e  THE LEUKOCYTE LOCATION PROCEDURE  Function  . . .  28 35  4.1  The C o n d i t i o n s o f the Search  35  4.2  Threshold Determination  36  4.3  The Scan P a t t e r n  38  THE SOFTWARE IMPLEMENTATION  41  5.1  "FOCUS"  .  41  5.2  "THRESH"  43  5.3  "JOYSTK"  45  5.4  "STRPNT"  45  ii  Page  6.  7.  SYSTEM EVALUATION  •  49  6.1  The T e s t System  .• • •  49  6.2  Contour T r a c i n g  50  6.3  The D i s c r e t e A r e a O p e r a t o r  51  6.4  Reliability  53  and E x e c u t i o n Time Measurements  CONCLUSIONS  58  APPENDIX I  60  APPENDIX I I  6 2  APPENDIX I I I  .  66  REFERENCES  •  iii  6  ?  LIST OF ILLUSTRATIONS Figure  Page  1.1.1  Sketch o f a normal b l o o d f i l m  3  1.2.1  Sketches o f t y p i c a l leukocytes  4  2.1.1  Photograph o f the microscope and hardware  9  2.2.1  I n t e r p r e t a t i o n o f p e r c e n t a g e m o d u l a t i o n . 100% m o d u l a t i o n a t GS = 5 i s shown i n (A) and 86.5% modul a t i o n a t GS = 4 i s shown i n (B)  10  S i g n a l to n o i s e r a t i o o f the image d i s s e c t o r as a f u n c t i o n o f A t , the d w e l l time  12  2.3.1  B l o c k diagram o f the o p t i c a l system  14  2.4.1  B l o c k diagram o f the computer i n t e r f a c e  15  2.5.1  Resolution  2.2.2  as a f u n c t i o n o f m a g n i f i c a t i o n  image d i s s e c t o r  f o r the  (A) and t h e microscope (B)  19  2.5.2  System r e s o l u t i o n as a f u n c t i o n o f m a g n i f i c a t i o n  3.2.1  A v i d e o scan l i n e when i n focus (A) and when out of focus (B)  3.2.2  C h a r a c t e r i s t i c s s e a r c h e d f o r i n a v i d e o scan l i n e  3.2.3  The v a r i a n c e p l o t t e d a g a i n s t focus c o n t r o l p o s i t i o n u n f i l t e r e d (A) and f i l t e r e d (B) w i t h a d i g i t a l f i l t e r window s i z e o f t e n  26  The v a r i a n c e f u n c t i o n f o r two d i f f e r e n t f i e l d s o f view showing peak v a r i a n c e s c a l i n g  28  S c a l i n g o f the v a r i a n c e f u n c t i o n due t o m a g n i f i c a t i o n shown a t 800X ( A ) , 1024X ( B ) , 1280X ( C ) , and 2000X ( D ) .  29  The e f f e c t o f no f i l t e r ( A ) , a r e d f i l t e r ( B ) , a b l u e f i l t e r ( C ) , and a green f i l t e r (D) on the v a r i a n c e f u n c t i o n e x t r a c t e d from an image o f c e l l s  . .  31  The v a r i a n c e f u n c t i o n e x t r a c t e d at f u l l i n t e n s i t y and a t h a l f i n t e n s i t y . The bottom curve i s the one a t h a l f intensity  32  The e f f e c t o f n o n - c r i t i c a l i l l u m i n a t i o n on t h e v a r i a n c e f u n c t i o n a t a m a g n i f i c a t i o n o f 2560X. The top curve represents c r i t i c a l i l l u m i n a t i o n  33  3.3.1  3.3.2  3.3.3  3.3.4  3.3.5  iv  . . .  20  . . .  25  24  Figure 4.1.1  4.1.2  4.3.1  Page S p e c t r a l d e n s i t y c u r v e o f t h e Z e i s s 46 78 06 wide-band pass green i n t e r f e r e n c e f i l t e r  35  Video scan l i n e s across a leukocyte w i t h w i t h o u t (A) c o n t r a s t e n h a n c i n g f i l t e r s  36  (B) and  S t a n d a r d s c a n p a t t e r n s used i n t h e d i f f e r e n t i a l l e u k o c y t e count. The s t r a i g h t - e d g e p a t t e r n ( A ) , t h e battlement p a t t e r n (B), the c r o s s - s e c t i o n a l p a t t e r n (C) , and t h e l o n g i t u d i n a l p a t t e r n (D)  39  4.3.2  Placement o f a d j a c e n t  39  4.3.3  R e l a t i o n s h i p between c u r r e n t and a d j a c e n t showing the placement o f t h e "dead" zone  s c a n areas scan  areas, 40  5.1.1  Flow d i a g r a m o f the f o c u s i n g program, "FOCUS"  42  5.1.2  The s c a n l i n e s used t o sample t h e v a r i a n c e  43  5.2.1  Flow diagram o f t h e t h r e s h o l d d e t e r m i n i n g "THRESH"  program, 44  5.4.1  Flow diagram o f "STRPNT"  46  5.4.2  The s e q u e n t i a l s e a r c h t e c h n i q u e used t o l o c a t e l e u k o c y t e s , d e m o n s t r a t i n g the minimum square f i t (A) of s i z e AGS, and t h e l o c a t i o n o f the s t a r t i n g p o i n t (B) by u s i n g GS  47  6.2.1  6.3.1 6.3.2  6.4.1 A.2.1  The c o n t o u r t r a c i n g a l g o r i t h m . The b l a c k dots a r e spaced a t GS and r e p r e s e n t the f o u r p o i n t s t h a t a r e i n t e r r o g a t e d to determine which d i r e c t i o n (the c i r c l e s ) the o p e r a t o r s h o u l d be moved The d i s c r e t e a r e a o p e r a t o r curvature  .50  used t o e x t r a c t boundary 51  Photographs o f s e v e r a l c e l l s , t h e i r c o n t o u r t r a c e s , and u n f i l t e r e d c u r v a t u r e f u n c t i o n s as e x t r a c t e d by t h e a r e a operator  52  The s c a n p a t t e r n used t o measure t h e time r e q u i r e d t o l o c a t e and p r o c e s s 100 c e l l s  54  A b l o c k diagram d e m o n s t r a t i n g a p o s s i b l e arrangement o f the system programs  63  v  LIST OF TABLES Table 1.2.1  2.5.1  6.4.1  6.4.2  A. 1.1  A.2.1  Page M o r p h o l o g i c a l and leukocytes  spectral characteristics  of 5  R e s u l t s o f the c a l i b r a t i o n of the image d i s s e c t o r resolution  17  R e s u l t s o f a t e s t designed to count l e u k o c y t e s a c r o s s an a r e a w i t h a t r u e count of 100 c e l l s  54  Times r e q u i r e d to l o c a t e and p r o c e s s 100 on t e n randomly s e l e c t e d b l o o d f i l m s  55  The optimum v a l u e s parameters  leukocytes  f o r the s i x m a g n i f i c a t i o n dependent 60  I n t e r n a l and e x t e r n a l r e f e r e n c e s to g l o b a l symbols and subroutines. "X" i n d i c a t e s i n t e r n a l l y d e f i n e d , "A" i n d i c a t e s e x t e r n a l access n e c e s s a r y , and " ( A ) " i n d i c a t e s e x t e r n a l access p r o b a b l e  vi  63  ACKNOWLEDGEMENT  I wish t o extend my s i n c e r e thanks  t o my s u p e r v i s o r , D r . J.S.  MacDonald, f o r h i s guidance and encouragement d u r i n g my p e r i o d o f s t u d y , and t o Dr. R. P e a r c e , o f t h e Department o f P a t h o l o g y , U.B.C, f o r h i s a d v i c e c o n c e r n i n g the m e d i c a l background I would a l s o l i k e the N a t i o n a l Research  of this  to g r a t e f u l l y  project.  acknowledge the a s s i s t a n c e o f  C o u n c i l o f Canada f o r i t s f i n a n c i a l s u p p o r t  s c h o l a r s h i p s and the major equipment g r a n t used to purchase  through  the m i c r o -  scope . Thanks a r e a l s o owing Mr. A. Leugner guidance  f o r h i s expert t e c h n i c a l  d u r i n g c o n s t r u c t i o n o f the hardware, and to Miss N. Duggan  f o r t y p i n g the f i n a l m a n u s c r i p t .  And f i n a l l y ,  I must g i v e c r e d i t t o my  w i f e , Nadine, who, through h e r c o n t i n u e d encouragement and u n d e r s t a n d i n g , has made i t p o s s i b l e f o r me to complete  vii  t h i s work.  1.  INTRODUCTION  The b i o m e d i c a l s c i e n c e s c h a r a c t e r i s t i c a l l y d e a l w i t h l a r g e volumes o f d a t a , which must be c o l l e c t e d , o r g a n i z e d , reduced, and g e n e r a l l y p r o c e s s e d i n many ways.  analyzed,  In p a r t i c u l a r , the a n a l y s i s o f  b i o l o g i c a l specimens c o n t a i n i n g c e l l s  i s known to be important  f o r the  m e d i c a l d i a g n o s i s o f many d i s e a s e s and chemical d i s o r d e r s i n the human body.  Standard m e d i c a l t e s t s  f o r the d i f f e r e n t i a l  diagnosis of disease  and t h e r a p y a r e r e q u i r e d t o be a c c u r a t e , economical most c a s e s , these to  and e f f i c i e n t .  t e s t s r e q u i r e a q u a l i f i e d and e x p e r i e n c e d  In  technician  examine the b i o l o g i c a l specimens under a h i g h power m i c r o s c o p e .  type o f v i s u a l e x a m i n a t i o n ing  the t e c h n i q u e s  i s s u b j e c t i v e and no l o n g e r e s s e n t i a l c o n s i d e r -  developed i n t h e f i e l d  s i t u a t i o n such as t h i s suggests tests  s h o u l d be The  invites  This  of p a t t e r n r e c o g n i t i o n .  that automation  A  o f some o f these r o u t i n e  attempted.  d i f f e r e n t i a l white b l o o d c e l l  automation.  count  i s one procedure  that  I t has been e s t i m a t e d t h a t t h i s r o u t i n e t e s t i n v o l v e s  manual counts on over 240,000 s l i d e s p e r day i n the U n i t e d S t a t e s alone (1).  I n J u l y 1972, T e c h n i c o n I n t e r n a t i o n a l o f Canada L i m i t e d announced TM  the Hemalog D  TM ( 2 ) . The Hemalog D  e n t i a l white blood c e l l t i n u o u s - f l o w equipment. of  i s d e s i g n e d t o perform  the d i f f e r -  count by i n c o r p o r a t i n g c y t o c h e m i s t r y i n t o  con-  C y t o c h e m i s t r y may be d e f i n e d as the i d e n t i f i c a t i o n  s p e c i f i c components w i t h i n the c o n f i n e s o f i n d i v i d u a l c e l l s by s t a i n TM  ing.  I n the Hemalog D  , the l i q u i d b l o o d sample i s d i v i d e d i n t o  p o r t i o n s and each p o r t i o n i s t r e a t e d w i t h a d i f f e r e n t s t a i n different  c o n s t i t u e n t s o f the sample.  to i d e n t i f y  Each p o r t i o n then passes  a view chamber where the s t a i n e d c e l l s  1  are sensed by  several  through  photo-sensors  2  which measure two v a r i a b l e s , l i g h t  l o s s and l i g h t s c a t t e r i n g .  By means  o f t h r e s h o l d i n g the l i g h t l o s s and l i g h t s c a t t e r i n g , the Hemalog D classifies  and counts the w h i t e b l o o d c e l l s i n the sample.  TM  The Hemalog  TM D  i s c u r r e n t l y undergoing c l i n i c a l environment  t e s t i n g and e v a l u a t i o n .  Some d i s a d v a n t a g e s o f t h i s system seem a p p a r e n t .  The Hemalog  TM D in  i s n o t m o d e l l e d a f t e r the normal p r o c e d u r e used by the t e c h n i c i a n s the c l i n c i a l  laboratory.  The i n i t i a l  d i f f e r e n t i a l white blood  cell  count i s u s u a l l y a s c r e e n i n g p r o c e d u r e , and i f a b n o r m a l i t i e s are d e t e c t e d , a q u a l i f i e d h e m a t o l o g i s t i s i n v i t e d t o v i s u a l l y examine the specimen. TM With the Hemalog D  t h e r e i s no means f o r v i s u a l e x a m i n a t i o n and t h e r e -  f o r e e x t r a expense and time a r e r e q u i r e d to p r e p a r e p a r t o f the f l u i d specimen  f o r such e x a m i n a t i o n .  specimens  i n the f l u i d  environment, of  C r o s s - c o n t a m i n a t i o n between c o n s e c u t i v e  channels w i l l need e v a l u a t i o n i n the c l i n i c a l  and w i l l p r o b a b l y be r e l a t e d t o the q u a l i t y and frequency  maintenance. A t l e a s t two a d d i t i o n a l systems  These  (17).  are c u r r e n t l y under  development.  a r e the P e r k i n - E l m e r Instrument (16) and the C o r n i n g L a r c System TM I n c o n t r a s t t o the Hemalog D  use v i s u a l  classification  , both o f these systems  reportedly  t e c h n i q u e s to p e r f o r m the d i f f e r e n t i a l w h i t e  b l o o d count, a l t h o u g h the methods used by these systems have n o t y e t been published. available  One o r both o f these systems  are expected t o be commercially  soon. The p r i m a r y c o n t e x t i n the d e s i g n o f the system t o be r e p o r t e d  h e r e has been the d i f f e r e n t i a l l e u k o c y t e count. analogous  However, due t o the  p r o p e r t i e s o f the system t o t h a t of the c u r r e n t  c l i n i c a l ap-  p r o a c h , the hardware and s o f t w a r e c o n t r o l p r o t i o n o f t h i s system i s a p p l i c a b l e to many o t h e r r o u t i n e t a s k s o t h e r than the d i f f e r e n t i a l l e u k o c y t e  3  count. 1.1  C l i n i c a l Procedure f o r the D i f f e r e n t i a l Leukocyte  An u n d e r s t a n d i n g o f the normal form a d i f f e r e n t i a l count i s h e l p f u l  Count  c l i n i c a l p r o c e d u r e used to p e r -  when automating t h i s  test.  Approxi-  3 mately 10 mm  o f b l o o d taken from a p a t i e n t i s smeared on a g l a s s  and s t a i n e d w i t h Wright's b l o o d s t a i n is  ( F i g . 1.1.1).  This prepared s l i d e  then mounted under a microscope to be examined by a t r a i n e d  tory technician.  slide  labora-  Commonly 1000X m a g n i f i c a t i o n and w h i t e l i g h t , o i l -  immersion microscopy i s used.  A t t h i s m a g n i f i c a t i o n , the t h r e e  c e l l u l a r components o f b l o o d are v i s i b l e :  principal  the l e u k o c y t e s (white b l o o d  cells),  the e r y t h r o c y t e s  lets).  With the a i d o f a m e c h a n i c a l s t a g e , the t e c h n i c i a n c o n t r o l s  position  ( r e d b l o o d c e l l s ) , and the thrombocytes  o f the s l i d e , i d e n t i f y i n g  countered.  and c l a s s i f y i n g  (platethe  each l e u k o c y t e en-  A t o t a l count f o r each c a t e g o r y o f l e u k o c y t e s i s s i m u l t a n -  eously recorded.  Normal p r o c e d u r e c a l l s f o r the i d e n t i f i c a t i o n  s i f i c a t i o n o f o n l y the f i r s t one hundred l e u k o c y t e s e n c o u n t e r e d .  and  clas-  Studies  have shown t h a t on a t e s t s l i d e w i t h a t r u e p r o p o r t i o n o f 50% n e u t r o p h i l s , a one hundred  cell  count produces an e x p e c t e d mean measure o f n e u t r o p h i l s  HEAD  TAIL  KLlm Too T h i c k  Fig.  1.1.1  Ideal Thickness  Sketch o f a normal b l o o d  Him Too T h i n  film.  4  from 40% hundred  to 60%, cell  a two hundred  count from 45.6%  number o f c e l l s ,  perhaps  cell  count from 43% t o 57%,  t o 54.4%  (3).  f i v e hundred o r one  n i z e d to o b t a i n an a c c u r a t e d i f f e r e n t i a l cell  count takes a t e c h n i c a i n  from f i v e  1.2  C h a r a c t e r i s t i c s of Leukocytes The p r i m a r y c l a s s i f i c a t i o n s  two  i n c l u d e s lymphocytes  A normal one  o f l e u k o c y t e s may  Sketches o f t y p i c a l and monocytes.  thousand, s h o u l d be  T a b l e 1.2.1.  F i g u r e 1.2.1.  Sketches o f t y p i c a l  hundred  be d i v i d e d  into  The g r a n u l a r c a t e g o r y i n c l u d e s The n o n g r a n u l a r c a t e g o r y  leukocytes,  S i z e , c o l o u r , n u c l e a r shape,  p e r c e n t a g e p o p u l a t i o n f o r each o f these c l a s s i f i c a t i o n s in  scruti-  t o t e n minutes.  e o s i n o p h i l s , n e u t r o p h i l s , and b a s o p h i l s .  1.2.1  five  T h i s suggests t h a t a l a r g e  count.  c a t e g o r i e s , g r a n u l a r and n o n g r a n u l a r .  Fig.  and a  l e u k o c y t e specimens  and  are summarized are shown i n  Many schemes have been d e r i v e d to c l a s s i f y l e u k o c y t e s  Dianister (microns)  Population  Lymphocyte  25-33  7-12  Monocyte  2-6  13-20  Grayish Blue  Neutrophil  60-70  10-12  Pink to Lilac  Eosinophil  1-4  10-14  Basophil  .25-.S  10-12  Rad Stippling Blue  (?)  Nucleus Shape  Granules Amount Aeount  Purple  Round  70-90?  Few  Light Purple  Indented  50?  Few  50?  Reddish Purple  Segmented 50?  Many  60-70?  Dark Blue  Tuo-Lobsd  30-40?  Many  Purple  Elongated  50?  Many  Cytoplasm Color Amount Clear Light 10-30? Blue  Cell Type  50?  5C?  Color  Table 1 . 2 . 1 Morphological and spectral characteristics of leukocytes.  •i i  6  by u s i n g some combination o f the f o l l o w i n g p r o p e r t i e s :  cytoplasmic  c o l o u r , shape and s i z e , n u c l e a r c o l o u r , shape and s i z e , and c o n c e n t r a t i o n of g r a n u l e s .  C o l o u r i s the o n l y p r o p e r t y that cannot be measured by t h e '  system developed h e r e .  Colour i d e n t i f i c a t i o n i s generally  expensive  to implement and slow to e v a l u a t e , e s p e c i a l l y when the c o l o u r s l i e s p e c trally  a d j a c e n t and a r e n o t too e a s i l y  defined.  I n the case o f a b l o o d  smear, c o l o u r i s a f f e c t e d by the c o m p o s i t i o n and pH o f the s t a i n  used,  the s t a i n i n g time, and the t h i c k n e s s o f the smear. 1.3  System D e s i g n O b j e c t i v e s The o b j e c t i v e has been t o d e s i g n and develop a computer  l e d system c a p a b l e o f l o c a t i n g m i c r o s c o p i c o b j e c t s and measuring p r o p e r t i e s as s i z e and shape o f these o b j e c t s .  controlsuch  A l l t e c h n i q u e s and a l -  gorithms have been d e s i g n e d to o p e r a t e " o n - l i n e " , t h a t i s , by u s i n g the image as " r e a d - o n l y " memory. reasons:  Such a method seems d e s i r a b l e f o r two  computer memory s i z e i s reduced s i n c e the image i n f o r m a t i o n  need n o t be s t o r e d i n memory, and a c c e s s time o f random p i c t u r e - p o i n t information i s generally The performance resolution.  faster  than w i t h e i t h e r a d i s c o r drum memory.  o b j e c t i v e has been maximum speed, w i t h o u t  sacrificing  Dynamic c o n t r o l o f the microscope has been an e s s e n t i a l  p a r t o f t h i s performance  objective.  A p p l i c a t i o n o f the system t o the d i f f e r e n t i a l l e u k o c y t e count has been p a r t i a l l y  implemented  as compared to normal  i n o r d e r to e v a l u a t e system  "manual" t e c h n i q u e s used by a t e c h n i c i a n .  the d i f f e r e n t i a l  l e u k o c y t e count i s not the o n l y s u i t a b l e  f o r the system.  Many r o u t i n e microscopy  and  the system  However,  application  techniques c o u l d be automated,  developed p r o v i d e s a good b a s i s  of a microscope.  efficiency,  f o r the dynamic  control  H i s t o l o g i c a l o r d e n s i t y p e r a r e a measurements c o u l d  7  e a s i l y be implemented 1.4  with this  system.  Other M i c r o s c o p i c - I m a g e P r o c e s s i n g Publications  b i n g hardware systems  cannot be enumerated  descri-  A l l the  h e r e , b u t none o f them are o n - l i n e  speed comparable  integrated  t o a t e c h n i c i a n p e r f o r m i n g the same  Many systems s u b s t i t u t e a p h y s i c a l memory d e v i c e to s t o r e the i n ^  f o r m a t i o n of the image: images  from a l l s c i e n c e s have i n c l u d e d a r t i c l e s  and s o f t w a r e t e c h n i q u e s f o r image p r o c e s s i n g .  systems o f f e r i n g task.  Systems  and s t o r e s  SCAD (4) scans c o l o u r e d photographs o f  the r e s u l t i n g d i g i t a l data on magnetic tape f o r sub-  sequent p r o c e s s i n g , CYTOSCAN (5) scans i n d i v i d u a l scope and s t o r e s  cell  the data on punched  scans p i c t u r e s o f c e l l  images  cells  through a m i c r o -  paper o r magnetic tape, FIDAC (6)  and t r a n s f e r s  the d a t a d i r e c t l y  p u t e r i n b l o c k form, CYDAC (7) scans a m i c r o s c o p i c image and  to a comtransfers  d a t a to magnetic tape, and-SPECTRE I I (7) p r o v i d e s p o i n t a d d r e s s a b l e i n f o r m a t i o n from a m i c r o s c o p i c image.  SPECTRE I I i s the c l o s e s t i n con-  cept t o the system developed h e r e , a l t h o u g h SPECTRE I I does n o t p r o v i d e dynamic  o r " h a n d s - o f f " c o n t r o l o f the m i c r o s c o p e .  exceptionally  SPECTRE I I i s a l s o  slow, r e q u i r i n g 120 seconds f o r a f u l l r a s t e r scan o f  256 by 256 p o i n t s .  Any  f u r t h e r comparison to these systems  by c h e c k i n g the r e f e r e n c e s .  can be made  8  2.  2.1  The  Computer V i s u a l - I n p u t The  Resolution  o f the  System  a Z e i s s U n i v e r s a l microscope  I.T.T.  ( F i g u r e 2.1.1).  system becomes a f u n c t i o n of the r e s o l u t i o n o f both  image d i s s e c t o r and  d e s i g n was  HARDWARE  computer v i s u a l - i n p u t system i s composed o f an  V i d i s s e c t o r camera and  the  THE  the m i c r o s c o p e .  to ensure t h a t the  A primary c o n s i d e r a t i o n i n  r e s o l u t i o n of the system was  variable  would always be c a p a b l e of r e s o l v i n g p e r t i n e n t image d e t a i l . be n o t e d t h a t any  c e l l r e c o g n i t i o n scheme p e r m i t t i n g  u t i o n w i l l p r o b a b l y o f f e r an i n c r e a s e and 2.2  the  It  and  should  a decrease i n r e s o l -  i n speed d u r i n g  data a c q u i s i t i o n  microscope c o n t r o l . The  Image D i s s e c t o r The  image d i s s e c t o r i s n o t h i n g  w i t h a s e n s i t i v e and o p t i c s are  adjusted  more than a  photomultiplier  e l e c t r o n i c a l l y movable photocathode a r e a . such t h a t an image i s formed on the  photocathode,  which emits e l e c t r o n s p r o p o r t i o n a l to the i n t e n s i t y at any These e l e c t r o n s  are  accelerated  and  focused  The  given  onto the d i s s e c t i n g  point. aperture  p l a n e f o r m i n g an e l e c t r o n image which i s c u r r e n t  d e n s i t y modulated  cording  T h i s e l e c t r o n image  may  be  to the o p t i c a l i n p u t  d e f l e c t e d e l e c t r o n i c a l l y across  i n s t a n t the p h o t o e l e c t r o n s are i n c i d e n t on and  i n t e n s i t y pattern.  the  the  aperture,  such t h a t a t  from a s m a l l , w e l l - d e f i n e d  aperture.  These e l e c t r o n s e n t e r  area o f the a  emerge as a c u r r e n t i n the output anode c i r c u i t .  t o r can be  v i s u a l i z e d as a means o f randomly a c c e s s i n g  information  from the  The  digital  ac-  any image  photomultiplier The  image d i s s e c -  point i n t e n s i t y  image. c o n t r o l used on  the  dissector i s well  documented  Figure 2.1.1  Photograph of the microscope and hardware.  10  SP  50 .  -10  -5  0  5  10  Dlotanco From Boundary (Units of GS)  - 1 2 - 8 - 4  0  K  8  12  Distance From Boundary (Units of CS)  F i g . 2.2.1  (8).  I n t e r p r e t a t i o n o f percentage m o d u l a t i o n . 100% m o d u l a t i o n a t GS = 5 i s shown i n (A) and 86.5% m o d u l a t i o n a t GS = 4 i s shown i n ( B ) .  With the d e f l e c t i o n  c o n t r o l p r o v i d e d , i t i s p o s s i b l e to randomly  a c c e s s o r address any one o f 1024 by 1024 p o i n t s on the image.  The d i s -  s e c t o r i s c u r r e n t l y equipped w i t h a tube h a v i n g a .005 i n c h round defining-aperture.  To a c h i e v e 100% m o d u l a t i o n between a d j a c e n t p o i n t s  a c r o s s the one i n c h square s u r f a c e o f the tube, a p p r o x i m a t e l y 200 by 200 p o i n t s a r e unique, r e p r e s e n t e d by an i n c r e m e n t a l step s i z e  (GS) o f  11  5 a d d r e s s i n g u n i t s ( F i g . 2.2.1).  However, t o i n c r e a s e r e s o l u t i o n , a s t e p  s i z e o f 4 i s assumed t o be the minimum, thereby e f f e c t i v e l y depth o f m o d u l a t i o n points. sidered  t o 86.5%, and c r e a t i n g a g r i d o f 256 by 256  unique.  dependent on the s i g n a l  sampling  addressable  T h e r e f o r e a maximum of 65,536 p o i n t s on the image w i l l be con-  The number o f grey l e v e l s o f i n t e n s i t y is  r e d u c i n g the  frequency  t h a t can be r e s o l v e d  to n o i s e r a t i o o f the v i d e o s i g n a l .  i n c r e a s e s , the s i g n a l to n o i s e r a t i o  As the  decreases  (9,10)  The s i g n a l t o n o i s e r a t i o i s g i v e n by the f o l l o w i n g e q u a t i o n :  g = 1.2 X 1 0  9  / J a 2At  (2.2.1)  Nrms where J = the average photocathode c u r r e n t d e n s i t y a = the a p e r t u r e  area  At = the element d w e l l time b e f o r e i n t e r r o g a t i o n . 2 -4 2 F o r the d i s s e c t o r tube i n use, J i s 1 ua/cm and a i s 1.26 X 10 cm . The r e s u l t s  of t h i s e q u a t i o n  are p l o t t e d i n F i g u r e 2.2.2.  d e s i r e d s i g n a l to n o i s e r a t i o ,  the t o t a l  t i o n i s a f u n c t i o n of three q u a n t i t i e s : tion  d e l a y time p r e c e d i n g i n t e r r o g a the s e t t l i n g  c i r c u i t r y , A t , and the s i g n a l p r o p a g a t i o n  used to l i m i t determined  the h i g h frequency  experimentally.  noise.  I t was  found  time o f the d e f l e c -  d e l a y o f the video  sufficient  t h a t a programmed  to a c h i e v e 5 - b i t g r e y - l e v e l r e s o l u t i o n .  t h a t w i t h i n the 50 m i c r o s e c o n d s ,  d e l a y o f 50 conversion  This implies  the f i l t e r e d v i d e o s i g n a l s e t t l e d to  w i t h i n the a n a l o g v o l t a g e e q u i v a l e n t to o n e - h a l f o f the l e a s t cant b i t o f q u a n t i z a t i o n .  filter  T h i s t o t a l d e l a y time i s b e s t  microseconds between D/A l o a d i n g and the b e g i n n i n g o f A/D was  To o b t a i n a  T h i s 50 microsecond  signifi-  d e l a y i s the nominal v a l u e  used i n a l l d i s c u s s i o n s and system measurements t o f o l l o w .  12  0  10  20  30  Dwell Tlma A t  Fig.  2.2.2  40  50  60  (microseconds)  S i g n a l to N o i s e r a t i o of the image d i s s e c t o r as a f u n c t i o n o f A t , the d w e l l time.  13  2.3  The M i c r o s c o p e The image d i s s e c t o r i s o p t i c a l l y  coupled to a Zeiss  Universal  m i c r o s c o p e such t h a t an image i s formed on the photocathode o f the d i s sector. For  F i g u r e 2.3.1  i s a b l o c k diagram o f the o p t i c a l  arrangement.  t r a n s m i t t e d l i g h t m i c r o s c o p y , a 60 watt 12 v o l t i l l u m i n a t o r i s used.  The power s u p p l y f o r the i l l u m i n a t o r i s r e q u i r e d to be w e l l and f i l t e r e d  regulated  t o remove any 60 c y c l e r i p p l e , s i n c e t h i s would appear as  n o i s e on the v i d e o s i g n a l from the d i s s e c t o r .  The microscope i s equipped  w i t h a m e c h a n i c a l s t a g e d r i v e n b y two s t e p p i n g motors.  The s t e p p i n g  f r e q u e n c y i s 200 hz and the s t e p s i z e i s 10 microns i n b o t h the X and Y directions.  The f i n e  focus c o n t r o l has a l s o been equipped w i t h a s t e p -  p i n g motor, p r o v i d i n g 2000 s t e p s p e r r e v o l u t i o n a t a s t e p p i n g f r e q u e n c y of  600 h z .  To move the s t a g e manually under program  f o c u s i n g , the system i n c l u d e s a j o y s t i c k . to  control with  dynamic  The j o y s t i c k can a l s o be used  d r i v e the f o c u s motor manually. V i b r a t i o n o r m e c h a n i c a l shock i n the neighbourhood o f the m i c r o -  scope can cause j i t t e r i n the image. s t r e n g t h e n i n g the o p t i c a l or  by mounting  jitter  2.4  Most c o n f u s i o n from  f o r by c a r e f u l programming, b u t i t i s b e s t to  to p r e v e n t the causes o f the j i t t e r .  The Computer The  of  c o u p l i n g between the microscope and d i s s e c t o r ,  these components on a shock t a b l e .  can be compensated  attempt  The system c o u l d be improved by  Interface  computer  used i n t h i s system i s a PDP-9 equipped w i t h 16K  memory and t h r e e Dectape u n i t s .  The i n t e r f a c e f o r the d i s s e c t o r has  been w e l l documented ( 8 ) , a l t h o u g h a few changes have been made. 2.4.1  i s a b l o c k diagram o f the hardware  configuration.  Figure  A l t h o u g h much  14  Imago Dissector  - Eyepiece . Optical Coupling  Eyepiece  »» A . Microscopo Head  Oil  O i l Immersion Eyepiece  Blood Filni  Conden3or  Scanning Stage  Light Path  Figure 2.3.1  Light Source  Block diagram of the o p t i c a l system.  LOAD D/A 10 DATA LINES  (TO DECTAPS UNITS  <-  10 aiTA LIKES LOAD D/A PDP-9 COMPUTER  X D/A CCNV.  IMAGE DISSECTOR  Y D/A CONV. AKALOG VIDEO SIGNAL  START A/D 6 DATA LINES  A/D CONV.  READ A/D  STEP X STEP X  CRT DISPLAY  SCANNING STAGE  STEP I STEP I  STEP CLOCKWISE STEP COUNTERCLOCKWISE  3 DATA LINES  TELETYPE  START.  8 A/D CONV.  RES!) LOAD MUX. CH.  Figure 2.4.1 Block diagram of the computer i n t e r f a c e .  CH.  Max.  MODE  JOYSTICK  16  time has been spent i n d e s i g n i n g and  c o n s t r u c t i n g the hardware, the  d e t a i l s of the c o n s t r u c t i o n w i l l not be e x p l a i n e d h e r e . t h e a l g o r i t h m s and  techniques  system are more i m p o r t a n t , o b t a i n e d i n assembling i n s t r u c t i o n s used adequately  developed  It is felt  f o r the a p p l i c a t i o n of  that  this  a l t h o u g h much p r a c t i c a l e x p e r i e n c e has been '  the hardware.  Appendix I I summarizes the  to c o n t r o l the hardware.  software  These d e s c r i p t i o n s s h o u l d  e x p l a i n the p o s s i b l e hardware f u n c t i o n s from  the u s e r ' s  p o i n t o f view. 2.5  The  System R e s o l u t i o n R e s o l u t i o n of the system i s a bounded f u n c t i o n of the  of  both  the image d i s s e c t o r and  solution  can be  determined,  the m i c r o s c o p e .  resolution  B e f o r e the combined r e -  the r e s o l u t i o n of each system component must  be e v a l u a t e d s e p a r a t e l y . O p t i c a l r e s o l u t i o n o f a microscope  N.A. where  , + N.A. , . cond obj  X = the wavelength o f N.A.  i s commonly e x p r e s s e d  microns  Q  (2.5.1)  illumination  , = the n u m e r i c a l a p e r t u r e o f the cond  N.A. ^J  = the n u m e r i c a l a p e r t u r e of the  condenser objective.  T h i s e q u a t i o n r e p r e s e n t s the h i g h e s t r e s o l v i n g power p o s s i b l e w i t h chosen l e n s e s and  type o f i l l u m i n a t i o n .  conditions of c r i t i c a l i l l u m i n a t i o n , is  f o c u s e d on  mately  the o b j e c t i n such  The  t h a t i s , when the source o f  a manner t h a t the beam f i l l s  u s u a l l y i m p l i e s t h a t N.A. J  r  the  e q u a t i o n o n l y a p p l i e s under  t w o - t h i r d s o f the a p e r t u r e of the o b j e c t i v e (11).  mination  as:  , i s e q u a l to N.A. cond  t a n t f e a t u r e about o p t i c a l r e s o l u t i o n i s that a l i m i t  light  approxi-  Critical  , ..  The  illu-  impor-  obj e x i s t s beyond which  OpticalMagnification  I  Magnification to CRT Display 1 micron Pover = . . . cm.  200  .0145 '  256  .0180  320 * 320 * 410 512 500 640  .0221 .0230  H5  ISO  Magnification to Image Dissector 1 micron Pover  .0046 .0057  46 57  Size of Field 1 Inage Dissector of View Eosoluticn  Optical Resolution  (microns)  GS =  551.76  .55  2.20  444=43 362.00 347.34  .44  1.76  o36  1.44  .35  1.40  .63  1.13  .68  1  GS =  4 1.2 1.2  221  .0070  230  .0073  73  287  .0091  .036S .0453 ,0561  .0113  225.36  117  217.63  .25  .0117  .22  .83  .0144  144  .18  .72  .0173  176.64  .178  572  .0182  182  .14  800 *  .0572  355 368 453 561  91 113  1024 12S0 2000 . 2560 3200  .0715  715  .0227  227-  111.92  .11  .56 .56 .44  .27  „C3S9 .1430  889 1430  .0282  282  1783  .2200  2200  454 566  .36 .224  .1783  .090 .056 .045 .036  .27  .0454 .0566 .0699  90.00  SCO *  *  .0237 .0355  70  699  273.72  142.64 139.84 55.94  44.87  36,36  .23  .14  Same magnification, but vith different objective. Note Optical Resolution.  Table 2.5.1  Results of the c a l i b r a t i o n of the image dissector resolution.  1.00  .180  .144  1.2  .68 .42 .42 .42 .27  .21 .21 .21  18  the m a g n i f y i n g power cannot be aperture p o s s i b l e  i s about 1.4,  ques are n e c e s s a r y to i n c r e a s e  p r o f i t a b l y increased. and the  The  best numerical  at t h i s v a l u e , o i l - i m m e r s i o n i n d e x of r e f r a c t i o n o f the  techni-  inter-lens  medium. A p p l y i n g the w i t h green l i g h t about  . 21u.  (A =  This  l e u k o c y t e which are highest 100X  objective  a one  and  a N.A.  o f 1.3,  corresponds w e l l w i t h the about  .25u  i n diameter.  image d i s s e c t o r has  a .005  optical resolution i s  s i z e of granules i n This  resolved  on  the  b r a t e the  Image measurements were made on  system. display  u n i t , and  a c r o s s the  one  been c a l i b r a t e d , the  image d i s s e c t o r T a b l e 2.5.1 c a t i o n and  the  defining-aperture  256  by  256  distinct  at 10 m i c r o n i n t e r v a l s was an e i g h t  used to  these measurements were c o n v e r t e d i n t o t h e i r  i n c h square photocathode.  Once the  c a l i b r a t i o n i s applicable only  summarizes the  r e s u l t s o f the  d i f f e r e n t s t e p s i z e s f o r the  t o r r e s o l u t i o n may  be  cali-  centimeter  2.5.1A f o r GS expressed  as:  as  This  the  eyepiece.  c a l i b r a t i o n based on  dissector.  = 4.  system  as l o n g  remains i n a c o n s t a n t p o s i t i o n r e l a t i v e to the  also p l o t t e d i n Figure  depth  d i s s e c t o r step s i z e i n t o a m e a n i n g f u l  a s l i d e scribed  has  the  the. image a r e a .  measure o f l e n g t h ,  equivalents  o c c u r s when  T h e r e f o r e at 86.5%  = 4 ( r e f e r to s e c t i o n 2.2),  In o r d e r to t r a n s l a t e  square CRT  the  also represents  i n c h round  i n c h square photocathode s u r f a c e .  can be  that  i s used.  o f m o d u l a t i o n w i t h GS points  .54u)  r e s o l u t i o n p o s s i b l e w i t h t h i s microscope and  The and  e q u a t i o n f o r o p t i c a l r e s o l u t i o n , i t i s found  magnifi-  These r e s u l t s  graph shows t h a t  are dissect  19  1.0  B  g •A  0.5  o.o  I  1000  I  I  2000  1  I  I  •  30O0  Pover of Kaenification  Fig.  2.5.1  R e s o l u t i o n as a f u n c t i o n of m a g n i f i c a t i o n f o r t h e image d i s s e c t o r (A) and the microscope ( B ) .  20  Fig.  2.5.2  System r e s o l u t i o n as a f u n c t i o n of  magnification.  21  Optical resolution dissector bounded  i s plotted  resolution  system  i n F i g u r e 2.5.IB.  are then combined  resolution.  Optical resolution  i n F i g u r e 2.5.2  and  t o produce the  22  3.  3.1  DEVELOPMENT OF THE AUTO-FOCUS  ALGORITHM  Background O p t i c a l systems must be p r o p e r l y  formance.  This i s e s p e c i a l l y  focused  to y i e l d optimum  t r u e f o r a high-power microscope where the  depth o f f i e l d may be l e s s than  .25 micron  (.11).  The most common method  o f e v a l u a t i n g image q u a l i t y i s s u b j e c t i v e human e v a l u a t i o n . w e l l d e f i n e d edges, c l e a r l y  d e f i n e d shapes, f i n e d e t a i l ,  for.  Auto-focusing  algorithms  that optimize  Sharp o r  and o v e r a l l  c r i s p n e s s o r c o n t r a s t o f the image are some o f the f e a t u r e s looks  per-  an  observer  image q u a l i t y must  d e f i n e the means used t o e x t r a c t measures o f these p r o p e r t i e s . Auto-focusing  algorithms  have been p r e v i o u s l y proposed, a l t h o u g h  most o f them a r e t o o complex o r too i n e f f i c i e n t on-line. be  Mendelsohn and M a y a l l  to e f f e c t i v e l y  implement  (12) have suggested that f o c u s i n g can  a c c o m p l i s h e d by maximizing the f u n c t i o n i=l <(> = n  I  (OD. -  ty)  f o r a l l OD. > ty '  (3.1.1)  where OD^ i s the grayness a t a p o i n t , n i s the number o f p o i n t s i n the image w i t h  OD^ > ty, and ty Is a r e f e r e n c e  arbitrarily  d e n s i t y i n the mid-range o f image g r e y n e s s . that  f i x e d a t an o p t i c a l  Mendelsohn and M a y a l l  admit  the c h o i c e o f tj; may n o t be s t r a i g h t f o r w a r d , b u t e x p l a i n t h a t the  choice  o f ty can c o n t r o l which component o f the image w i l l be i n focus  when a l l components do n o t l i e i n the same h o r i z o n t a l p l a n e . r i t h m may a l s o be d i f f i c u l t  to evaluate  a t h i g h speed.  This  algo-  Conceptually,  each e v a l u a t i o n of the f u n c t i o n ty d u r i n g m a x i m i z a t i o n i n v o l v e s a scan of the complete f i e l d  o f view.  The a u t o - f o c u s i n g  algorithm  to be p r e s e n -  t e d h e r e e l i m i n a t e s one dimension o f the s c a n , and t h e r e f o r e s h o u l d  offer  23  s u f f i c i e n t speed f o r e f f e c t i v e cessing 3.2  the A l g o r i t h m  A s i n g l e v i d e o scan one-dimensional f u n c t i o n .  line  Figure  a c r o s s an image may be viewed as a 3.2.1A shows a scan l i n e a c r o s s a  o f view when the image i s i n f o c u s .  C h a r a c t e r i s t i c a l l y , as an image  i s put out o f f o c u s , t h e i n t e n s i t y i n f o r m a t i o n on the scan r a t e s as shown i n F i g u r e the f o c u s ,  f o r i n a scan  good c o n t r a s t r a t i o ,  tion  By examining the image w h i l e  l i n e may be e s t a b l i s h e d .  features searched  relation,  3.2.IB.  line deteriovarying  c o r r e l a t i o n between image q u a l i t y and c e r t a i n c h a r a c t e r i s t i c s  i n the scan  has  pro-  system.  Developing  field  implementation i n an o n - l i n e image  F i g u r e 3.2.2  shows  l i n e , namely, sharp edge  and r e n d i t i o n o f f i n e d e t a i l .  the primary definition,  Based on t h i s  a f o c u s i n g a l g o r i t h m i s proposed t h a t u t i l i z e s a f u n c t i o n t h a t  a maximum when the scan  l i n e contains  these  characteristics.  to be used i s s t a t i s t i c a l v a r i a n c e o f the i n t e n s i t y l e v e l s  the scan  line.  adjusting  the focus  control u n t i l  the v a r i a n c e  func-  along  reaches a maximum.  of a s e t of measurements i s a measure of the v a r -  i a t i o n o f the s i n g l e measurements w i t h i n t h a t s e t . i n t e n s i t y l e v e l s o f the i n - f o c u s scan the o u t - o f - f o c u s variation,  The  I t i s proposed t h a t f o c u s i n g may be accomplished by  The v a r i a n c e  estimator  cor-  scan  line.  line  I t follows  I n F i g u r e 3.2.1, the  c o n t a i n more v a r i a t i o n  t h a t v a r i a n c e , a measure o f  c o u l d be used to judge the q u a l i t y o f the image. 2 o f the v a r i a n c e , s , i s t r a d i t i o n a l l y  s  2  I  (x. - x )  than  The  unbiased  d e f i n e d as f o l l o w s ( 1 8 ) .  2  i  N-1  where i = 1 to N; x. = an i n d i v i d u a l sample; x =  Ix  (3.2.1) x. .N  24  200  nu«*ctor  I Coordinate  F i g . 3.2.1  To s i m p l i f y  400  600  M j i e c t o r X Coordinate  A video scan l i n e when i n focus (A) and when out o f focus ( B ) .  2 the c a l c u l a t i o n s r e q u i r e d to e x t r a c t s , i t i s more conven-  2 i e n t to use the b i a s e d estimator o f s , which i s defined (18) as f o l l o w s . (x  2  ±  -x)  2  i  (3.2.2)  N where i • 1 to N  • an i n d i v i d u a l sample  25  60 /  . Fine Detail  AO  > o  I Sharp Edges  20  -P m  _1_  JL  200  ^00  600  Dissector 1 Coordinate Fig.  Although  3.2.2  C h a r a c t e r i s t i c s searched scan l i n e .  f o r i n a video  the magnitudes o f the e s t i m a t o r s d i f f e r ,  i t i s permissable  to  use e i t h e r e s t i m a t o r as both e q u a t i o n s have a maximum w i t h the same subset of data.  E q u a t i o n 3.2.2  y (x. .  s  i  = x  2  may  - 2x x. + i  x ) 2  x.  + 7  1  -2 - 2x 9  N  N 3.2.3  algebraically.  N  I  Equation  be reduced  was  V  N  Nx  2  -2 + x  <  used i n a l l the measurements t h a t f o l l o w .  ( 3  ' 2  3 )  Numerically  t h i s f u n c t i o n i s e v a l u a t e d by s i m p l e r e c u r s i v e e v a l u a t i o n as each  intensity  26  200 -  0 1  '  '  -SO  i  -60  i -40  i  i  i  i  -20  0  20  40  '  i  — i  60  80  Nuriber o f Focua Stops  Number o f Focus Steps  Fig.  3.2.3  The v a r i a n c e p l o t t e d a g a i n s t focus c o n t r o l p o s i t i o n u n f i l t e r e d (A) and f i l t e r e d (B) w i t h a d i g i t a l f i l t e r window s i z e of t e n .  27  l e v e l , x., becomes a v a i l a b l e :  accumulate the two t o t a l s T x .  and J x . ,  2  2 and when the s c a n is. c o m p l e t e , square the second t o t a l and d i v i d e by N , s u b t r a c t i n g the r e s u l t from the f i r s t t o t a l d i v i d e d by N . F i g u r e 3.2.3A shows the v a r i a n c e of i n t e n s i t y p l o t t e d a g a i n s t the  f i n e f o c u s adjustment f o r a t y p i c a l c l u s t e r o f c e l l s .  The  "zero"  p o i n t on the h o r i z o n t a l a x i s r e p r e s e n t s the f o c u s c o n t r o l p o s i t i o n where the  image was j u d g e d as b e i n g o f the b e s t q u a l i t y .  the' same c u r v e a f t e r d i g i t a l low-pass f i l t e r i n g . peak d e t e c t i o n by smoothing the c u r v e .  F i g u r e 3.2.3B shows Filtering  simplifies  The d i g i t a l f i l t e r used s i m p l y  averages the v a r i a n c e from s e v e r a l a d j a c e n t p o i n t s and a s s i g n s the average to  the c e n t r a l p o i n t .  v a r i a n c e att.the k ^  To s t a t e t h i s i n t h e form o f an e q u a t i o n , the  p o i n t i s d e f i n e d as f o l l o w s :  I k v, =  l  v. (3.2.4)  N  where N = the d i g i t a l f i l t e r window s i z e (^1) v. = the v a r i a n c e a t the i  th.  x  point  and i v a r i e s from k - — t o N  k + — -1 f o r N even and from i  k N  N - 1  —  ^  ,  ,  to k H  N - 1  —  . for  odd.  S e v e r a l s u b j e c t i v e e v a l u a t i o n s o f image q u a l i t y  verified  t h a t the v a r i a n c e peaks when the image i s i n f o c u s , t h e r e b y j u s t i f y i n g the use o f t h i s a l g o r i t h m .  At f i r s t ,  i t may be d i s c o n c e r t i n g t o f i n d  that  an a u t o m a t i c system can f o c u s more c r i t i c a l l y than the human o p e r a t o r . One o f the t e s t s conducted r e q u i r e d a p e r s o n to m a n u a l l y f o c u s the m i c r o s c o p e t o produce the b e s t image.  The a u t o m a t i c f o c u s would then  28  be  a c t i v a t e d and many times  image was  an improvement of the manually  noticeable. Focus by v a r i a n c e m a x i m i z a t i o n  ment, p r o v i d e s ging,  good a v e r a g i n g o v e r  compensates f o r o p t i c a l c u r v a t u r e .  F a c t o r s A f f e c t i n g the V a r i a n c e Several factors alter  function. these  i s n u m e r i c a l l y simple  the f i e l d o f view, and  a l o n g s e v e r a l non-adjacent s c a n l i n e s , 3.3  In o r d e r  discussions  these  conditions affecting  01  contents  I  • I  and  -20  -10  0  I  10  Hunbor of Focua Steps  Fig.  3.3.1  variance  techniques  controlled.  During  The  1  20  variance  effectively,  the f o l l o w i n g  assumed t h a t a l l v a r i -  the v a r i a n c e f u n c t i o n are h e l d  constant,  " z e r o " p o i n t on the h o r i z o n t a l a x i s  o f the f i e l d  |  avera-  Function  the p o i n t where image q u a l i t y was The  i n such  the b e s t o v e r a l l image i s o b t a i n e d .  f a c t o r s , i t s h o u l d be  unless i t i s s t a t e d otherwise. represents  imple-  the c h a r a c t e r i s t i c shape o f the  understood  concerning  to  By maximizing the  to a p p l y v a r i a n c e m a x i m i z a t i o n  f a c t o r s must be  a b l e s and  adjusted  judged to be  of view a f f e c t  L  o  I  I  -20  "best".  the v a r i a n c e f u n c t i o n .  J  -10  1  0  1  10  1-  20  Number of Focus Stopo  The v a r i a n c e f u n c t i o n of two d i f f e r e n t of view showing peak v a r i a n c e s c a l i n g .  fields  29  Uvaber  Number o f Focus Stops •  Fig.  3.3.2  o f Focus Stops  S c a l i n g o f the v a r i a n c e f u n c t i o n due to m a g n i f i c a t i o n .shown a t 800X ( A ) , 1024X (B),  1280X  ( C ) , and 2 0 0 0 X  (D).  30  Different  objects  c o n t a i n v a r y i n g amounts o f i n f o r m a t i o n , and s o , as  the f i e l d o f view i s changed, one can e x p e c t t h a t the peak v a r i a n c e will  a l s o change ( F i g u r e 3.3.1).  I t i s p o s s i b l e t h a t a new f i e l d  value  o f view  may have a peak v a r i a n c e  t h a t i s below the s e n s i t i v i t y o f the v a r i a n c e  maximization algorithm.  F o r such cases where a maximum v a r i a n c e  may n o t be d e t e c t a b l e , o r may n o t even e x i s t , should maintain information peak v a l u e  the m a x i m i z a t i o n  c o n t r o l by r e c o g n i z i n g t h i s s i t u a t i o n .  content  o f the o b j e c t s i n the f i e l d  o f the v a r i a n c e  3.3.2.  T h e r e f o r e , the  o f view determines the  c o n t r o l s the s c a l i n g on the h o r i z o n t a l As m a g n i f i c a t i o n i s i n c r e a s e d , the  depth o f f i e l d o f the o p t i c s d e c r e a s e s ,  and t h e r e f o r e the range i n which  image i n f o r m a t i o n i s r e c e i v e d a l s o d e c r e a s e s , t i o n , a narrower, shape.  algorithm  function.  System m a g n i f i c a t i o n a x i s as shown i n F i g u r e  value  g i v i n g the v a r i a n c e  func-  S i n c e peak d e t e c t i o n i s the o b j e c t i v e , the focus  c o n t r o l s t e p s i z e s h o u l d be made s m a l l e r as m a g n i f i c a t i o n i s i n c r e a s e d , e f f e c t i v e l y spreading is  difficult  the v a r i a n c e  to g e n e r a l i z e t h i s  dependent on the n u m e r i c a l s p e c i f i c o b j e c t i v e s used  scaling property,  aperture  (11).  f u n c t i o n over the h o r i z o n t a l a x i s .  It  as the depth o f f i e l d i s  (N.A.) and e x i t p u p i l s i z e o f the  However, a convenient  estimate  i s t o say  t h a t as m a g n i f i c a t i o n i s doubled, the focus step s i z e should be reduced by  1/3 to 1/2 (See Appendix I c o n c e r n i n g  determined  experimentally. Optical f i l t e r s  a r e u s u a l l y chosen t o enhance the c o n t r a s t  of the image i n a m e a n i n g f u l way. also a f f e c t 3.3.3  FOCSTP f o r n o m i n a l v a l u e s ) as  the s p r e a d  However, the f i l t e r  and peak v a l u e  o f the v a r i a n c e  demonstrates how d i f f e r e n t f i l t e r s  e x t r a c t e d from an image o f c e l l s .  affect  selection w i l l function.  the v a r i a n c e  Figure  function  As a f i l t e r narrows the v a r i a n c e  31  ol  i  -80  - i  -40  I  i  1  0  40  80  Nunbov of Fotroo Steps  Fig.  3.3.3  L.  oI  I  _g  0  1  1  0  1 — - — i  40  80  Kunber of Focuc Steps  The e f f e c t of no f i l t e r ( A ) , a r e d f i l t e r ( B ) , a b l u e f i l t e r ( C ) , and a green f i l t e r (D) on the v a r i a n c e f u n c t i o n e x t r a c t e d from an image o f c e l l s .  »•  32  function,  the f i l t e r  i s being  more s e l e c t i v e .  the image a r e l o s i n g c o n t r a s t . the v a r i a n c e being  An image c l o s e l y r e p r e s e n t s  plane o f thickness. from the p l a n e s  filter  increases  I f the f i l t e r  surrounding  function w i l l  the o b j e c t a t o n l y one  i s chosen such t h a t image  vides s u f f i c i e n t  variance  a l s o be s y m m e t r i c a l .  Generally,  then  the b e s t  f u n c t i o n and p r o -  c o n t r a s t i n the i n t e r e s t i n g areas o f the image.  i n t e n s i t y o f i l l u m i n a t i o n produces a s c a l i n g a f f e c t on the  function.  As background i l l u m i n a t i o n i s h a l v e d ,  o f i n t e n s i t y between a l i g h t t h a t the v a r i a n c e  0  the d i f f e r e n c e  and dark o b j e c t i s a l s o h a l v e d .  Considering  i s r e l a t e d t o the squares o f these d i f f e r e n c e s ,  I  i  -60  Fig.  information  the p l a n e o f i n t e r e s t i s s y m m e t r i c a l ,  produces a w e l l - d e f i n e d s y m m e t r i c a l v a r i a n c e  The  the peak v a l u e o f  f u n c t i o n , the c o n t r a s t o f s p e c i f i c p a r t s o f the image i s  enhanced.  the v a r i a n c e  As a f i l t e r  That i s , some p a r t s o f  3.3.4  —i -40  -—i  '  -20  0  Number of  Focu3  1  20  1  40  1  60  Steps  The v a r i a n c e f u n c t i o n e x t r a c t e d a t f u l l i n t e n s i t y and a t h a l f i n t e n s i t y . The bottom curve i s the one at h a l f i n t e n s i t y .  33  halving of  the i n t e n s i t y  the f u l l v a l u e Critical  of  critical  First,  reduces the v a r i a n c e to approximately  illumination  ( r e f e r to S e c t i o n 2.5 the v a r i a n c e  maximum o p t i c a l r e s o l u t i o n o c c u r s and  or e l s e a s h i f t  definition  f u n c t i o n i n two ways.  under c o n d i t i o n s o f  3.2.3  illumination conditions.  critical  Second,  the  two  f o r the v a r i a n c e s h o u l d peak a t the same time  i n the peak v a r i a n c e w i l l  c i d e n t o n l y under c o n d i t i o n s o f c r i t i c a l shows the e f f e c t  f o r the  t h e r e f o r e the v a r i a n c e f u n c t i o n s h o u l d have a h i g h e r  peak v a l u e under c r i t i c a l terms i n e q u a t i o n  quarter  ( F i g u r e 3.3.4).  illumination) affects  illumination,  one  of c r i t i c a l  occur.  T h i s peaking  illumination.  Figure  is coin3.3.5  i l l u m i n a t i o n at a h i g h m a g n i f i c a t i o n .  t u n a t e l y , at lower powers o f m a g n i f i c a t i o n t h i s phenonmena i s not  0 I  _J -60  -40  »_i -20  i 0  i 20  i 40  1 60  Foras  u  Hunber of Focus Steps  Fig.  3.3.5  The e f f e c t of n o n - c r i t i c a l i l l u m i n a t i o n on the v a r i a n c e f u n c t i o n at a m a g n i f i c a t i o n of 2560X.' The top curve r e p r e s e n t s c r i t i c a l i l l u m i n a t i o n .  34  pronounced. All  these f a c t o r s  f o r during implementation  can e a s i l y be c o n s i d e r e d and compensated  o f a u t o - f o c u s i n g by v a r i a n c e m a x i m i z a t i o n .  Through an u n d e r s t a n d i n g o f these p r o p e r t i e s , a g e n e r a l i z e d a l g o r i t h m can be developed pendent.  maximization  t h a t i s b o t h m a g n i f i c a t i o n and o b j e c t i n d e -  35  4.  4.1  THE LEUKOCYTE LOCATION PROCEDURE  The C o n d i t i o n s o f the Search E r y t h r o c y t e s outnumber l e u k o c y t e s  by approximately  600:1 on  a normal b l o o d smear, and t h e r e f o r e l o c a t i n g o r i s o l a t i n g l e u k o c y t e s can be  a time-consuming task.  field  As m a g n i f i c a t i o n i n c r e a s e s , the a r e a o f the  o f view d e c r e a s e s and t h e r e f o r e the p r o b a b i l i t y o f a l e u k o c y t e  existing  i n any p a r t i c u l a r  field  o f view a l s o d e c r e a s e s .  This  implies  t h a t a l o n g e r time i s r e q u i r e d t o l o c a t e a l e u k o c y t e . Most l e u k o c y t e  l o c a t i o n algorithms  colour properties of a blood Since  f i l m s t a i n e d with Wright's s t a i n  (4,13).  the image d i s s e c t o r cannot d i s t i n g u i s h between c o l o u r s and i s  only s e n s i t i v e to i n t e n s i t y , to  take advantage o f t h e  enhance the c o n t r a s t r a t i o  300  i t i s h e l p f u l to s e l e c t o p t i c a l o f the l e u k o c v t e s  400 ' 500  600  700  filters  so thev mav be  separated  800 900  Wavelength A i n nm.  Fig.  4.1.1  from the e r y t h r o c y t e s . light The  By making use o f the heavy a b s o r p t i o n  i n the s t a i n e d l e u k o c y t e n u c l e u s ,  s p e c t r a l d e n s i t y curve  a l l magnifications this  S p e c t r a l d e n s i t y curve o f the Z e i s s 46 78 06 wide-band pass green i n t e r f e r e n c e f i l t e r .  o f the f i l t e r  the l e u k o c y t e s  of green  may be l o c a t e d .  t h a t gave the b e s t r e s u l t s a t  i s shown i n F i g u r e 4.1.1.  When choosing  system, i t must be r e a l i z e d t h a t the e r y t h r o c y t e s  a filter for  a r e r e q u i r e d to  36  show i n the image, o r e l s e the a u t o - f o c u s i n g a l g o r i t h m not  function properly.  with  and w i t h o u t  F i g u r e 4.1.2 shows scan  c o n t r a s t enhancing f i l t e r s .  t r o l l e d i l l u m i n a t i o n and u n i f o r m dense n u c l e u s 4.2  i s a satisfactory  Threshold  Under c o n d i t i o n s o f con-  s t a i n i n g , s e a r c h i n g f o r the o p t i c a l l y criterion  forlocating  Fig.  4.1.2  Dissector I Coordinate  D e t e c t i o n seems b e s t done i n two stages  the n u c l e i o f a l l c l a s s e s o f l e u k o c y t e s f i r s t stage  t h r e s h o l d f o r the p a r t i c u l a r  The cells  are n o t o f the same d e n s i t y .  cell  The second s t a g e r e f i n e s found  the f i r s t  so that measurements can be  cell. first  t h r e s h o l d i s the most c r i t i c a l  d e s i g n a t e d by t h i s  leukocytes.  since  i n v o l v e s the e s t a b l i s h i n g o f a crude t h r e s h o l d t h a t i s  to include a l l leukocytes.  made on t h i s  dense" i s r e q u i r e d t o  Video scan l i n e s a c r o s s a l e u k o c y t e w i t h (B) and w i t h o u t (A) c o n t r a s t enhancing f i l t e r s .  the l e u k o c y t e s .  expected  the n u c l e u s .  Determination  Dissector X Coordinate  The  3) w i l l  l i n e s across a leukocyte  Some q u a n t i t a t i v e measure o f " o p t i c a l l y  locate  (Chapter  An a b s o l u t e  threshold should, with threshold value  i n the sense t h a t  a h i g h p r o b a b i l i t y , be  does n o t work as the n u c l e a r  37  i n t e n s i t y v a r i e s s i g n i f i c a n t l y from s l i d e view to f i e l d be  o f view.  to s l i d e  and from f i e l d o f  T h i s suggests t h a t the f i r s t  a f u n c t i o n o f the mean i n t e n s i t y o f the c u r r e n t  the s e a r c h  enters  i n t o a new f i e l d  i n t e n s i t y sampled over v a r i o u s constant,  threshold  field  should  o f view.  As  o f view, the average background  areas o f the image i s m u l t i p l i e d by a  d e f i n e d as the c o n t r a s t r a t i o ,  to produce the t h r e s h o l d  value.  To p u t t h i s i n t h e form o f an e q u a t i o n , the „ _ _. ^. .Contrast R a t i o =  Threshold —:  f o r Leukocytes —; —:  , . •_ (4.2.1) 1 X  1  Average Background I n t e n s i t y The  t h r e s h o l d d e f i n e d i n t h i s way works w e l l as l o n g as the c o n t r a s t  r a t i o has been c a r e f u l l y  chosen.  tance p r o p e r t i e s o f each b l o o d  f i l m , i t i s necessary  c o n t r a s t r a t i o f o r each s l i d e . on  the new s l i d e ,  using  this  T h i s i s done by l o c a t i n g a  this leukocyte. updated c o n t r a s t  A fixed contrast ratio  The whole  slide  transmit-  to r e d e f i n e the  and by a d j u s t i n g the c o n t r a s t r a t i o u n t i l  threshold includes cessed  However, due t o the v a r y i n g  leukocyte the r e s u l t i n g  can then be p r o -  ratio.  cannot be used f o r a l l s l i d e s s i n c e a  wide v a r i a t i o n i n the s t a i n i n g d e n s i t y and g e n e r a l q u a l i t y o f the b l o o d films exists. cessed with  A p p r o x i m a t e l y 25% o f the f i l m s cannot be p r o p e r l y  a fixed contrast r a t i o .  The b l o o d on the t e s t s l i d e s had  been smeared by hand and then s t a i n e d a u t o m a t i c a l l y . a v a i l a b l e automatic blood  spinners  P e r k i n - E l m e r Coleman Model 90 Blood to p r e p a r e s l i d e s  claimed lent,  There a r e now  and s t a i n e r s , f o r example, the S p i n n e r (16).  These u n i t s are used  f o r i n s t r u m e n t s which a r e n o t able  adjustments i n p e r c e p t i o n  t h a t the e x p e r i e n c e d  t o make the s u b t l e  human can make.  It i s  t h a t such a u t o m a t i c a l l y p r e p a r e d s l i d e s are u n f a i l i n g l y  containing  an a b s o l u t e  pro-  randomly d i s t r i b u t e d , u n i f o r m l y  minimum o f m e c h a n i c a l d i s t o r t i o n  spread  (16,17).  cells  excel-  that have  S l i d e s produced  38  by such an a u t o m a t i c p r o c e s s a fixed  contrast r a t i o  process  a l l such p r e p a r e d The  l e u k o c y t e has  s h o u l d be  c o u l d be  determined and  been found.  A "suspected"  below the f i r s t  contour  t r a c i n g o f the n u c l e u s  to a c c u r a t e l y contour This  leukocyte  t h r e s h o l d and  measurements o f t h i s s u s p e c t e d  required.  used s u c c e s s f u l l y  i s d e f i n e d as  i s of a certain  leukocyte  i s assumed t o be  may one  be  a leukocyte 4.3  The  may  the  the  value  intensity  cytoplasmic  shows f o u r of the s t a n d a r d  d i f f e r e n t i a l count ( 1 4 ) .  the  cell  m a t e r i a l of  The  types  across  the smear i s not  been made, and  (3).  one  since  the  uniform:  the margins and  lymphocytes predominate i n the middle of the f i l m p r e s e t p a t t e r n has  used  l o n g i t u d i n a l p a t t e r n i s the  n e u t r o p h i l s and monocytes predominate at  flexible  scan p a t t e r n s  S e v e r a l p a t t e r n s have been s t a n d a r d i z e d  d i s t r i b u t i o n of l e u k o c y t e  pletely  In  determined i n t h i s manner.  4.3.1  most commonly used.  of any  and  Scan P a t t e r n Figure  in  be  size.  tests.  t r a c e an o b j e c t , a r e f i n e d t h r e s h o l d  f o r b o t h the n u c l e a r and  an  required,  of these  t h r e s h o l d i s u s u a l l y d e f i n e d to be  Thresholds  to  suspected  l e v e l o c c u r r i n g at the maximum g r a d i e n t of i n t e n s i t y a c r o s s boundary.  that  slides.  Various  is  i t i s expected  second t h r e s h o l d v a l u e i s r e q u i r e d once a  object that f a l l s  order  s t u d i e d , and  the t a i l ,  No  and  commitment  the p a t t e r n s e l e c t i o n i s com-  ( R e f e r to Appendix I I c o n c e r n i n g  "PATTRN").  However, independent of the p a t t e r n used, some care must be taken to ensure t h a t c e l l s are n o t lie  i n adjacent  processed  areas  f i e l d s of view. o f the  field  counted twice  s i n c e p a r t s of them  A "dead" zone needs to be  left  of view as shown i n F i g u r e 4.3.2.  between This  39  A  B  »  >  C  D  Fig.  4.3.1  S t a n d a r d scan p a t t e r n s used i n the d i f f e r e n t i a l l e u k o c y t e count. The s t r a i g h t edge p a t t e r n ( A ) , the b a t t l e m e n t p a t t e r n ( B ) , the c r o s s - s e c t i o n a l p a t t e r n ( C ) , and the l o n g i t u d i n a l p a t t e r n (D).  "> o  •K "-V-i  Current Search Area  3f  Dead Zone 1 A d j a c e n t Search Aroa  Fig.  4.3.2  Placement of a d j a c e n t scan a r e a s .  40  "dead" zone s h o u l d i d e a l l y be e q u a l t o the s i z e of the l a r g e s t expected.  In the case o f white b l o o d c e l l s ,  gest type, w i t h s h o u l d a l s o be  a s i z e r a n g i n g from 13  area i s processed  and  the whole c e l l w i l l  of  m i s s i n g any  of view.  lar-  T h i s dead zone As  the  center  a c e l l i s l o c a t e d which o v e r l a p s onto the frame,  then  frame ( F i g u r e 4.3.3).  the monocyte i s the  to 19 microns.  c o n s i d e r e d to frame each f i e l d  cell  be v i s i b l e and p r o c e s s i n g can  I f these  c r o s s onto  c o n s i d e r a t i o n s are made, the  leukocytes i s very small  the  probability  indeed.  Dead Zone  y Current F i e l d of View  Fig.  4.3.3  R e l a t i o n s h i p between c u r r e n t and a d j a c e n t scan a r e a s , showing the placement of the dead zone.  41  5.  THE SOFTWARE  IMPLEMENTATION  Four g l o b a l r o u t i n e s , each h a v i n g been w r i t t e n t o c o n t r o l  a distinct  f u n c t i o n , have  the microscope and l o c a t e the l e u k o c y t e s .  r o u t i n e s have been w r i t t e n i n such a way t h a t they a r e l o g i c a l l y pendent o f m a g n i f i c a t i o n . is  a c h i e v e d by a l t e r i n g  the v a l u e s a s s i g n e d t o the s i x v a r i a b l e s desThe n e x t  chapter w i l l  was t e s t e d u s i n g such  techniques  as contour  5.1  d e s c r i b e how the system t r a c i n g and c u r v a t u r e measure-  operator.  "FOCUS" As  maximization.  a l r e a d y d i s c u s s e d , a u t o - f o c u s i n g i s accomplished A flow diagram o f the v a r i a n c e m a x i m i z a t i o n  c a l l e d "FOCUS", i s shown i n F i g u r e 5.1.1. is' a f u n c t i o n o f d i g i t a l  algorithm,  E x e c u t i o n time o f t h i s r o u t i n e  D i g i t a l f i l t e r i n g i s accomplished  by s t o r i n g the v a r i a n c e v a l u e s i n m a t r i x  form, and as the d i g i t a l  s i z e i n c r e a s e s , the l e n g t h o f t h i s m a t r i x a l s o i n c r e a s e s .  the p o i n t r e p r e s e n t e d by the c e n t r a l e n t r y i n the m a t r i x .  m a g n i f i c a t i o n i s i n c r e a s e d the depth o f f i e l d  decreases,  the number o f d i s c r e t e s t e p s d e f i n i n g a l o g i c a l  filter  The average  v a l u e o f t h i s m a t r i x i s c o n s i d e r e d t o r e p r e s e n t the f i l t e r e d  clockwise  by v a r i a n c e  f i l t e r window s i z e , m a g n i f i c a t i o n , and the  i n i t i a l p o s i t i o n o f the f o c u s c o n t r o l .  at  inde-  Compensation f o r any change i n m a g n i f i c a t i o n  c r i b e d i n Appendix I .  ment by an a r e a  These  variance As the  and t h e r e f o r e  clockwise or counter-  i n c r e m e n t a l focus adjustment d e c r e a s e s .  T h i s decrease  i n the  i n c r e m e n t a l d i s t a n c e t h a t the focus motor must be d r i v e n h e l p s t o speed up e x e c u t i o n .  T h i s i s the o n l y r o u t i n e t h a t executes  higher magnifications.  I f f u t u r e a p p l i c a t i o n permits  m a g n i f i c a t i o n , the focus motor s h o u l d be equipped to  p r o v i d e the p r o p e r  incremental step  size.  more q u i c k l y a t the use o f a s i n g l e  with different  gearing  42  I n i t i a l i z e VARIANCE matrix f o r d i g i t a l filtering. ( "|  Step fccus motor CW by FCCSTP.  [  Got nev variance.  X  Update and evaluate VARIANCE matrix.  Step CCW by TCCSTP and get new variance,  _  y  Update and evaluate VARIAUCE matrix.  Stop 2 X POCGT? CH _to_Varianco peak.  F i g . 5.1.1  Flow Diagram of the focusing program, "FOCUS"  43  Field of View  y  Fig.  5.1.2  The  variance  The scan  l i n e s used to sample the v a r i a n c e .  i s normally  shown i n F i g u r e 5.1.2.  sampled a c r o s s  The c r i t i c a l  d i g i t a l f i l t e r window s i z e ,  Scan Lines  the s e a r c h  area as  g l o b a l f a c t o r s a r e VARNUM, the  and FOCSTP, the number o f p h y s i c a l focus  s t e p s e q u i v a l e n t t o one l o g i c a l focus  increment i n t h i s  algorithm  c(Refer to Appendix I f o r nominal v a l u e s ) . 5.2  "THRESH" "THRESH" i s the r o u t i n e t h a t c a l c u l a t e s the t h r e s h o l d  of i n t e n s i t y plified  used t o s e g r e g a t e  from the background.  flow diagram o f THRESH i s shown i n F i g u r e 5.2.1.  l i n e across  t e s t e d t o see i f i t f a l l s o u t s i d e these  limits,  The average o f these  between p r e d e f i n e d  a message i s d i s p l a y e d a s k i n g  the d e v i a t i o n o f i n t e n s i t y  level  two p o i n t s i s then  limits.  e i t h e r i n c r e a s e o r decrease the i l l u m i n a t i o n .  A sim-  A s i n g l e scan  the s e a r c h a r e a i s sampled t o o b t a i n the i n t e n s i t y  of the two b r i g h t e s t p o i n t s .  is  leukocytes  level  During  I f the i n t e n s i t y the o p e r a t o r t o system  design,  due to changes i n specimen t h i c k n e s s  and  c o n c e n t r a t i o n o f s t a i n was u n d e r e s t i m a t e d , and t h e r e f o r e the system  Zero B l and D2. C=1000.  Sot  PCot tho i n t e n s i t y of . a p o i n t on tho l i n o Y - C.  Tont tho i n t e n s i t y of t h i s point.  Test i f the l i n e T = C h&s been completely scanned.  Average B l and B2 and t o s t s i z e of average.  Store negative o f average i n TLSTEL.  Display noasage i n t e n s i t y too bright f o r a timed interval.  D i s p l a y message i n t e n s i t y too d u l l f o r timed i n t e r v a l .  Sot C = 700. B l and B2.  Fig.  5.2.1  Zero  Flow diagram o f the t h r e s h o l d d e t e r m i n i n g program "THRESH"  45  c o u l d be improved i f THRESH had some means o f remotely i l l u m i n a t i o n when the i n t e n s i t y exceeds c e r t a i n l i m i t s .  c o n t r o l l i n g the However, i f the  average i n t e n s i t y i s a c c e p t a b l e , the t h r e s h o l d i s c a l c u l a t e d by m u l t i p l y i n g the average background i n t e n s i t y by the c o n t r a s t r a t i o , . CR.  The  n e g a t i v e o f t h i s v a l u e i s s t o r e d i n TLEVEL f o r g l o b a l a c c e s s . 5.3  "JOYSTK" "JOYSTK" i s the h a n d l e r  tem.  f o r the j o y s t i c k a t t a c h e d t o the. s y s -  Two modes o f j o y s t i c k c o n t r o l are a v a i l a b l e ,  or s l i d e p o s i t i o n  control with  focus c o n t r o l o n l y ,  dynamic a u t o - f o c u s i n g .  The mode i s s e l e c -  t e d by means o f a l a b e l l e d t w o - p o s i t i o n s w i t c h on the j o y s t i c k u n i t .  The  p r o p o r t i o n a l p o s i t i o n and d i r e c t i o n t h a t the j o y s t i c k i s moved c o n t r o l s the v e l o c i t y driven.  and d i r e c t i o n  that either  the stage o r focus motor i s  The j o y s t i c k h a n d l e r must be i n i t i a l i z e d b e f o r e each use by  c a l l i n g JOYINT.  A f t e r i n i t i a l i z a t i o n , JOYSTK s h o u l d be c a l l e d  from a program loop as l o n g as c o n t r o l i s d e s i r e d . which JOYSTK i s c a l l e d w i l l  affect  the response  repeatedly  The frequency  with  speed o f the j o y s t i c k  action. 5.4  "STRPNT" "STRPNT" i s the r o u t i n e t h a t a c t u a l l y l o c a t e s the l e u k o c y t e s .  As each l e u k o c y t e i s found, l e u k o c y t e are passed interprets  these  c o o r d i n a t e s d e f i n i n g the l o c a t i o n o f the  t o an e x t e r n a l s u b r o u t i n e  c a l l e d CNTOUR.  CNTOUR  c o o r d i n a t e s as d e f i n i n g the l o c a t i o n o f a c e l l , and  uses the c o o r d i n a t e s  to contour  t r a c e and c l a s s i f y  the c e l l .  p o i n t o f view o f CNTOUR, STRPNT merely produces a data stream  From the of c e l l  coordinates. STRPNT b e g i n s  s e a r c h i n g a new f i e l d o f view i n the bottom  46  rinHl«.iir.o pcati and jstn^a control p&rnImotors.  ,  :T::Z...  _J I n i l i n l l i o couutor to j j c a l l auto-focus,  :L. :.:z.  C a l l TOCU3 and THRESH 1 Get point i n t e n s i t y ar.d incrcrant scan coordinates by AGS.  j Perform AGS -"] minimum square | teat,  Tost i f current f i o l d ' of view baa been scanned ^completely,  183  Move to c e l l boundary by GS increments.  ~  J.  C a l l CHIOUB t o process the c o l l . Two returns are p o s s i b l e  1  Noroal\ Roturu \ Access* P/:TTRH andmovo Btage t o noxt f i e l d of viev.  Error^ Return  Store c e l l coordinates Bo t h i s c e l l can ba i d e n t i f i e d as found.  •  T o s t - i f tho end o f PATTRIJ has been roached.  Incrcmont c o l l count and t e s t i f enough c o l l s have been found.  To a Done?  Increment focus count and t o s t I f timo t o auto-focus•  i g . 5.4.1  Flow diagram of "STRPNT"  [Normal\ Exit  47  l e f t hand corner, t e s t i n g one row at a time (Figure 5.4.2A).  Sequential  points are spaced apart by an incremental step size c a l l e d AGS.  When  a point i s found that f a l l s below the threshold, an area test i s made where a minimum square of size AGS i s used.  I f a l l four corners of  this square f a l l below the threshold, the edge of the object i s searched for.  The edge i s detected by stepping l e f t with a step size GS u n t i l  the f i r s t point above the threshold i s found (Figure 5.4.2B).  GS i s  assumed to be the incremental step size to be used by CNTOUR i n the c l a s s i f i c a t i o n work.  The coordinates of the edge point are stored i n  A Fig.  5.4.2  The s e q u e n t i a l s e a r c h technique used t o l o c a t e l e u k o c y t e s , demonstrating the minimum square f i t (A) o f s i z e AGS, and the l o c a t i o n o f the s t a r t i n g p o i n t (B) by u s i n g GS.  SXD2 and SYD2 and the s u b r o u t i n e these  coordinates  If  the c o o r d i n a t e s  of  view f o r o t h e r  A f t e r the f i e l d  B  and STRPNT w i l l are accepted, cells,  CNTOUR i s c a l l e d . verify  CNTOUR may  reject  the v a l i d i t y o f SXD2 and SYD2.  STRPNT w i l l  continue  searching  n o t i n g the p o s i t i o n o f the c e l l  o f view has been s e a r c h e d ,  already  the f i e l d found.  STRPNT moves the s l i d e a c -  c o r d i n g to the d i r e c t i o n s s t o r e d i n PATTRN ( R e f e r t o Appendix I I ) which d e s c r i b e how the stage  s h o u l d be moved.  48  STRPNT a l s o c a l l s FOCUS to m a i n t a i n  the image i n f o c u s .  I d e a l l y , FOCUS s h o u l d be c a l l e d each time the f i e l d s i n c e t h e r e i s no assurance the s t a g e i s moved.  t h a t the image w i l l s t i l l be i n focus  However, i t was found  focus when e i t h e r one o f two events cyte was found and t h i s  o f view i s changed,  field  t h a t i t was a c c e p t a b l e t o  occurred:  when a s u s p e c t e d  times  leuko-  o f view had not been f o c u s e d , o r when  f o c u s i n g had n o t o c c u r r e d f o r a f i x e d number o f stage moves. of  after  The number  t h a t the s t a g e can be moved b e f o r e f o c u s i n g i s needed i s depen-  dent on m a g n i f i c a t i o n , and as m a g n i f i c a t i o n i n c r e a s e s , f o c u s i n g must o c c u r more o f t e n . STRPNT has two e x i t s t o the c a l l i n g program. normal one, o c c u r s when the s p e c i f i e d exit  cell  count  One e x i t , the  i s reached.  The o t h e r  o c c u r s when the scan p a t t e r n s t o r e d i n PATTRN i s exhausted  the c e l l  count  i s reached.  before  49  6.  6.1  The  Test  System  Auto-focusing of the system. t a i n e d , and  SYSTEM EVALUATION  was  s u b j e c t i v e l y evaluated  from the  S e v e r a l i n d i v i d u a l o p i n i o n s o f image q u a l i t y were  c o r r e l a t i o n between peak v a r i a n c e  and  e s t a b l i s h e d under a wide range o f c i r c u m s t a n c e s . improve the focus o n l y v e r i f i e d the b e s t  apart  image q u a l i t y  I f the c o n s i d e r a t i o n s d i s c u s s e d i n an  efficeint  focusing.  A complete s o f t w a r e II was  was  t h a t v a r i a n c e m a x i m i z a t i o n produced  average image q u a l i t y .  c o n s i s t e n t means o f  ob-  Attempts to manually  Chapter 3 are implemented, v a r i a n c e m a x i m i z a t i o n p r o v i d e s and  rest  package m o d e l l e d a f t e r  w r i t t e n to t e s t the e f f i c i e n c y o f the system.  the one  i n Appendix  T h i s package  o f f e r e d s e v e r a l d i s t i n c t modes as l i s t e d below:  The  the parameters l i s t e d i n Appendix I on the b a s i s o f the m a g n i f i c a t i o n used.  1.  Initialization - Initializes  2.  Scan L i n e D i s p l a y - D i s p l a y s the i n t e n s i t y l e v e l s encount e r e d on a s i n g l e scan l i n e i n the X direction. The Y c o o r d i n a t e i s c o n t r o l l e d through the t e l e t y p e keyboard.  3.  Threshold  4.  J o y s t i c k C o n t r o l - A c t i v a t e s c o n t i n u a l c o n t r o l of focus and stage p o s i t i o n through the j o y s t i c k .  5.  Contour T r a c i n g and Curvature Measurement - L o c a t e s a s p e c i f i e d number of l e u k o c y t e s , and contour t r a c e s t h e i r n u c l e i . The contour t r a c e s and c u r v a t u r e f u n c t i o n s may be d i s p l a y e d f o r viewing.  I n t e n s i t y Map - D i s p l a y s a l l the p o i n t s i n the f i e l d of view t h a t l i e e i t h e r above or below the t h r e s h o l d . T h i s mode i s used to p i c k the c o n t r a s t r a t i o , CR, which i s used to d e f i n e t h e • t h r e s h o l d .  l a s t mode i s the most important  one  f o r determining  the  efficienty  50  of the system.  The  remainder of t h i s  ques used f o r contour t r a c i n g and cuss the r e l a t i v e speed one 6.2  Contour  c e n t r o i d of any  The  dots d e s i g n a t e  these  dis-  techniques.  the t r a c i n g a l -  A boundary p o i n t i s d e f i n e d as the a r r a y p o i n t s  d e f i n e d as GS  lying  forming a minimum square,  i n t h i s context.  The  four  the a r r a y p o i n t s i n the minimum square c o n f i g u r a t i o n .  two,  o r t h r e e o f the  threshold i n t e n s i t y .  t h r e s h o l d , the 6.2.1  techni-  c e n t r o i d of the minimum square i s d e f i n e d as a boundary p o i n t  whenever one, the  the  measurement, and w i l l  conducted o n - l i n e u s i n g  four of  the s i d e of the square b e i n g black  describe  Tracing  g o r i t h m shown i n F i g u r e 6.2.1. the  curvature  will  can expect u s i n g  Contour t r a c i n g was  at  chapter  o f the square f a l l below  I f none o r a l l o f the p o i n t s f a l l below  c e n t r o i d i s not  i n d i c a t e the  four corners  a boundary p o i n t .  The  the  arrows i n F i g u r e  f o u r p o s s i b l e d i r e c t i o n s i n which the next boundary  3  O . A  D3  2  O -< D2  e DA  ©  « O  > o /, OD1  O  1  Fig.  6.2.1  p o i n t i s sought.  The c o n t o u r t r a c i n g a l g o r i t h m . The b l a c k dots are spaced at GS and r e p r e s e n t the f o u r p o i n t s t h a t are i n t e r r o g a t e d to determine which d i r e c t i o n (the c i r c l e s ) the o p e r a t o r s h o u l d be moved.  For a counterclockwise  i n t e r r o g a t e d i n a counterclockwise rogated  order.  t r a c e , the The  first  f o u r d i r e c t i o n s are direction inter-  depends upon which o f the minimum square p o i n t s are below  the  51  threshold.  O t h e r t r a c i n g a l g o r i t h m s may  T h i s t r a c i n g a l g o r i t h m was tor 6.3  found  d e s c r i b e d i n (15).  implemented so t h a t the d i s c r e t e a r e a  opera-  d e s c r i b e d i n the next s e c t i o n c o u l d be used to measure c u r v a t u r e . The  D i s c r e t e Area The  Operator  d i s c r e t e a r e a o p e r a t o r shown i n F i g u r e 6.3.1  measure boundary c u r v a t u r e . dary p o i n t as determined of  be  The  by  the  was  used to  area o p e r a t o r i s c e n t e r e d over a bouncontour  tracing algorithm.  The c e n t r o i d  the a r e a o p e r a t o r assumes a v a l u e o f +386, and each a r r a y p o i n t h a v i n g  an i n t e n s i t y below the t h r e s h o l d assumes the v a l u e as shown i n F i g u r e 6.3.1.  A l l o t h e r a r r a y p o i n t s assume a zero v a l u e .  v a l u e s o f the a r r a y p o i n t s and o  o  -3  -7  -7  -3  o  o  o  o  o  o  o  c  -37  •-37  -17  o  a  o  o  o  o.  o  o  o  o  o  o  -7  -37  o  o  -3  -17  o  6  at  -81 •-81 x •»386 o o -81 -81 o  o  o  o  -37 •-37  -3  -7  -7  curvature  -3  -37  -7  -37  -7  -17  -3 -1  -3  The d i s c r e t e a r e a o p e r a t o r used to e x t r a c t boundary c u r v a t u r e .  the boundary p o i n t .  The  values associated with  f o l l o w a Gaussian w e i g h t e d p r o f i l e modelled  f u n c t i o n can be generated  contour has been t r a c e d .  At  the a r r a y p o i n t s  a f t e r the p h y s i o l o g y o f  the c o n c e n t r i c r e c e p t i v e f i e l d s o f a c a t ' s eye A curvature  the  -1  -17-  -1  6.3.1  o„  o  o  Fig.  o  -3  -37  t o t a l of  c e n t r o i d g i v e a measure o f the  -1  -7  The  (15). e f f i c i e n t l y once the  the f i r s t boundary p o i n t , a l l 36 p o i n t s o f  the a r e a o p e r a t o r are i n t e r r o g a t e d and  s t o r e d i n m a t r i x form.  Further  Fig. 6.3.2  Photographs of several cells, their contour traces, and unfiltered curvature functions as extracted by the area operator.  boundary p o i n t s i n v o l v e the i n t e r r o g a t i o n of o n l y s i x p o i n t s , which represent  a new  row  these new  points represent  the m a t r i x must be  o r column of the m a t r i x .  cells.  t i o n of leukocytes  c u r v a t u r e has  t r a c e s and  c l a s s i f i c a t i o n of leukocytes still  remains to be  curvature  been shown to be  and  functions of  an  effective  (1,5,8,12).  completed w i t h  purpose o f implementing o n - l i n e contour  Classifica-  t h i s system.  curvature  been to demonstrate t h a t such o n - l i n e p r o c e s s i n g a b l e and  o r column t h a t  depends on which d i r e c t i o n the c e n t r o i d o f  shows contour  Nuclear  parameter i n the  row  s h i f t e d i n o r d e r to l i e on the next boundary p o i n t .  F i g u r e 6.3.2 several  The  techniques  techniques  e f f i c i e n t means o f automating the d i f f e r e n t i a l  are  The has  suit-  leukocyte  count. 6.4  Reliability  and E x e c u t i o n  Time Measurements  S e v e r a l performance t e s t s over a c e r t a i n number of  cells  were conducted to e x t r a c t a mean measure o f the system performance. These t e s t s i n v o l v e d l o c a t i n g o r i s o l a t i n g a s p e c i f i e d number o f contour  t r a c i n g these The  bility  first  cells,  t e s t was  and  generating  designed  t h e i r curvature  was  chosen t h a t had  was  then arranged  a t r u e count of 100  t h a t would scan  the number o f c e l l s  m i s s e d and why.  p i c k e d up.  T h i s may  A scan  relia-  pattern  on i t . A program  t h i s p a t t e r n ten times, p r i n t i n g traced.  This  t e s t was  t y p i c a l r e s u l t s are shown i n Table 6.4.1.  3 o b v i o u s l y missed a c e l l , but c e l l was  leukocytes  a c t u a l l y i s o l a t e d and  t e d s e v e r a l times and  functions.  to o b t a i n a measure of the  of c o r r e c t l y i s o l a t i n g or l o c a t i n g leukocytes.  cells,  i t was  impossible  out  conducPass  to determine which  Pass 8 i n d i c a t e d an e x t r a c e l l had  have been a l a r g e p l a t e l e t and  been  a more s t r i n g e n t  54  Pass §  True C e l l Count  As Counted  1  100  100  2  100  100  3  100  A  100  99 100  5 6  100  100  100  100  7  100  100  8  100  101  9 10  100  100  100  100  T a b l e 6.4.1  Error Count  -1  +1  R e s u l t s o f a t e s t designed to count l e u k o c y t e s a c r s o s an a r e a w i t h a t r u e count of 100 c e l l s .  area c r i t e r i o n could probably e l i m i n a t e this e r r o r . count from t h i s t e s t i s 100  c e l l s , and  The mean c e l l  the s t a n d a r d d e v i a t i o n i s +  .11  cells. The  second  t e s t was  d e s i g n e d to g i v e an a b s o l u t e measure o f  the time r e q u i r e d to i s o l a t e and p r o c e s s 100 shown i n F i g u r e 6.4.1  was  used i n t h i s  g e n e r a l l y l o n g enough t h a t 100  Fig.  the p a t t e r n was j u s t be be  6.4.1  c e l l s would be  The  A scan p a t t e r n as  scan p a t t e r n was  found b e f o r e the end o f  The scan p a t t e r n used to measure the time r e q u i r e d to l o c a t e and p r o c e s s 100 c e l l s .  reached.  repeated.  test.  cells.  I f t h i s was  At the end o f a 100  r e t u r n e d t o the i n i t i a l  position.  not  the case, the p a t t e r n would  c e l l p a s s , the s l i d e would always Ten s l i d e s were s e l e c t e d a t  Tino i n Seconds f o r 100 C o l l Scan © M a g n i f i c a t i o n Below  Slide Nuiabor  1  A  B  C  A+3+C  D  S  F  160  167  153  162  160  153  163  G  E  I  160  205  193  194  199  128  135  132  132  247  247  D+E+F  G-i-H-s-I  2  105'  103  107  105  130  123  127  123  3  183  180  185  133  194  195  193  196  250  244  4  101  100  101  101  120  117  121  119  167  162  163  164  5  120  116  116  117  124  125  130  126  194  196  195  195  6  265  269  272  269  283  290  286  239  306  311  309  309  7  99  100  100  1C0  113  115  114  115  125  132  123  127  8  120  123  126  123  130  129  129  129  171  169  167  169  9  230  226  223  228  259  261  260  260  296  299  299  293  10  140  139  143  141  148  150  147  I43  175  179  181  179  Average T i m s  Table  100 X 16 X 1 . 2 5  40 X 16 X 2  40 X 16 X 1 . 6  6.4.2  153  Average T i a a  Times r e q u i r e d t o l o c a t e and process blood f i l m s .  167  100 l e u k o c y t e s  Average T i n e  202  on.ten randomly s e l e c t e d  56  random and the test performed across the same area of each s l i d e three times at three d i f f e r e n t magnifications. summarized i n Table 6.4.2.  The results of this test are  The contrast ratio for each s l i d e was  chosen as described at the end of Section  4.2.  The results of this second test are most interesting.  Notice  that the mean time required to complete the test increased i^ith magnif i c a t i o n as expected.  However, the increase i n time i s not very large.  As magnification i s doubled, the probability of locating a leukocyte i n any one p a r t i c u l a r f i e l d of view i s halved.  Supposedly this should  cause a doubling of the time required to run the test.  This does not  occur since focusing takes less time at the higher magnifications. Focusing takes longer at the lower magnifications because the variance function i s stretched over the h o r i z o n t a l axis and peak detection requires that the focusing motor be driven greater distances.  I f the  gearing of the focus motor were changed for each magnification such that the step s i z e of the focus motor would be equivalent to the number of incremental steps required, the mean time for this test at the lower magnifications would decrease.  Therefore once the required magnifica-  t i o n i s determined, i t w i l l be advantageous to t a i l o r  the focus control  hardware to this magnification. The d i s t r i b u t i o n of the times recorded for the second test i s not Gaussian, and therefore i t i s d i f f i c u l t of the deviation of the r e s u l t s . + 5 7.95  Therefore  magnifications i n d i c a t i n g  the meaning  However, at 1024X the deviation i s  seconds, at 1280X i t i s + 61.85  + 64.20 seconds.  to interpret  seconds, and at 2000X i t i s  the deviation also increases for higher  that there i s a two-dimensional spread as  magnification i s increased.  57  Some measure o f s u c c e s s o f t h i s system d e s i g n can be o b t a i n e d by comparing t h e r e s u l t s o f t h i s  t e s t to the time a t e c h n i c a i n r e q u i r e s  to p e r f o r m a d i f f e r e n t i a l l e u k o c y t e c o u n t .  A t e c h n i c i a n r e q u i r e s from  f i v e t o t e n minutes t o p e r f o r m a 100 c e l l d i f f e r e n t i a l l e u k o c y t e c o u n t . The  times f o r the second t e s t do n o t i n c l u d e complete  classification  of t h e c e l l s , a l t h o u g h c u r v a t u r e f u n c t i o n s f o r t h e n u c l e i o f the c e l l s have been e x t r a c t e d .  Keeping  t h i s i n mind, i t i s c o n s e r v a t i v e l y  e s t i m a t e d t h a t one t e n t h o f a second p e r c e l l s h o u l d be s u f f i c i e n t t o complete the c l a s s i f i c a t i o n .  Based on t h i s a s s u m p t i o n , t h e system  d e v e l o p e d i s a t l e a s t t w i c e as f a s t as the t e c h n i c i a n p e r f o r m i n g the same t a s k .  F u r t h e r advantages a r e a l s o o b v i o u s when i t i s c o n s i d e r e d  t h a t the t e c h n i c i a n n o r m a l l y cannot work a t the m i c r o s c o p e f o r more than an h o u r a t a time due t o eye s t r a i n .  Based on t h i s second  test  a l o n e , t h e t e c h n i q u e s used i n t h i s s y s t e m a r e c o m p a r a t i v e l y e f f i c i e n t .  4  58  7.  The and  system p r o v i d e s  i s adaptable  to v a r i o u s  system was d e s i g n e d  The  g e n e r a l purpose c o n t r o l o f a microscope  types o f o n - l i n e image p r o c e s s i n g .  t o develop e f f i c i e n t  mate the d i f f e r e n t i a l to p e r f o r m o t h e r  CONCLUSIONS  count.  types  logical  techniques  algorithms  table.  to eventually  The system can be e q u a l l y w e l l  auto-  configured  o f b i o l o g i c a l image p r o c e s s i n g . algorithms  forcontrolling  the f u n c t i o n s o f the  microscope have i n themselves worked s u c c e s s f u l l y . these  The  i n their interaction with  The e f f i c i e n c y o f  the hardware has been a c c e p -  By t i g h t e n i n g the s p e c i f i c a t i o n s o f the hardware and by t a i l o r i n g  the hardware to a s p e c i f i c m a g n i f i c a t i o n , f u r t h e r gains can be made i n system e f f i c i e n c y . this  c o u l d be done.  I t i s w o r t h w h i l e t o review some o f the ways i n which The s e t t l i n g  time o f the D/A c o n v e r t e r s  and a m p l i -  f i e r s i n t h e d e f l e c t i o n c i r c u i t r y o f the image d i s s e c t o r can be improved.  C u r r e n t l y 50 microseconds p l u s the A/D c o n v e r s i o n  to i n t e r r o g a t e a p o i n t .  time i s r e q u i r e d  I t s h o u l d be p o s s i b l e to reduce t h i s time to  25 microseconds as the d i s s e c t o r r e q u i r e s a d w e l l time o f o n l y 11 m i c r o seconds t o m a i n t a i n grey  a s i g n a l t o n o i s e r a t i o o f 64 r e p r e s e n t i n g  level resolution.  With t h i s  improvement, a 20% to 35% r e d u c t i o n  i n the times o f the second t e s t i n Chapter 6 c o u l d be e x p e c t e d 6.4.2).  five-bit  (Table  Another s u b s t a n t i a l time s a v i n g can be made by g e a r i n g the  f o c u s i n g motor to a s p e c i f i c m a g n i f i c a t i o n .  Focusing  time c o u l d be  reduced by as much as 15% t o 25% a t the lower m a g n i f i c a t i o n s .  This  would imply  test  about a 10% r e d u c t i o n i n the r e s u l t s o f the second  i n Chapter 6. Currently blood  the wide t o l e r a n c e s found i n the q u a l i t y o f the  f i l m s has f o r c e d u p d a t i n g  o f the c o n t r a s t r a t i o  f o r each new s l i d e  59  processed.  T h i s method would be  are a u t o m a t i c a l l y  i m p r a c t i c a l i n a system where the  i n s e r t e d under the microscope.  The  f i l m s produced from an a u t o m a t i c b l o o d s p r e a d e r and investigated.  of a f i x e d contrast r a t i o t h i s i s not  each new of a  t r u e , the  c o n t r a s t r a t i o w i l l have to be i s no  the  q u i t e u n i f o r m , and  can be e x p e c t e d to work w e l l .  s l i d e s i n c e there  means o f g u a r a n t e e i n g  be  trans-  the  use  Alternatively updated f o r the q u a l i t y  slide. I t would be  advantageous i n a p r a c t i c a l system to  some means o f d i g i t a l l y a s m a l l range. tolerable trast  f i l m s s h o u l d be  blood  s t a i n e r should  In such an a u t o m a t i c a l l y c o n t r o l l e d p r o c e s s ,  m i t t a n c e p r o p e r t i e s o f the  if  q u a l i t y of  slides  c o n t r o l l i n g the i n t e n s i t y o f i l l u m i n a t i o n w i t h i n  Presently  a variance  i n image i n t e n s i t y of 12.5%  though s o f t w a r e compensation and  r a t i o method o f t h r e s h o l d One  the f l e x i b i l i t y o f the  c l a s s i f i c a t i o n s h o u l d be  is possible.  lower power l e n s e s  r e q u i r e d to  t h a t a decrease  c o s t l e s s , microscope adjustments such as c r i t i c a l ,  execution  v i b r a t i o n and  image as much.  classify  A decrease i n m a g n i f i c a t i o n o f f e r s s e v e r a l  as condenser placement are not speed i n c r e a s e s , and  From the e x p e r i e n c e  acquired  the depth o f f i e l d shock do not  increases,  disturb  of 1280X i s e x p e c t e d to be  t r a d e o f f s mentioned above.  the  from contour t r a c i n g and  measurement, the l e n s arrangement of 40X2X16 p r o d u c i n g  magnification  this  to determine the minimum  A decrease i n r e q u i r e d r e s o l u t i o n i m p l i e s  in magnification  curvature  con-  determination.  s p a t i a l r e s o l u t i o n o r amount o f i n f o r m a t i o n  advantages:  is  of the major o b j e c t i v e s o f the f u t u r e a p p l i c a t i o n o f  system to l e u k o c y t e  leukocytes.  provide  a  a good compromise between  the  60  APPENDIX I  The way  t h a t they  programs to c o n t r o l are l o g i c a l l y  the microscope are w r i t t e n i n such  independent o f m a g n i f i c a t i o n .  It  a  was  p o s s i b l e to compensate f o r the a f f e c t s o f m a g n i f i c a t i o n by d e f i n i n g s i x v a r i a b l e s which assume d i f f e r e n t v a l u e s  for different magnifications.  These v a r i a b l e s are d e s c r i b e d below. GS - The  i n c r e m e n t a l s t e p s i z e f o r the X and Y c o o r d i n a t e s o f  the image d i s s e c t o r to be used when p e r f o r m i n g r e c o g n i t i o n work on the c e l l s . GS = 4 r e p r e s e n t s the s t e p s i z e f o r maximum r e s o l u t i o n . AGS  - The i n c r e m e n t a l s t e p s i z e f o r the X and Y c o o r d i n a t e s o f the image d i s s e c t o r to be used when s e a r c h i n g f o r c e l l s . G e n e r a l l y AGS i s chosen by d e t e r m i n i n g the s i z e of the minimum square w i t h s i d e AGS t h a t w i l l f i t on a l l l e u k o c y t e s at the m a g n i f i c a t i o n i n use. AGS commonly becomes two to s i x times GS.  Variable  Fover of Magnification 102/+X  1280X  2000X  (40X16X1.6)  (40X16X2)  (100X16X1.25)  GS  4  4  4  AGS  20  20  30  CR  16  20  22  STPEQ  6  5  3  VARNUM  .5-  5  5  -3  -1  FOCSTP  Table A.1.1  -4  The optimum v a l u e s f o r the s i x m a g n i f i c a t i o n dependent parameters.  CR - A s i x - b i t binary, number c o n s i d e r e d to be a f r a c t i o n . The average background i n t e n s i t y i s m u l t i p l i e d by t h i s f r a c t i o n to produce the t h r e s h o l d used t o l o c a t e o r i s o l a t e the l e u k o c y t e s . STPEQ - The number of 10 micron s t e p s n e c e s s a r y to p o s i t i o n the stage on the n e x t f i e l d of view. For example, STPEQ = 6 w i l l cause the s t a g e to move 60 microns  61  each time  the f i e l d o f view i s changed.  VARNUM - The number o f d i s c r e t e v a r i a n c e v a l u e s t h a t f a l l under the window o f the d i g i t a l f i l t e r when a u t o - f o c u s i n g . FOCSTP - The n e g a t i v e o f the number o f s t e p s the f i n e focus motor w i l l be advanced each time the a u t o - f o c u s i n g algorithm r e q u i r e s the focus c o n t r o l to be l o g i c a l l y moved. The n o m i n a l v a l u e s  f o r each o f these v a r i a b l e s a r e l i s t e d  A.1.1 a c c o r d i n g to m a g n i f i c a t i o n .  i n Table  62  APPENDIX I I  Four s u b r o u t i n e s have been w r i t t e n t o c o n t r o l the microscope and  l o c a t e the l e u k o c y t e s .  These programs may be arranged  system as shown i n F i g u r e A.2.1 although Besides  i n a complete  o t h e r arrangements are p o s s i b l e .  the m a g n i f i c a t i o n parameters d e f i n e d i n Appendix I , o t h e r  a b l e s are i n v o l v e d i n s u b r o u t i n e  communication.  vari-  These v a r i a b l e s a r e  d e s c r i b e d below. TLEVEL - A n e g a t i v e number r e p r e s e n t i n g the t h r e s h o l d used to l o c a t e l e u k o c y t e s . SXD2, SYD2 - A b s o l u t e c o o r d i n a t e s f o r the image d i s s e c t o r d e f i n i n g the l o a t i o n o f the n u c l e u s o f a l e u c o cyte. The next p o i n t to the r i g h t a t an i n c r e ment o f GS l i e c w i t h i n the c e l l n u c l e u s . WCNT - The t o t a l number o f c e l l s  to be found.  VARI - The v a l u e o f the f i l t e r e d v a r i a n c e f u n c t i o n a t the current focus.setting. Three s u b r o u t i n e s  must be p r o v i d e d by the user w i t h the  following functions. DELAY - P r o v i d e s the d e l a y time r e q u i r e d f o r the d i s s e c t o r to s e t t l e b e f o r e the i n t e n s i t y o f a p o i n t i s i n t e r rogated. CNTOUR - I t i s assumed t h a t t h i s user w r i t t e n s u b r o u t i n e w i l l c l a s s i f y the c e l l a f t e r the l o c a t i o n c o o r d i n a t e s SXD2 and SYD2 a r e passed to i t . CRT - A c o l l e c t i o n o f s u b r o u t i n e s r e s p o n s i b l e f o r d i s p l a y i n g messages i n v a r i o u s forms and s t r u c t u r e on the Type 30D d i s p l a y u n i t a t t a c h e d t o the PDP-9. Any type o f message h a n d l i n g can be s u b s t i t u t e d for this device. T a b l e A.2.1 summarizes the i n t e r n a l and e x t e r n a l r e f e r e n c e s to the g l o b a l symbols and s u b r o u t i n e s . fined.  "X" i n d i c a t e s i n t e r n a l l y de-  "A" i n d i c a t e s e x t e r n a l access n e c e s s a r y .  access probable.  This table should  "(A)" indicates external  a s s i s t i n u s i n g the system programs  63  USER'S PROGRAM - I n i t i a l i z a t i o n and C o n t r o l Tho s i x m a g n i f i c a t i o n dopondont v a r i n b l o 3 f o r tho SYSTEM Programs Euat bo i n i t i a l i z e d before Rny SYSTEM Program i s c o l l e d .  •4*  Lce Jo  P. r  "1  3  JOYIHT I n i t i a l i z e s JOYSTK. C a l l onco before JOYSTK i o usod.  ••8-' Roto  SYSTEM PROORAMS  G  q Hi  . JCYSTK Provided nanual c o n t r o l of microscope through the j o y s t i c k . Call r e c u r s i v o l y froQ Dome program l o o p t o maintain c o n t r o l .  O  P  o  "0 u  3  -g  .q r-l  p.  o H  M -l>  o5 <S 3  STRPNT Searches f o r s p e c i f i e d number of c o l l s . Moves s t a g e , c a l l s FOCUS and THRESH, Passe3 c a l l coordinates to soisa user subroutine c a l l e d CNTOUR vhere r e c o g n i t i o n work takes p l a c e ,  T  FOCUS Automatically  focuse3  tho microscope on the c o l l s i n the c u r r e n t f i e l d of view.  THRESH Datonnino3 the t h r e s h o l d f o r the nucleus of the leukocyte. Monitors intensity level. Displays messogo i f i n t e n s i t y exceeds c e r t a i n l i m i t s .  USER'S PROGRAMS  CRT Device handler f o r the Type 30D CRT d i s p l a y unit. (This h a n d l e r has boon w r i t t e n )  »  THRESH and FOCUS may  Fig.  A.2.1  CNTOUR C a l l e d by STRPUT. Here i s vhero user w r i t e 3 r e c o g n i t i o n r o u t i n e s f o r the l e u k o c y t e s . There are tvo r e t u r n s t o STRPNT: 1. C o l l coordinates okay. 2. C o l l coordinates bad.  a l s o bo c a l l e d by the User.  A b l o c k diagram demonstrating a p o s s i b l e arrangement of the system programs.  64  Variable Name  Hame of Pro gran Where Variables are Defined e Monitor  GS  X  AGS  X  ca  X  JOISTK  FCCUS  STRFNT  THRESH  A  A  CNTOUR*  A  (A)  A  (A)  A  (A)  A  TLEVEL  X  STPEQ  X  FOCSTP  X  A  VAHNUM  X  A  A  SXD2  X  A  STD2  X  A  WCNT  X  A  VARI  X  Subroutine Namo DELAY.  X  JOYSTK  A  A  A  •JCYINT  A  X  FOCUS  (A)  A'  A  VAR  X  THRESH  (A)  STRPNT  A  X  A X  CNTOUR  A  CRT  *  (A).  X  X  A .  (A)  (A)  User written prograns,  «» CRT message display routines.  Table  A.2.1  This handler i s available.  I n t e r n a l and e x t e r n a l r e f e r e n c e s symbols  and s u b r o u t i n e s .  "X"  internally  d e f i n e d , "A" i n d i c a t e s  necessary,  and " ( A ) " i n d i c a t e s  probable.  to g l o b a l  indicates external  external  access  access  65  and  i n understanding The  i n "PATTRN".  the program  pattern  that  i s used t o move the s t a g e about i s s t o r e d  STRPNT a c c e s s e s  s h o u l d be moved.  this table  The s t r u c t u r e  changing the e n t r i e s  listings.  t o determine how the stage  o f the p a t t e r n may be changed by  i n PATTRN.  The f o l l o w i n g  structure  i s used f o r  PATTRN: PATTRN  IOT COUNT IOT COUNT ooo 0  "IOT"  i s one o f t h e f o u r i n s t r u c t i o n s  possible be  directions,  to move the stage i n the f o u r  and "COUNT" i s the number o f 10 micron s t e p s t o  taken i n the d i r e c t i o n  o f the IOT.  F o r example, t o completely  scan a r e c t a n g u l a r p a t t e r n o f s i z e 100 microns by 2000 microns a t ' t h e top l e f t hand c o r n e r , PATTRN would appear as PATTRN  SSXP  starting  follows:  / +X d i r e c t i o n  200  SSYM  / -Y d i r e c t i o n  10  SSXM  / -X d i r e c t i o n  200  SSYP  / +Y  direction  10  0  / Buffer  There i s no l o g i c a l  restriction  be  the end o f the i n s t r u c t i o n  used to i n d i c a t e  terminator  on the s i z e o f PATTRN. _ A zero must buffer.  66  APPENDIX I I I  The  IOT i n s t r u c t i o n s  f o r the s p e c i a l hardware a r e l i s t e d  below.  Mnemonic  Code  Description  STCL  707042  Start  remote scan o f d i s s e c t o r .  SPCL  707021  Stop remote scan o f d i s s e c t o r .  LDX  707002  Load X c o o r d i n a t e o f d i s s e c t o r accumulator (10 b i t s ) .  from the  LDY  707022  Load Y c o o r d i n a t e o f d i s s e c t o r  from the  accumulator  (10 b i t s ) .  ADCV  707041  S t a r t A/D c o n v e r s i o n on d i s s e c t o r  ADSF  707061  Skip i f d i s s e c t o r  ADRB  707076  Read r e s u l t o f the d i s s e c t o r A/D c o n v e r s i o n i n t o the accumulator (6 b i t s ) .  SSXP  707202  Step  A/D c o n v e r s i o n i s done.  the s t a g e i n the X p o s i t i v e  (10 micron  signal.  direction  step).  SSXM  707241  Step  the stage i n t h e X n e g a t i v e  direction.  SSYP  707204  Step  the stage i n the Y p o s i t i v e  direction.  SSYM  707242  Step  the s t a g e i n the Y n e g a t i v e  direction.  SSKP  707201  Skip i f the scanning  FCW  707264  Step  the focus motor c l o c k w i s e .  FCCW  707262  Step  the focus motor c o u n t e r c l o c k w i s e .  FSKP  707261  Skip i f the focus motor s t e p i s done.  LMUX  707244  Load j o y s t i c k m u l t i p l e x e r channel accumulator  stage step i s done.  from the  (3 b i t s - 8 channels) .  JSTR  707224  S t a r t A/D c o n v e r s i o n o f j o y s t i c k  signal.  JDNE  707221  Skip i f j o y s t i c k A/D c o n v e r s i o n i s done.  JGET  707222  Read r e s u l t o f j o y s t i c k A/D c o n v e r s i o n the accumulator (6 b i t s ) .  into  67  REFERENCES  1.  Young, Ian T., "The C l a s s i f i c a t i o n o f White B l o o d C e l l s " , IEEE Transactions on B i o m e d i c a l E n g i n e e r i n g , V o l . BME-19, No. 4, pp. 291-298, J u l y , 1972.  2.  Saunders, A l e x M., "Development o f Automation o f D i f f e r e n t i a l Counts by use o f C y t o c h e m i s t r y " , C l i n i c a l Chemistry, V o l . 18, No. 8, pp. 783-788, August, 1972.  3.  D a c i e , J . C , Lewis, S.M., P r a c t i c a l Haematology, J . & A. C h u r c h i l l L t d . , London, 1968.  4.  Young, Ian T., "Automatic Leukocyte R e c o g n i t i o n " , Automated C e l l I d e n t i f i c a t i o n and C e l l S o r t i n g , Academic P r e s s , New York, 1970.  5.  Weid, George L., Bahr, Gunther F., B a r t e l s , P e t e r H., "Automatic A n a l y s i s o f C e l l Images by T i c a s " , Automated C e l l I d e n t i f i c a t i o n and C e l l S o r t i n g , Academic P r e s s , New York, 19 70.  6.  L e d l e y , R.S., "Automatic P a t t e r n R e c o g n i t i o n f o r C l i n i c a l M e d i c i n e " , P r o c e e d i n g s o f IEEE, V o l . 57, No. 11, pp. 2007-2020, November, 1969.  7.  S t e i n , P.G., L i p k i n , L.E., S h a p i r o , H.M., " S p e c t r e I I : A G e n e r a l Purpose M i c r o s c o p e Input f o r a Computer", S c i e n c e , V o l . 166, No. 3903,. pp. 328-335, October, 1969.  8.  C o s s a l t e r , John G., "A Computer V i s u a l - I n p u t System f o r the Autom a t i c R e c o g n i t i o n o f B l o o d C e l l s " , M.A.Sc. T h e s i s , The U n i v e r s i t y o f B r i t i s h Columbia, Department o f E l e c t r i c a l E n g i n e e r i n g , 1970.  9.  E b e r h a r d t , E.H., "Noise i n Image D i s s e c t o r Tubes", Research Memo No. 337, I.T.T. I n d u s t r i a l L a b o r a t o r i e s , I n d i a n a , 1960.  10.  E b e r h a r d t , E.H., " S i n g a l - t o - N o i s e R a t i o i n Image D i s s e c t o r s " , Research Memo No. 386, I.T.T. I n d u s t r i a l L a b o r a t o r i e s , I n d i a n a , 1960.  11.  Hardy, A r t h u r C , P e r r i n , F r e d H., The P r i n c i p l e s o f O p t i c s , McGrawH i l l Book Company, I n c . , New York, 1932.  12.  Mendelsohn, Mortimer L., M a y a l l , B r i a n H., "Computer O r i e n t e d A n a l y s i s o f Human Chromosones - I I I . Focus", Computers i n B i o l o g y and M e d i c i n e , V o l . 2, pp. 137-150, October, 1972.  13.  Young, I a n T., " B i o l o g i c a l Image P r o c e s s i n g - Automated Leukocyte R e c o g n i t i o n " , M.I.T. Q u a r t e r l y P r o g r e s s Report, No. 89, A p r i l , 1968.  14.  MacGregor, R.C., S c o t t , R.W., Loh, G.L., "The D i f f e r e n t i a l Leukocyte Count", J o u r n a l o f Pathology and B a c t e r i o l o g y , V o l . 51, pp. 337-368, 1940.  68  15.  Bennett, John R., "On the Measurement o f the C u r v a t u r e of the Boundaries o f Two-Dimensional Q u a n t i z e d Shapes", Ph.D. T h e s i s , The U n i v e r s i t y o f B r i t i s h Columbia, Department o f E l e c t r i c a l E n g i n e e r i n g , January, 1972.  16c  Ingram, M., "The P e r k i n - E l m e r Instrument f o r Automatic A n a l y s i s o f Blood C e l l Images", C l i n i c a l Chemistry N e w s l e t t e r , V o l . 4, Mo. 2, pp. 33-37, Winter, 19 72.  17.  The L a r c System by C o r n i n g , pamphlet d i s t r i b u t e d by C o r n i n g f i c Instruments, May, 1973.  18.  L i , Jerome C.R., Michigan, 1969.  Scienti-  S t a t i s t i c a l I n f e r e n c e , Edwards B r o t h e r , I n c . ,  

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