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Algorithms for detecting and segmenting nucleated blood cells Poon, Steven Sui-Sang 1989

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ALGORITHMS FOR DETECTING AND SEGMENTING NUCLEATED BLOOD CELLS b y STEVEN SUI -SANG POON, P . E n g . A . S c . , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF A P P L I E D SCIENCE i n THE FACULTY OF GRADUATE STUDIES Depa r tmen t o f E l e c t r i c a l E n g i n e e r i n g We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t 1989 © S t e v e n S u i - S a n g P o o n , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of SLGar/e/C/jL &J& The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract i i The a n a l y s i s o f t h e d i f f e r e n t t y p e s o f c e l l s i n b l o o d i s r o u t i n e l y u s e d i n t o d a y ' s m e d i c a l p r a c t i c e t o g i v e an i n d i c a t i o n o f a p e r s o n ' s s t a t e o f h e a l t h . Many i m a g i n g s y s t e m s and a l g o r i t h m s have b e e n d e v e l o p e d o v e r t h e l a s t 30 y e a r s i n an a t t e m p t t o au tomate t h i s p r o c e s s . Some o f t h e s e s y s t e m s c a n now d i s t i n g u i s h t h e d i f f e r e n c e b e t w e e n n o r m a l and a b n o r m a l c e l l s b u t t h e d i f f e r e n t i a t i o n among t he v a r i o u s t y p e s o f a b n o r m a l c e l l s i s s t i l l u n d e r g o i n g a c t i v e r e s e a r c h . A new s y s t e m , t he C e l l A n a l y z e r Imag ing S y s t e m , h a s b e e n d e v e l o p e d t o a c q u i r e and p r o c e s s images f r om a m i c r o s c o p e . I n t h i s w o r k , some new a l g o r i t h m s have b e e n d e v e l o p e d u s i n g t h i s s y s t e m t o d e t e c t and segment n u c l e a t e d c e l l s i n W r i g h t ' s s t a i n e d b l o o d smears f o r c l a s s i f i c a t i o n and s u b - c l a s s i f i c a t i o n o f t he n o r m a l and a b n o r m a l c e l l t y p e s . The i n i t i a l s t e p s a r e t o o b t a i n h i g h q u a l i t y images by g r e a t l y r e d u c i n g n o i s e as w e l l as b y c o r r e c t i n g d i s t o r t i o n s , a b e r r a t i o n s and s h a d i n g e f f e c t s p r e s e n t i n t h e a c q u i r e d i m a g e s . S p e c t r a l i n f o r m a t i o n f r o m t h e images i s t h e n u t i l i z e d t o d e t e c t and segment n u c l e a t e d c e l l s f r o m t h e r e s t o f t h e s c e n e ( n o n - n u c l e a t e d c e l l s and b a c k g r o u n d ) . A l l n u c l e a t e d c e l l s as w e l l as t h o s e w h i c h a r e j u s t t o u c h i n g a r e s e l e c t e d and s e p a r a t e d i n t o i n d i v i d u a l c e l l s . The r e s u l t i n g s i n g l e c e l l s a r e f u r t h e r segmen ted i n t o t he r e g i o n s o f n u c l e u s and c y t o p l a s m . S i m p l e f e a t u r e s a r e t h e n e x t r a c t e d f r o m the segmen ted c e l l s and t h e s e f e a t u r e s a r e compared t o d e t e r m i n e i f any c l u s t e r i n g o f a p a r t i c u l a r c l a s s o f c e l l e x i s t s . R e s u l t s show t h a t t h e s e a l g o r i t h m s c a n d e t e c t , segment and c l a s s i f y d i f f e r e n t t y p e s o f n o r m a l and a b n o r m a l e r r o r s i n s e g m e n t a t i o n a c c o u n t s f o r a n a l y z e d . i i i n u c l e a t e d b l o o d c e l l s . The m a j o r a p p r o x i m a t e l y 6% o f t h e c e l l s i v Table of Contents Page A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i A c k n o w l e d g e m e n t s • . v i i i 1. I n t r o d u c t i o n 1 2 . A u t o m a t e d B l o o d A n a l y z e r ; 6 2 . 1 Image C y t o m e t r y Sys tems 6 2 . 2 F l o w C y t o m e t r y Sys tems 7 2 . 3 G e n e r a l I m a g i n g Sys tem D e s i g n . . . . 8 2 . 4 C e l l A n a l y z e r I m a g i n g Sys tem 11 3 . S e g m e n t a t i o n T e c h n i q u e s 15 3 .1 O v e r v i e w 15 3 . 2 T h r e s h o l d i n g o r C l u s t e r i n g 15 3 . 3 Edge D e t e c t i o n 18 3 . 4 R e g i o n E x t r a c t i o n 21 4 . B l o o d C e l l A n a l y s i s A l g o r i t h m s 23 4 . 1 O v e r v i e w 23 4 . 2 Image A c q u i s i t i o n 25 4 . 3 Image C a l i b r a t i o n 27 4 . 4 R e c o g n i t i o n o f N u c l e a t e d C e l l 32 4 . 5 Bounda ry D e t e c t i o n o f S i n g l e C e l l s 38 4 . 6 N u c l e u s and C y t o p l a s m S e g m e n t a t i o n 40 4 . 7 S i m p l e F e a t u r e E x t r a c t i o n 45 V 5 . D i s c u s s i o n and R e s u l t s 48 5 . 1 D a t a C o l l e c t i o n 48 5 . 2 D e t e c t i o n A c c u r a c y 49 5 . 3 S e g m e n t a t i o n A c c u r a c y 52 5 . 4 F e a t u r e C a l c u l a t i o n A c c u r a c i e s 61 5 . 5 C e l l C l a s s i f i c a t i o n 63 6 . C o n c l u s i o n and F u t u r e S u g g e s t i o n s 67 6 . 1 O v e r v i e w 67 6 . 2 S y s t e m P e r f o r m a n c e 68 6 . 3 F u t u r e P l a n s 68 6 . 4 Summary o f A u t h o r ' s C o n t r i b u t i o n s 70 7 . B i b l i o g r a p h y 72 A p p e n d i x A . C l a s s i f i c a t i o n Codes f o r B l o o d C e l l Types 79 v i List of Tables Page I. Segmentation Errors i n Non-Touching Nucleated C e l l s 58 I I . Percentage Errors i n Non-Touching Nucleated C e l l s 58 I I I . Segmentation Errors i n Touching Nucleated C e l l s . . . 59 IV. Percentage Errors i n Non-Touching Nucleated C e l l s 59 V. Percentage Segmentation Errors i n Nucleated C e l l s 60 v i i List of Figures Page 1. T y p i c a l W r i g h t ' s S t a i n e d Smear 3 2 . B l o c k D i a g r a m o f a T y p i c a l Image C y t o m e t r y S y s t e m 9 3 . L a y o u t o f C e l l A n a l y z e r I m a g i n g Sys tem 12 4 . B l o c k D i a g r a m o f C e l l A n a l y z e r I m a g i n g S y s t e m 13 5 . S e l e c t i n g a T h r e s h o l d i n a B i - m o d a l H i s t o g r a m 17 6 . Use o f C l u s t e r i n g i n O b j e c t D i s c r i m i n a t i o n 19 7 . B l o c k D i a g r a m o f t he P r o c e d u r e s t o A n a l y z e B l o o d C e l l s 24 8 . N o i s e R e d u c t i o n Due t o Image A v e r a g i n g 28 9 . N o i s e R e d u c t i o n U s i n g B a c k g r o u n d S u b t r a c t i o n 31 1 0 . C l u s t e r P l o t s o f S p e c t r a l Images o f B l o o d C e l l s 33 1 1 . S e g m e n t a t i o n o f N u c l e a t e d C e l l s 35 1 2 . S m o o t h i n g t h e N u c l e a t e d C e l l Mask . . 3 7 1 3 . B o u n d a r y D i r e c t i o n Codes 38 14 . S e p a r a t i o n o f T o u c h i n g C e l l s 41 1 5 . H i s t o g r a m o f t h e Segmented N u c l e a t e d C e l l s 42 1 6 . P r o c e s s t o Segment N u c l e u s and C y t o p l a s m 44 1 7 . M i n o r and M a j o r E r r o r s i n S e p a r a t i n g T o u c h i n g C e l l s . . 51 1 8 . M i n o r C y t o p l a s m E r r o r s 54 1 9 . M i n o r N u c l e u s E r r o r s 55 2 0 . M a j o r C y t o p l a s m E r r o r s 56 21 M a j o r N u c l e u s E r r o r s 57 2 2 . F e a t u r e C a l c u l a t i o n A c c u r a c i e s 62 23 C l u s t e r P l o t o f t h e Mean I n t e n s i t i e s 64 24 C l u s t e r P l o t o f t h e P e r i m e t e r and R a t i o o f A r e a s 65 Acknowledgements v i i i I w o u l d l i k e t o t h a n k a l l t h e s t a f f and s t u d e n t members o f t he B . C . C a n c e r R e s e a r c h C e n t r e who have a s s i s t e d me i n t h i s t h e s i s . I w o u l d s p e c i f i c a l l y want t o t h a n k D r . B o n n i e M a s s i n g and D r . Kamer T e z c a n f o r t h e i r t i m e and e x p e r t i z e i n c l a s s i f y i n g more t h a n 1000 b l o o d c e l l s . I w o u l d a l s o l i k e t o e x p r e s s my g r a t i t u d e t o J o y c e Mak f o r h e r t i m e and p a t i e n c e i n h e l p i n g me w i t h t h i s d o c u m e n t a t i o n . F i n a l l y , I w o u l d l i k e t h a n k my s u p e r v i s o r , D r . Rabab Ward , and my c o - s u p e r v i s o r s , D r . B r a n k o P a l c i c and D r . M i c h a e l B e d d o e s , f o r t h e i r g e n e r o s i t y i n s h a r i n g t h e i r a d v i c e and t i m e as w e l l as t h e i r p a t i e n c e i n a l l o w i n g me t o c o m p l e t e t h i s work t o my s a t i s f a c t i o n . I am p a r t i c u l a r l y i n d e b t t o D r . B r a n k o P a l c i c f o r t h e use o f h i s f a c i l i t i e s a t t h e B . C . C a n c e r R e s e a r c h C e n t r e i n w h i c h I p e r f o r m my e x p e r i m e n t s and r e s e a r c h . Chapter 1 Introduction One o f t h e most u s e f u l i n d i c a t o r s o f a p e r s o n ' s s t a t e o f h e a l t h i n t o d a y ' s m e d i c a l p r a c t i c e i s t h e e x a m i n a t i o n o f b l o o d c e l l s . The r e a s o n i s t h a t t h e p r o d u c t i o n o f t he v a r i o u s t y p e s o f b l o o d c e l l s ( e r y t h r o c y t e s , l e u k o c y t e s and p l a t e l e t s ) i n t h e body i s h i g h l y s e n s i t i v e t o s t r e s s , i n j u r i e s , d i s e a s e s , p o i s o n s , i o n i z i n g r a d i a t i o n and o t h e r n o x i o u s s t i m u l i ( Z u c k e r - F r a n k l i n e t a l . , 1 9 8 8 ; Begemann and R a s t e t t e r , 1 9 7 9 ) . The e r y t h r o c y t e s , commonly known as r e d b l o o d c e l l s , s y n t h e s i z e h e m o g l o b i n w h i c h i s r e s p o n s i b l e f o r t r a n s p o r t i n g o x y g e n and c a r b o n d i o x i d e t o and f r o m v a r i o u s p a r t s o f t h e b o d y . The l e u k o c y t e s o r w h i t e b l o o d c e l l s a r e r e s p o n s i b l e f o r d e s t r o y i n g and i n a c t i v a t i n g i n f e c t i v e c e l l s as w e l l as f o r p r o d u c i n g a n t i b o d i e s and o t h e r a g e n t s f o r t he b o d y ' s immune s y s t e m . The p l a t e l e t s p r e v e n t b l e e d i n g b y a c t i n g as a g e n t s t o c l o t t he b l o o d on t he s u r f a c e o f t h e wound. Thus t he a b n o r m a l i t y i n t h e number o f e a c h t y p e o f c e l l i n t h e b l o o d samp le as w e l l as t h e i r r e g u l a r i t y i n t h e m o r p h o l o g y o f t h e c e l l s a r e i m p o r t a n t i n d i c a t i o n s o f t he i l l c o n d i t i o n s u n d e r w h i c h t h e y were p r o d u c e d ( Z u c k e r - F r a n k l i n e t a l . , 1 9 8 8 ; Begemann and R a s t e t t e r , 1 9 7 9 ) . T h i s t h e s i s i s a s t u d y w h i c h l e a d s t o t he d e v e l o p m e n t o f a l g o r i t h m s f o r d e t e c t i n g and s e g m e n t i n g t h e v a r i o u s t y p e s o f b l o o d c e l l s i n s i n g l e l a y e r d e p o s i t s o f b l o o d t a k e n f r om p a t i e n t s ' . I t i s t h o u g h t t h a t t h e c o m p o s i t i o n o f t h e m i x t u r e o f t he b l o o d c e l l s i s l i n k e d w i t h t he h e a l t h o f t h e p a t i e n t . 2 A common t e c h n i q u e o f a n a l y s i s i s t o v i s u a l l y examine t he c e l l s i n a b l o o d smear t h r o u g h a m i c r o s c o p e ( F i g u r e 1 ) . The smear i s p r o d u c e d by s p r e a d i n g a t h i n f i l m o f b l o o d o v e r a g l a s s s l i d e w h i c h i s t h e n s t a i n e d s u c h t h a t v a r i o u s f e a t u r e s o f t h e c e l l a r e enhanced f o r v i s u a l i n t e r p r e t a t i o n u n d e r t h e m a g n i f i e d f i e l d o f a m i c r o s c o p e . W r i g h t ' s s t a i n , d e v e l o p e d i n t h e 1 8 0 0 s , h a s become the s t a n d a r d s t a i n f o r b l o o d smear a n a l y s i s ( Z u c k e r - F r a n k l i n e t a l . , 1 9 8 8 ; Begemann and R a s t e t t e r , 1 9 7 9 ) . The e r y t h r o c y t e s have no n u c l e u s and when u s i n g W r i g h t ' s s t a i n , a p p e a r as l i g h t r e d i n c o l o u r . The l e u k o c y t e s have b o t h n u c l e u s and c y t o p l a s m and a r e s t a i n e d d a r k and l i g h t b l u e r e s p e c t i v e l y . The p l a t e l e t s h a v e o n l y c y t o p l a s m m a t e r i a l and a r e s t a i n e d l i g h t b l u e . The v i s u a l i n t e r p r e t a t i o n and c l a s s i f i c a t i o n o f t h e s e smears a r e s u b j e c t i v e , t e d i o u s , t i m e - c o n s u m i n g and s u s c e p t i b l e t o human e r r o r . I n t h e d i a g n o s i s and c l a s s i f i c a t i o n o f l e u k e m i a , t h e r e h a v e b e e n a t t e m p t s made b y t h e F r e n c h - A m e r i c a n - B r i t i s h (FAB) c o o p e r a t i v e g roup ( B e n n e t t e t a l . , 1 9 7 6 ; M i l l e r e t a l . , 1981) t o s t a n d a r d i z e t h e c r i t e r i a f o r t he d i v i s i o n o f t h e m y e l o i d and l y m p h o i d t y p e s as w e l l as s u b - c l a s s i f i c a t i o n w i t h i n t h e s e t y p e s s u c h t h a t a p p r o p r i a t e t r e a t m e n t c a n be p r e s c r i b e d . A l t h o u g h t h e r e i s g e n e r a l a c c e p t a n c e o f t h e c l a s s i f i c a t i o n scheme, t h e e x p e n s e o f t h i s manua l a n a l y s i s and t h e i n c o n s i s t e n c i e s i n c l a s s i f i c a t i o n b y o b s e r v e r s among d i f f e r e n t i n s t i t u t i o n s , as w e l l as t h o s e i n t h e same i n s t i t u t i o n w a r r a n t some f o r m o f o b j e c t i v e e v a l u a t i o n w h i c h c a n be u s e d f o r d i a g n o s i s . T h i s c a n be a c h i e v e d b y q u a n t i t a t i v e measuremen ts o f t h e s e c e l l s u s i n g a sem i o r f u l l y a u t o m a t e d p r o c e d u r e . S i n c e a l a r g e number o f c e l l s i s g e n e r a l l y m e a s u r e d , a f u l l y a u t o m a t e d 3 Figure 1 TYPICAL WRIGHT'S STAINED BLOOD SMEAR A t y p i c a l blood smear contains a) red blood c e l l s , b) white blood c e l l s , c) p l a t e l e t s , and d) debris. This photograph was taken at 40x microscope magnification. 4 s y s t e m must e v e n t u a l l y be d e v e l o p e d i f t h i s a p p r o a c h i s t o be p r a c t i c a l l y i m p l e m e n t e d . The m a j o r d e f i c i e n c y o f t h e c o m m e r c i a l l y a v a i l a b l e c e l l a n a l y s i s sys tems i s t h e i r i n a b i l i t y t o d i f f e r e n t i a t e t h e v a r i o u s t y p e s o f a b n o r m a l c e l l s . These c o m m e r c i a l i m a g i n g s y s t e m s were d e s i g n e d o v e r t h e l a s t t e n t o t w e n t y y e a r s , and l a c k t he q u a l i t y o f t h e t r a n s d u c e r s and o p t i c a l components as w e l l as t h e p r o c e s s i n g power w h i c h became a v a i l a b l e i n t he l a t e e i g h t i e s . I n a d d i t i o n , t h e a l g o r i t h m s w h i c h i n v o l v e t h e d e t e c t i o n , s e g m e n t a t i o n , f e a t u r e e x t r a c t i o n and c e l l c l a s s i f i c a t i o n o f d i f f e r e n t t y p e s o f c e l l s a r e s t i l l u n d e r g o i n g r e s e a r c h . The mos t d i f f i c u l t and a l s o t he most c r u c i a l s t e p i n an a u t o m a t e d b l o o d smear c l a s s i f i c a t i o n a l g o r i t h m i s t h a t o f c o r r e c t l y s e g m e n t i n g t h e image i n t o i t s m a i n c o m p o n e n t s . These a r e t h e n u c l e u s and t h e c y t o p l a s m o f t he n u c l e a t e d c e l l s , t h e c y t o p l a s m o f t h e n o n - n u c l e a t e d c e l l s , and t he b a c k g r o u n d . B e c a u s e o f n e u r o l o g i c a l p r o c e s s i n g w h i c h o c c u r s i n t he e y e , a human o b s e r v e r c a n e a s i l y d e t e r m i n e and o u t l i n e t h e v a r i o u s p a r t s ( n u c l e u s and c y t o p l a s m ) o f t h e c e l l i n t h e image . A m a c h i n e , h o w e v e r , h a s t o r e s o r t t o d i g i t a l p r o c e s s i n g t e c h n i q u e s t o r e l a t e t h e s e t o f p i x e l s w h i c h make up t he image , i n t o d i s t i n c t r e g i o n s . N o n e t h e l e s s , a mach ine c a n q u a n t i t a t i v e l y d e s c r i b e t h e f e a t u r e s e x t r a c t e d f r o m t h e s e d e f i n e d r e g i o n s , a t a s k w h i c h t h e human c a n o n l y e s t i m a t e u s i n g q u a l i t a t i v e means . Any e r r o r s i n t r o d u c e d i n s e g m e n t a t i o n w i l l p r o p a g a t e t o f e a t u r e e x t r a c t i o n , o b j e c t c l a s s i f i c a t i o n and w i l l p o s s i b l y l e a d t o a m i s i n t e r p r e t a t i o n o f t h e c e l l s i n t he s c e n e . T h e r e f o r e , c o r r e c t 5 s e g m e n t a t i o n i s o f a pa ramoun t i m p o r t a n c e . Howeve r , t he d e v e l o p m e n t o f t e c h n i q u e s f o r a c c u r a t e and c o n s i s t e n t s e g m e n t a t i o n o f c e l l s i s n o t a t r i v i a l t a s k . T h i s t h e s i s i s c o n c e r n e d w i t h t h e s o f t w a r e d e v e l o p m e n t o f new a l g o r i t h m s f o r r e c o g n i t i o n , s e g m e n t a t i o n , f e a t u r e e x t r a c t i o n , and a n a l y s i s o f n u c l e a t e d b l o o d c e l l s u s i n g a new image c y t o m e t r y s y s t e m , t he C e l l A n a l y z e r w h i c h was d e s i g n e d and a s s e m b l e d t o measure t h e p r o p e r t i e s o f c e l l s b a s e d on t h e i r i m a g e s . These a l g o r i t h m s a r e c o m p u t a t i o n a l l y l e s s e x p e n s i v e and ma tch i f n o t s u p e r c e d e t h e a c c u r a c y o f t h o s e d e v e l o p e d f o r o t h e r c y t o m e t r y s y s t e m s . The body o f t he t h e s i s b e g i n s w i t h t h e d i s c u s s i o n o f t h e c h a r a c t e r i s t i c s o f image c y t o m e t r y s y s t e m s i n C h a p t e r two . T h i s s e c t i o n e v a l u a t e s t he s y s t e m s c u r r e n t l y u s e d f o r a n a l y z i n g b l o o d c e l l s and d e s c r i b e s t h e s y s t e m w h i c h i s u s e d t o a n a l y z e t h e b l o o d c e l l s i n t h i s t h e s i s . The d i s c u s s i o n o f some e x i s t i n g s e g m e n t a t i o n t e c h n i q u e s i s o u t l i n e d i n C h a p t e r t h r e e . T h i s s e c t i o n g i v e s an i n d i c a t i o n o f t h e d i f f e r e n t methods u s e d by o t h e r r e s e a r c h e r s t o segment t h e c e l l s i n t h e i m a g e . P a r t s o f t h e s e methods a r e i n c o r p o r a t e d i n t o t he a l g o r i t h m s p r e s e n t e d i n C h a p t e r f o u r . New a p p r o a c h e s f o r a n a l y z i n g n u c l e a t e d b l o o d c e l l s , s u c h as t h e m a n i p u l a t i o n o f t h e s p e c t r a l i n f o r m a t i o n i n s e g m e n t i n g n u c l e a t e d c e l l s , t he s e p a r a t i o n o f t o u c h i n g c e l l s u s i n g t h e o b j e c t c h a i n code i n f o r m a t i o n and t h e a n g l e o f t he t a n g e n t t o t h e o b j e c t b o u n d a r y , and t h e f i l t e r i n g o f t he image u s i n g an edge e n h a n c i n g a v e r a g e f i l t e r , a r e i n t r o d u c e d i n t h i s c h a p t e r . The p e r f o r m a n c e o f t h e s e a l g o r i t h m s i s e x a m i n e d i n C h a p t e r f i v e . Chapter 2 Automated Blood Analyzer 6 2 . 1 Image C y t o m e t r y Sys tems The n e c e s s i t y f o r a u t o m a t i o n h a s l e d t o t h e a p p l i c a t i o n o f mach ine v i s i o n a n d r o b o t i c s t o m i c r o s c o p y i n t h e e a r l y 1950s w i t h t h e i n t r o d u c t i o n o f t h e b l o o d c e l l a n a l y z e r s (Young and R o b e r t s , 1 9 5 1 ; W a l t o n , 1 9 5 2 ) . S i n c e t h e n , a t t e m p t s have been made t o au tomate o t h e r a r e a s o f m e d i c i n e s u c h as t h e s c r e e n i n g o f c e r v i c a l c e l l s ( e g . B e n g t s s o n e t a l . , 1 9 7 9 ; T u c k e r e t a l . , 1 9 7 9 ; Shoemaker e t a l . , 1982) and t h e a n a l y s i s o f chromosomes ( e g . P h i l i p and L u n d s t e e n , 1 9 8 5 ; P r e s t o n , 1 9 7 6 ) . A l l t h e s e s y s t e m s i n c o r p o r a t e some t y p e o f a s e n s o r f o r t r a n s f o r m i n g t h e m i c r o s c o p i c s c e n e i n t o a d i g i t a l i m a g e , some r o b o t i c s f o r b r i n g i n g t h e a r e a o f i n t e r e s t on t h e m i c r o s c o p e s l i d e t o t h e s e n s o r ' s v i e w , and a c o m p u t e r f o r a n a l y z i n g t h e d a t a and s u p e r v i s i n g t he e n t i r e p r o c e s s . T h i s t e c h n o l o g y h a s b r o u g h t a c c u r a c y , u n i f o r m i t y , r e p r o d u c i b i l i t y , and a c o n t r o l l e v e l o f q u a l i t y t o t h e p e r f o r m a n c e o f t h e s c r e e n i n g p r o g r a m s . The s u c c e s s o f t h e s e a u t o m a t e d c y t o m e t r y s y s t e m s c a n be s e e n b y t h e c o m m e r c i a l p r o d u c t i o n o f l e u k o c y t e a n a l y z e r s b y f i v e d i f f e r e n t compan ies ( t h r e e f r o m t h e U n i t e d S t a t e s and two f r om J a p a n ) i n t h e l a s t two d e c a d e s ( P r e s t o n , 1987 ; Imgram and P r e s t o n , 1970 ; M e g l a , 1 9 7 3 ; N o r g r e n , K u l k a r n i and Graham, 1 9 8 1 ) . These a n a l y z e r s ( e g . Hemat rak b y S m i t h K l i n e Beckman , LARC b y C o r n i n g G l a s s W o r k s , and d i f f 3 b y C o u l t e r E l e c t r o n i c s ) have s u p e r i o r p e r f o r m a n c e o v e r humans i n c l a s s i f y i n g t he 7 s l i d e s as n o r m a l o r a b n o r m a l b u t l a c k t h e c a p a b i l i t i e s i n d i f f e r e n t i a t i n g t h e d i f f e r e n t t y p e s o f a b n o r m a l c e l l s . B e c a u s e o f t h i s d e f i c i e n c y and e c o n o m i c a l r e a s o n s , t he t h r e e c o m p a n i e s i n t h e U n i t e d S t a t e s h a v e s t o p p e d t h e i r p r o d u c t i o n by 1 9 8 6 . D e s p i t e t h e l a c k o f i n d u s t r i a l i n t e r e s t , t h e r e i s s t i l l a c t i v e r e s e a r c h i n a u t o m a t i n g t he a n a l y s i s o f a b n o r m a l b l o o d c e l l s b y g r o u p s s u c h as P a l c i c and J a g g i (1989) i n C a n a d a , B a c u s and G r a c e (1987) i n t h e U n i t e d S t a t e s , Aus e t a l (1986) and Haussraann and L i e d t k e (1984) i n Germany, and L a n d e w e e r d e t a l (1983) i n t h e N e t h e r l a n d s . 2 . 2 F l o w C y t o m e t r y Sys tems The a n a l y s i s o f c e l l s u s i n g t h e f l u i d - f l o w t e c h n o l o g y ( F u l w y l e r , 1 9 6 5 ; T y r e r and P r e s s m a n , 1987) s t a r t e d i n t he 1 9 6 0 s . F l u i d s u s p e n s i o n i s made i n t o t i n y d r o p l e t s and c e l l s i n t he d r o p l e t s a r e p a s s e d t h r o u g h an i n t e r r o g a t i o n o r i f i c e a t h i g h speeds (5000 c e l l s / s ) . A c e l l c a n be e x a m i n e d w h i l e i t p a s s e s t h r o u g h a f i e l d o f v i e w i n a p p r o x i m a t e l y f o u r m i c r o s e c o n d s . L a s e r l i g h t i s shone a t t h e d r o p l e t s , w h i c h may c o n t a i n t he c e l l , and t h e t r a n s m i t t a n c e and f l u o r e s c e n c e a t v a r i o u s w a v e l e n g t h s o f l i g h t a r e m e a s u r e d . The d a t a i s t r a n s f e r r e d t o a c o m p u t e r and p r o c e s s e d . T h e s e s y s t e m s p r e s e n t l y a r e n o t c a p a b l e o f d i f f e r e n t i a t i n g as many t y p e s o f w h i t e b l o o d c e l l s as good image c y t o m e t r y s y s t e m s . Howeve r , t h e d e v e l o p m e n t o f new i m m u n o l o g i c a l l y b a s e d b i o c h e m i c a l r e a g e n t s , w h i c h t a g t h e d i f f e r e n t c l a s s e s o f b l o o d c e l l s , may overcome t h i s p r o b l e m . W i t h t h e s e new m a r k e r s , t he f l o w s y s t e m s c o u l d be i d e a l f o r s c r e e n i n g s a m p l e s q u i c k l y and r e c o g n i z i n g t h e r a r e a b n o r m a l c e l l s 8 a c c u r a t e l y . S i n c e t h e y do n o t a l l o w v i s u a l o b s e r v a t i o n o f t he c e l l s d e t e c t e d , a b l o o d smear must be made when an a b n o r m a l samp le i s d e t e c t e d f o r v e r i f i c a t i o n p u r p o s e s . 2 . 3 G e n e r a l I m a g i n g S y s t e m D e s i g n A l t h o u g h many image c y t o m e t r y s y s t e m s have b e e n d e v e l o p e d , t h e y a r e v e r y s i m i l a r i n d e s i g n and o p e r a t i o n . The b a s i c s y s t e m c o n s i s t s o f an i l l u m i n a t i o n s o u r c e , m i c r o s c o p e o p t i c s , m o t o r i z e d m e c h a n i c a l s t a g e , c a m e r a , d i g i t i z i n g c i r c u i t r y , image memory, d i s p l a y m o n i t o r , and p r o c e s s o r s ( F i g u r e 2 ) . The s t a g e w h i c h h o l d s t h e s a m p l e s i s c a p a b l e o f m o v i n g o b j e c t s i n t h e X and Y d i r e c t i o n t o t h e d e t e c t o r s f i e l d o f v i e w . Z d i r e c t i o n i s p r o v i d e d f o r f o c u s s i n g p u r p o s e s . L i g h t i s t r a n s m i t t e d f r om t h e i l l u m i n a t i o n s o u r c e t h r o u g h t he samp le t o t h e camera d e t e c t o r . The d e t e c t e d image i s t r a n s f o r m e d i n t o a d i g i t a l image b y t h e i n t e r f a c i n g c i r c u i t r y . The r e s u l t i n g d i g i t a l image i s s t o r e d i n t he compu te r memory f r o m where i t c a n be d i s p l a y e d on t he m o n i t o r a n d / o r p r o c e s s e d and a n a l y z e d by t h e c o m p u t e r . The m a j o r d i f f e r e n c e among t h e s e i m a g i n g s y s t e m s i s t h e t r a n s d u c e r e m p l o y e d and t h e method u s e d i n s c a n n i n g . M o s t s y s t e m s u s e a two d i m e n s i o n a l d e t e c t o r s u c h as t h o s e f o u n d i n t u b e cameras o r a two d i m e n s i o n a l a r r a y c h a r g e c o u p l e d d e v i c e (CCD) cameras ( e g . J a g g i e t a l . , 1 9 8 8 ; T u c k e r , 1 9 7 9 ) . The image i s c a p t u r e d by t h e d e t e c t o r w h i l e t he s t a g e s c a n s t h e s l i d e b y m o v i n g t he samp le f r o m one f rame t o t h e n e x t . V e r y few s y s t e m s u s e a l i n e a r d e t e c t o r s s u c h as d i o d e o r CCD a r r a y s ( e g . 9 Camera A / D Interface I Microscope I Stage Controller Illumination Source Image Memory Host Computer F i g u r e 2 BLOCK DIAGRAM OF A TYPICAL IMAGE CYTOMETRY SYSTEM A t y p i c a l s y s t e m c o n s i s t s o f an i l l u m i n a t i o n s o u r c e , m i c r o s c o p e , m o t o r i z e d s t a g e , d i g i t i z i n g c i r c u i t r y , image memory, d i s p l a y m o n i t o r and p r o c e s s o r s . 10 J a g g i and P a l c i c , 1 9 8 5 ; B e n g t s s o n e t a l . , 1979 ; T u c k e r e t a l . , 1 9 8 7 ) . I n t h e s e s y s t e m s , a n image i s o b t a i n e d b y m o v i n g t he samp le a c r o s s t h e s e n s o r and p i e c i n g t o g e t h e r t h e image l i n e s . The a d v a n t a g e o f l i n e a r s c a n n e r s i s t h a t t h e y have more s e n s o r e l e m e n t s t h a n e i t h e r a row o r a c o l u m n o f t h e two d i m e n s i o n a l c o u n t e r p a r t and hence p r o v i d e a w i d e r f i e l d o f v i e w . A l s o , t h e e l e m e n t s o f t h e s e n s o r a r e d i g i t i z e d and s t o r e d as d i g i t a l d a t a , and n o t i n a v i d e o ( a n a l o g u e ) f o r m a t w h i c h r e q u i r e s r e - s a m p l i n g t o c o n v e r t t o a d i g i t a l f o r m . The m a j o r d i s a d v a n t a g e i s t h a t a p r e c i s e m e c h a n i c a l s c a n n i n g i s r e q u i r e d f o r h i g h r e s o l u t i o n o f t h e t w o - d i m e n s i o n a l i m a g e s . The re a r e s y s t e m s w h i c h use a c o m b i n a t i o n o f t he l i n e a r and m a t r i x ( v i d e o ) d e t e c t o r s ( e g . Graham and N o r g e n , 1 9 8 0 ) . O b j e c t s d e t e c t e d b y t h e l i n e a r a r r a y a r e moved i n t o t he f i e l d o f v i e w o f t h e two d i m e n s i o n a l d e t e c t o r f o r h i g h r e s o l u t i o n image a c q u i s i t i o n . T h e r e a r e a l s o s y s t e m s w h i c h u s e a one e l e m e n t d e t e c t o r s u c h as a p h o t o m u l t i p l i e r o r p h o t o d i o d e ( e g . Ing ram and P r e s t o n , 1 9 7 0 ; Shoemaker e t a l . , 1 9 8 2 ) . A r o t a t i n g p o l y g o n i s u s e d t o d e f l e c t t he l a s e r s p o t t o s c a n t h e o b j e c t i n one d i m e n s i o n w h i l e t h e s t a g e i s moved t o s c a n t h e image i n t h e o t h e r d i m e n s i o n . The q u a l i t y o f t h e i n p u t image i n i m a g i n g i s dependen t n o t o n l y on t h e t y p e o f t r a n s d u c e r s u s e d b u t a l s o on t he o p t i c a l components and e l e c t r o n i c ( d i g i t i z i n g ) c i r c u i t r y . The most i m p o r t a n t component i n t h e o p t i c a l s y s t e m i s t h e o b j e c t i v e l e n s o f t h e m i c r o s c o p e w h i c h d e t e r m i n e s t h e m a g n i f i c a t i o n o f t he samp le and h e n c e g o v e r n s t he s a m p l i n g d e n s i t y o f t h e i m a g e . These l e n s e s a r e n o t p e r f e c t and g e n e r a l l y i n t r o d u c e d i s t o r t i o n s , a b e r r a t i o n s , and s h a d i n g e f f e c t s . M o s t cameras t o d a y a r e 11 b u i l t f o r t h e t e l e v i s i o n b r o a d c a s t communi ty where t h e d e t e c t e d image on t h e t r a n s d u c e r i s c o n v e r t e d t o a n a n a l o g s i g n a l . T h i s s i g n a l i s l a t e r d i g i t i z e d f o r m a c h i n e a n a l y s i s r e s u l t i n g i n a l o s s o f i n f o r m a t i o n due t o r e s a m p l i n g and q u a n t i z a t i o n e r r o r s . Cameras w h i c h d i r e c t l y d i g i t i z e t h e s e n s o r image i n t o d i g i t a l d a t a f o r mach ine a n a l y s i s a r e e x p e n s i v e and do n o t c o n f o r m t o a f i x e d s t a n d a r d o f d a t a t r a n s m i s s i o n w h i c h r e s u l t s i n t he d e p e n d a n c y on a p a r t i c u l a r m a n u f a c t u r e r f o r f u t u r e u p g r a d e s . D i s t o r t i o n s i n t r o d u c e d by t he o p t i c s , t r a n s d u c e r and d i g i t i z i n g c i r c u i t r y must be compensa ted f o r b e f o r e t h e image c a n be a n a l y z e d . 2 .4 C e l l A n a l y z e r I m a g i n g Sys tem The C e l l A n a l y z e r I m a g i n g Sys tem was u s e d t o d e v e l o p and t e s t a l g o r i t h m s u s e d f o r s e g m e n t i n g t h e b l o o d smears d e s c r i b e d i n t h i s t h e s i s . The C e l l A n a l y z e r was o r i g i n a l l y d e s i g n e d a t t h e B r i t i s h C o l u m b i a C a n c e r R e s e a r c h C e n t r e f o r t h e measurement o f l i v e , u n s t a i n e d c e l l s t o s t u d y t h e i r p r o p e r t i e s as a f u n c t i o n o f t i m e a n d / o r t r e a t m e n t s ( P a l c i c , J a g g i and N o r d i n , 1987) . The d e s c r i p t i o n o f t h i s i n i t i a l d e s i g n i s d e s c r i b e d e l s e w h e r e ( J a g g i and P a l c i c , 1985 ; J a g g i , Poon and P a l c i c , 1 9 8 6 ) . A b l o c k d i a g r a m o f t h e c u r r e n t s y s t e m i s shown i n F i g u r e 3 and 4 . A two-d i m e n s i o n a l CCD c a m e r a , f rame g r a b b i n g and image p r o c e s s i n g b o a r d , and c o l o u r d i s p l a y m o n i t o r was added t o t h e o r i g i n a l s y s t e m f o r measurements o f s t a i n e d c e l l s ( J a g g i e t a l . , 1 9 8 8 ) . I n a d d i t i o n t o l i v e c e l l e x p e r i m e n t s , t h i s s y s t e m i s c u r r e n t l y b e i n g u s e d f o r t h e d e v e l o p m e n t o f a u t o m a t i c s e g m e n t a t i o n and f e a t u r e e x t r a c t i o n a l g o r i t h m s o f s t a i n e d Microscope • Camera 12 Power Supply and Controllers RGB Display Computer Monitor e nrm i i i i i f f lmii i f l PC/AT Computer Keyboard F i g u r e 3 LAYOUT OF CELL ANALYZER IMAGE SYSTEM The l a y o u t o f t h e m a j o r components o f t h e C e l l A n a l y z e r I m a g i n g Sys tem c o n s i s t s o f a m i c r o s c o p e , c a m e r a , camera c o n t r o l u n i t , RGB m o n i t o r , c o m p u t e r , c o m p u t e r m o n i t o r , k e y b o a r d , and a r a c k c o n t a i n i n g t he power s u p p l y f o r t h e l i g h t s o u r c e and t h e c o n t r o l l e r s t o move t h e s t a g e i n t he x , y and z p o s i t i o n s . 13 RGB CCD Camera RGB Display T Microscope I Frame Grabber and Image Processor Stage Controller Stabilized Light Source Computer Monitor I PC/AT Computer F i g u r e 4 BLOCK DIAGRAM OF THE CELL ANALYZER IMAGING SYSTEM The m a j o r components o f t h e s y s t e m a r e t h e s t a b i l i z e d l i g h t s o u r c e f o r i l l u m i n a t i n g t he s a m p l e , a s t a g e t o move t he o b j e c t i n t o t h e m i c r o s c o p e s f i e l d o f v i e w , t h e m i c r o s c o p e o p t i c s t o m a g n i f y t h e i m a g e , t h e camera t o a c q u i r e t h e i m a g e s , d i g i t i z i n g c i r c u i t r y t o t r a n s f o r m t h e v i d e o s i g n a l i n t o d i g i t a l f o r m a t w h i c h c a n be s t o r e d i n t o image memory and d i s p l a y e d on an RGB m o n i t o r o r m a n i p u l a t e d b y a c o m p u t e r . 14 b l o o d and c e r v i c a l c e l l s as w e l l as e x p e r i m e n t s o f o t h e r c e l l s and t i s s u e s w h i c h r e q u i r e an i n t e r a c t i v e u s e o f an i m a g i n g s y s t e m . The C e l l A n a l y z e r s y s t e m i s p a r t i c u l a r l y u s e f u l f o r d e v e l o p m e n t a l w o r k . S i n c e many s t a i n e d s l i d e s c o n t a i n m u l t i - s p e c t r a l i n f o r m a t i o n , a 3 - c h i p CCD camera (Sony DXC-3000A) i s emp loyed t o c a p t u r e images i n t he t h r e e p r i m a r y c o l o u r s o f t h e s p e c t r u m s : r e d , g r e e n and b l u e . A f r ame g r a b b i n g and i m a g i n g b o a r d ( M a t r o x MVP-AT) i s u s e d t o d i g i t i z e , s t o r e , p r o c e s s and d i s p l a y t h e i m a g e . T h i s b o a r d a c c e p t s t h r e e i n p u t c h a n n e l s , one f o r e a c h s p e c t r u m , and s t o r e s e a c h image i n one o f t h e f o u r 512x512x8 f rame b u f f e r s . The h a r d w a r e p r o c e s s i n g f e a t u r e s o f t h i s b o a r d i n c l u d e h i s t o g r a m p r o c e s s o r , 3x3 c o n v o l u t i o n s , and 3x3 m o r p h o l o g i c a l o p e r a t o r s . O t h e r p r o c e s s i n g and a n a l y s i s f u n c t i o n s a r e p e r f o r m e d b y t h e h o s t IBM P C / A T c o m p u t e r . F o r b l o o d smear a n a l y s i s , a n o b j e c t i v e l e n s ( P l a n Apoch roma t 40x w i t h a 0 . 9 5 n u m e r i c a l a p e r t u r e , a i r ) i s u s e d i n c o n j u n c t i o n w i t h a ma tched c o n d e n s e r l e n s and a TV r e l a y l e n s ( l x ) g i v i n g a s p a t i a l r e s o l u t i o n o f 0 . 3 3 m i c r o n s i n b o t h x and y d i r e c t i o n s . A l l t h r e e c o l o u r images o f t h e camera a r e u s e d , e a c h o f w h i c h h a s a p h o t o m e t r i c r e s o l u t i o n o f 8 b i t s o r 256 g r e y l e v e l s . Chapter 3 Segmentation Techniques 15 3.1 O v e r v i e w Many s e g m e n t a t i o n t e c h n i q u e s have b e e n d e v e l o p e d o v e r t he p a s t s e v e r a l d e c a d e s ( e g . F u and M u i , 1981; D a v i s , 1975). These methods c a n be c a t e g o r i z e d i n t o t h r e e d i f f e r e n t c l a s s e s : i ) c h a r a c t e r i s t i c f e a t u r e t h r e s h o l d i n g o r c l u s t e r i n g , i i ) edge d e t e c t i o n , and i i i ) r e g i o n e x t r a c t i o n . A s i n g l e a l g o r i t h m g e n e r a l l y c a n n o t segment a p a r t i c u l a r s c e n e and h e n c e a c o m b i n a t i o n o f s e g m e n t a t i o n p r o c e s s e s i s o f t e n u s e d . G e n e r a l l y t h e s e p r o c e s s e s p e r f o r m w e l l i n some a p p l i c a t i o n s b u t may f a i l i n o t h e r s . D e t a i l e d d e s c r i p t i o n s o f t h e d i f f e r e n t c l a s s e s o f known s e g m e n t a t i o n a l g o r i t h m s a r e d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n s . 3 .2 T h r e s h o l d i n g o r C l u s t e r i n g T h r e s h o l d i n g i s a common t e c h n i q u e u s e d i n s e g m e n t i n g r e g i o n s i n a s c e n e . The p r o c e s s a s s i g n s d i s t i n c t l a b e l s t o a r e a s b a s e d on some p r o p e r t i e s o f t h e image . A p r o p e r t y may be a c h a r a c t e r i s t i c f e a t u r e s u c h as t h e image g r a y l e v e l s o r may be o f l o c a l n a t u r e s u c h as t h e g r a d i e n t o r L a p l a c i a n o f t he g r a y l e v e l s . I n a l l c a s e s , a s p e c i f i e d r a n g e o f v a l u e s o f a g i v e n p r o p e r t y i s u s e d t o d e f i n e t h e p i x e l s i n t h e image w h i c h b e l o n g t o t he same r e g i o n . O f t e n , a h i s t o g r a m o f an image p r o p e r t y i s u s e d t o d e t e r m i n e t h e t h r e s h o l d s f o r e a c h r e g i o n . T h e s e 16 h i s t o g r a m s a r e g e n e r a l l y smoothed t o remove n o i s e . C a r e must be t a k e n , h o w e v e r , t o a v o i d s m o o t h i n g o u t s m a l l b u t v a l i d m i n i m a o r max ima. A t h r e s h o l d i n g t e c h n i q u e w h i c h c a n be a p p l i e d t o g r a y l e v e l h i s t o g r a m s i s t h e mode m e t h o d . T h i s t y p e o f h i s t o g r a m g i v e s an i n d i c a t i o n o f t he number o f p i x e l s w h i c h h a v e t h e same g r a y l e v e l i n t h e image ( F i g u r e 5 ) . T h u s , e a c h p e a k (mode) o f t h e h i s t o g r a m r e p r e s e n t s a r e a s o f s i m i l a r i n t e n s i t y l e v e l . A b o u n d a r y i s p l a c e d a t t he v a l l e y b e t w e e n p e a k s t o s e p a r a t e t h e r e g i o n s . The r a t i o n a l e f o r c h o o s i n g s u c h p o i n t s i s t o m i n i m i z e t h e p r o b a b i l i t y o f m i s c l a s s i f y i n g e a c h r e g i o n . S i n c e t he number o f p i x e l s a t t he v a l l e y compared t o t h e p e a k s i s r e l a t i v e l y s m a l l , m i s p l a c e m e n t o f t h e t h r e s h o l d f r o m t h e e x a c t l o c a t i o n h a s r e l a t i v e l y l i t t l e e f f e c t on t h e r e s u l t i n g i m a g e . F o r e x a m p l e , Wermser , Haussmann and L i e d k e (1984) u s e d t h i s t e c h n i q u e t o segment t h e b l o o d c e l l s f r o m t h e b a c k g r o u n d o f t h e image . A d i f f e r e n t t e c h n i q u e i s u s e d f o r t h r e s h o l d i n g g r a d i e n t h i s t o g r a m s . S i n c e t h e s e h i s t o g r a m s r e p r e s e n t t h e sum o f t h e m a g n i t u d e o f g r a d i e n t s a t a g i v e n g r a y l e v e l , t he b o u n d a r y i s p l a c e d a t t h e h i g h e s t p o i n t ' i n t he h i s t o g r a m . T h i s p o i n t s i g n i f i e s t h e l o c a t i o n o f t h e l a r g e s t d i f f e r e n c e s ( t h e e d g e s ) o f t he image . T h i s method wo rks w e l l w i t h some images b u t f a i l s i n o t h e r s . F o r images where t h e r e a r e many s i m i l a r i n t e n s i t y p i x e l s w i t h a s m a l l g r a d i e n t , t h e i r sum may ove rmask t he sum g e n e r a t e d a t t h e edge o f r a r e o b j e c t s and h e n c e g e n e r a t e a wrong t h r e s h o l d l e v e l . 17 F i g u r e 5 SELECTING A THRESHOLD IN A BI-MODAL HISTOGRAM The t h r e s h o l d b o u n d a r y i s p l a c e d a t a v a l l e y b e t w e e n t h e p e a k s . The e r r o r i n m i s p l a c i n g t he b o u n d a r y i s l e s s a t t he v a l l e y (x-^) t h a n a t o t h e r p o i n t s (X2). 18 C l u s t e r i n g e x t e n d s t h e t e c h n i q u e o f t h r e s h o l d i n g t o t h e m u l t i -d i m e n s i o n a l s p a c e . T h i s t e c h n i q u e i s u s e d when p o o r d i s c r i m i n a t i o n e x i s t s e m p l o y i n g a s i n g l e f e a t u r e b u t d i s t i n c t r e g i o n s c a n be d e t e c t e d u s i n g h i s t o g r a m s o f two o r more c h a r a c t e r i s t i c f e a t u r e s ( F i g u r e 6 ) . Any f e a t u r e w h i c h i s u s e f u l f o r s e g m e n t i n g a r e g i o n , s u c h as t h e g r a y l e v e l s o f images s e e n t h r o u g h d i f f e r e n t s p e c t r a l f i l t e r s , g r a d i e n t s , t e x t u r e f e a t u r e s , e t c . , c a n be u s e d . Haussmann and L i e d t k e (1984) u s e t h e g r e e n and b l u e image components o f t he image t o s e p a r a t e t he n u c l e a t e d c e l l s f r o m t h e r e d b l o o d c e l l s . A l g o r i t h m s f o r c l u s t e r a n a l y s i s have been a v a i l a b l e f o r l o c a t i n g the d e c i s i o n b o u n d a r y b e t w e e n r e g i o n s i n a m u l t i -d i m e n s i o n a l s p a c e (Amadasun and K i n g , 1 9 8 8 ; Umesh, 1 9 8 8 ) . To r e d u c e t he amount o f c o m p u t a t i o n s r e q u i r e d i n t h e a n a l y s i s , t he s m a l l e s t number o f f e a t u r e s w h i c h c a n d i s c r i m i n a t e t h e r e g i o n s i s e m p l o y e d . T h r e s h o l d i n g and c l u s t e r i n g t e c h n i q u e s a r e g l o b a l o p e r a t o r s w h i c h use some a g g r e g a t e p r o p e r t i e s o f d i f f e r e n t f e a t u r e s . T h e s e f e a t u r e s a r e v e r y d e p e n d e n t on t h e t y p e o f r e g i o n s w h i c h a r e segmen ted i n t h e image . A l t h o u g h t h e segmen ted r e g i o n s a r e c l o s e d , some images may r e q u i r e s m o o t h i n g t o e l i m i n a t e t h e n o i s y b o u n d a r i e s . S i n c e no s p a t i a l i n f o r m a t i o n i s u s e d i n t h e s e l e c t i o n o f t h e t h r e s h o l d , t h e r e s u l t i n g r e g i o n s may n o t be c o n t i g u o u s . 3 . 3 Edge D e t e c t i o n Edge d e t e c t i o n a l g o r i t h m s use t h e i n f o r m a t i o n o f edge p o i n t s t o d e t e r m i n e t h e b o u n d a r y b e t w e e n o b j e c t s . The edge p o i n t s a r e l o c a t e d F i g u r e 6 USE OF CLUSTERING IN OBJECT DISCRIMINATION O b j e c t s i n g roup A c a n be e a s i l y s e p a r a t e d f r om t h o s e i n g roup B i n t h e t w o - d i m e n s i o n a l ( x , y ) f e a t u r e p l o t . T h i s i s n o t t h e c a s e i n e i t h e r o f the o n e - d i m e n s i o n a l f e a t u r e p l o t s . 20 where t h e r e i s a n a b r u p t change i n g r a y l e v e l s i n t h e i m a g e . I n t h i s t e c h n i q u e , t h e e l e m e n t s w h i c h a r e c a n d i d a t e s t o b e l o n g t o an edge a r e f i r s t e x t r a c t e d and t h e n c o m b i n e d t o f o r m t h e b o u n d a r y . The e x t r a c t i o n o f edge p i x e l s r e q u i r e s a measure w h i c h c o r r e s p o n d s t o t he change i n g r a y v a l u e o f t h e p i x e l w i t h i t s s u r r o u n d i n g . V a r i o u s m e t h o d s , s u c h as t h e g r a d i e n t , S o b e l , K i r s c h , and P r e w i t t o p e r a t o r s ( R o s e n f e l d and K a k , 1982 ; Young and F u , 1 9 8 6 ) , h a v e b e e n d e v e l o p e d f o r t h i s p u r p o s e . These o p e r a t o r s c a n be i m p l e m e n t e d as a s e r i e s o f image c o n v o l u t i o n s where t he w e i g h t s i n t he c o n v o l u t i o n k e r n e l i s d i f f e r e n t f o r e a c h f i l t e r . The r e s u l t i n g v a l u e g i v e s a n i n d i c a t i o n o f the s t r e n g t h o f t h e changes a r o u n d e a c h p i x e l . The edge p o i n t s a r e t h e n e x t r a c t e d b y t h r e s h o l d i n g t he p r o c e s s e d i m a g e . M a r r (1982) h a s d e v e l o p e d a L a p l a c i a n o f a G a u s s i a n edge f i l t e r . I n t h i s me thod , t h e z e r o - c r o s s i n g s o f t h e f i l t e r c o r r e s p o n d t o t he edges o f t h e s t r u c t u r e s w h i c h h a v e a s p a c e c o n s t a n t g r e a t e r t h a n ( o r a l o w e r s p a t i a l f r e q u e n c y t h a n ) a s e l e c t e d v a l u e u s e d i n t h e G a u s s i a n b l u r r i n g p r o c e s s . The re a r e s e v e r a l p r o b l e m s w i t h edge d e t e c t i o n t e c h n i q u e s . T h i s i s b e c a u s e t h e t r a n s i t i o n f r o m one r e g i o n o f t h e image t o t h e o t h e r somet imes o c c u r s o v e r s e v e r a l p i x e l s and i s t h e n n o t a b r u p t e n o u g h . The c o n t o u r s p r o d u c e d f r o m t h r e s h o l d i n g edge i n f o r m a t i o n a r e g e n e r a l l y more t h a n one p i x e l w i d e and n o t n e c e s s a r i l y c l o s e d . H e n c e , some p o s t -p r o c e s s i n g u s i n g t h i n n i n g and c o n t o u r - c l o s i n g a l g o r i t h m i s r e q u i r e d . A n o t h e r p r o b l e m i s t h a t t h e t e x t u r e o f some r e g i o n s a r e s i g n i f i c a n t enough t o be t h r e s h o l d e d as edge p o i n t s r e s u l t i n g i n e r r o n e o u s image 21 s e g m e n t a t i o n . N e v e r t h e l e s s , t h e r e s u l t s f r o m t h e edge d e t e c t i o n t e c h n i q u e s c a n be u s e d i n c o n j u n c t i o n w i t h o t h e r methods i n d e t e r m i n i n g p a r t i c u l a r r e g i o n s . 3 . 4 R e g i o n E x t r a c t i o n A n o t h e r a p p r o a c h t o s e g m e n t a t i o n i s t o g roup p i x e l s w i t h s i m i l a r p r o p e r t i e s , s u c h as g r a y l e v e l s , t e x t u r e , c o l o r i n f o r m a t i o n , e t c . , i n t o r e g i o n s . These r e g i o n e x t r a c t i o n t e c h n i q u e s c a n be s e p a r a t e d i n t o t h r e e c a t e g o r i e s : r e g i o n m e r g i n g , r e g i o n s p l i t t i n g , and a c o m b i n a t i o n o f r e g i o n m e r g i n g and s p l i t t i n g . I n r e g i o n m e r g i n g o r g r o w i n g t e c h n i q u e s , t he image i s i n i t i a l l y d i v i d e d i n t o many s m a l l r e g i o n s s u c h as a p i x e l o r a s m a l l n e i g h b o u r h o o d o f p i x e l s . V a r i o u s p r o p e r t i e s t h a t r e f l e c t t he c h a r a c t e r i s t i c s o f t he o b j e c t a r e computed f o r e a c h r e g i o n . The c h a r a c t e r i s t i c s o f e a c h r e g i o n a r e compared w i t h i t s n e i g h b o u r i n g r e g i o n s . I f t h e p r o p e r t i e s o f t he a d j a c e n t r e g i o n s a r e s i m i l a r , t h e s e r e g i o n s a r e c o m b i n e d o r merged i n t o o n e . T h i s p r o c e s s i s i t e r a t e d by r e c o m p u t i n g t h e o b j e c t membersh ip p r o p e r t i e s f o r e a c h e n l a r g e d r e g i o n and m e r g i n g t h e r e g i o n s w h i c h have s i m i l a r c h a r a c t e r i s t i c s . The s e g m e n t a t i o n i s c o m p l e t e d when a l l a d j a c e n t r e g i o n s have s i g n i f i c a n t l y d i f f e r e n t p r o p e r t i e s s u c h t h a t no merge c a n f u r t h e r be made. The r e g i o n s p l i t t i n g o r d i v i d i n g t e c h n i q u e s b e g i n w i t h t h e e n t i r e image i n s t e a d o f many s m a l l r e g i o n s . A p r e d i c a t e d e s c r i b i n g t h e v a r i o u s 22 p r o p e r t i e s o f t h e o b j e c t i s e v a l u a t e d f r o m the e n t i r e r e g i o n . A n examp le i s t o d e t e r m i n e i f a l l p i x e l s i n t h e r e g i o n h a v e g r a y l e v e l s w h i c h do n o t d i f f e r b y a c e r t a i n amount . I f t h e p r e d i c a t e i s n o t s a t i s f i e d , t h e r e g i o n i s d i v i d e d i n t o s m a l l e r r e g i o n s and t h e p r e d i c a t e f o r e a c h o f t h e s u b - r e g i o n s i s r e c o m p u t e d . The p r o c e s s c o n t i n u e s u n t i l t h e p r e d i c a t e s f o r a l l r e g i o n s a r e s a t i s f i e d . The s p l i t and merge t e c h n i q u e u s e s a c o m b i n a t i o n o f r e g i o n m e r g i n g and s p l i t t i n g t o o b t a i n r e g i o n s o f s i m i l a r p r o p e r t i e s . R e g i o n s a r e merged when a d j a c e n t r e g i o n s have s i m i l a r p r o p e r t i e s and a r e s p l i t when t h e p r e d i c a t e d e s c r i b i n g t he p r o p e r t y i s n o t s a t i s f i e d . L i e d t k e e t a l . (1987) u s e d t h i s t e c h n i q u e on b l o o d c e l l s t o e x t r a c t t h e p r i m i t i v e s u s e d i n h i s s e g m e n t a t i o n me thod . R e g i o n e x t r a c t i o n t e c h n i q u e s u t i l i z e t h e l o c a l p r o p e r t i e s o f t h e image d i r e c t l y . A l t h o u g h t h e y p r o d u c e c l o s e d and c o n t i g u o u s r e g i o n s , t he d rawback i s t h a t t h e s e a l g o r i t h m s a r e c o m p u t a t i o n a l l y i n t e n s i v e . 23 Chapter 4 B lood Ce l l Analysis Algori thms 4 . 1 O v e r v i e w A p r o g r a m was d e s i g n e d and w r i t t e n t o i n c o r p o r a t e t h e a l g o r i t h m s w h i c h a r e d e v e l o p e d t o a u t o m a t i c a l l y segment n u c l e a t e d b l o o d c e l l s . The a p p r o a c h f o r a n a l y z i n g t h e s e c e l l s i n v o l v e s t he f o l l o w i n g s e v e n s t e p s : i ) a c q u i s i t i o n o f i m a g e s , i i ) p r e - p r o c e s s i n g t h e a c q u i r e d i m a g e s , i i i ) d e t e c t i o n o f p o s s i b l e c e l l s i n t h e s c e n e , i v ) s e g m e n t a t i o n o f t h e c e l l s i n t h e s c e n e , v ) p o s t - p r o c e s s i n g t he segmen ted r e g i o n s , v i ) e x t r a c t i o n o f f e a t u r e s , and v i i ) c l a s s i f i c a t i o n o f t h e o b j e c t ( F i g u r e 7 ) . The f i r s t two s t e p s , a c q u i r i n g and p r e - p r o c e s s i n g t h e i m a g e s , a r e c r i t i c a l s i n c e h i g h q u a l i t y i n p u t images w i l l s i m p l i f y and r e d u c e t he amount o f p r o c e s s i n g r e q u i r e d i n t h e l a t e r s t a g e s o f t he a n a l y s i s . The m a n i p u l a t i o n o f s p e c t r a l i n f o r m a t i o n i s u s e d i n t he f o l l o w i n g s t e p t o d e t e c t t h e c e l l s i n t he s c e n e . A l l n u c l e a t e d c e l l s a r e s e l e c t e d f r o m the b a c k g r o u n d and n o n - n u c l e a t e d r e d b l o o d c e l l s . Once t h e c e l l l o c a t i o n s a r e f o u n d , a l l s i n g l e c e l l s a r e a c c o u n t e d f o r and a l s o any c e l l s w h i c h a r e j u s t t o u c h i n g a r e s e p a r a t e d and t h e b o u n d a r y o f e a c h i s o l a t e d c e l l i s d e t e r m i n e d . The n e x t s t e p i s t o segment t h e i s o l a t e d c e l l s i n t o two r e g i o n s : n u c l e u s and c y t o p l a s m . P o s t - p r o c e s s i n g o f t he d e f i n e d r e g i o n s i s r e q u i r e d t o f i n e - t u n e t he mask o f e a c h r e g i o n . F e a t u r e s a r e t h e n c a l c u l a t e d b a s e d on t h e d e f i n e d r e g i o n b o u n d a r i e s . The c e l l s i n t h e s c e n e a r e t h e n c l a s s i f i e d b a s e d on t h e v a l u e s o f t h e f e a t u r e s . 24 IMAGE ACQUISITION 1 IMAGE PRE-PROCESSING OBJECT DETECTION I IMAGE SEGMENTATION I IMAGE POST-PROCESSING i • ~ FEATURE EXTRACTION i OBJECT CLASSIFICATION F i g u r e 7 BLOCK DIAGRAM OF THE PROCEDURES TO ANALYZE BLOOD CELLS A l g o r i t h m s a r e e m p l o y e d i n t h e a c q u i s i t i o n and p r e - p r o c e s s i n g s e c t i o n s t o e n s u r e t h a t t h e images u s e d i n t h e a n a l y s i s a r e c o r r e c t e d f o r . E r r o r s i n t h e d e t e c t i o n and s e g m e n t a t i o n s e c t i o n s s h o u l d be m i n i m i z e d t o l i m i t t h e i r p r o p a g a t i o n t o t h e l a t e r p a r t s o f t h e a n a l y s i s . F e a t u r e s are e x t r a c t e d f r o m t h e segmented r e g i o n s and t he c l a s s i f i c a t i o n o f t he o b j e c t i s made b a s e d on t h e f e a t u r e v a l u e s . 25 The most d i f f i c u l t p o r t i o n o f t h e a n a l y s i s i s t o d e f i n e t h e r e g i o n s o f t h e c e l l . U n l i k e f e a t u r e e x t r a c t i o n where o n l y m a t h e m a t i c a l a l g o r i t h m s w i l l s u f f i c e , s e g m e n t a t i o n a l s o r e q u i r e s p r i o r k n o w l e d g e o f t he g e o m e t r i c a l , m o r p h o l o g i c a l , and t o p o l o g i c a l p r o p e r t i e s o f t h e c e l l s i n t he s c e n e as w e l l as a h e u r i s t i c a p p r o a c h f o r a n a l y z i n g t h e p r o b l e m . S i n c e o b j e c t c l a s s i f i c a t i o n i s b a s e d on f e a t u r e v a l u e s w h i c h a r e d e r i v e d f r o m t h e segmen ted r e g i o n s , t he s e g m e n t a t i o n i s c r u c i a l f o r t h e c o r r e c t i n t e r p r e t a t i o n o f t he c e l l s i n t he s c e n e . 4 . 2 Image A c q u i s i t i o n S i n c e e a c h r e g i o n o f t h e b l o o d c e l l i s s t a i n e d a d i f f e r e n t c o l o u r and m u l t i - s p e c t r a l a n a l y s i s i s u s e d , two o r more images t a k e n w i t h d i f f e r e n t c o l o u r f i l t e r s must be o b t a i n e d . Camera c a l i b r a t i o n must be p e r f o r m e d a t t h e b e g i n n i n g o f e a c h e x p e r i m e n t t o e n s u r e c o r r e c t c o l o u r r e g i s t r a t i o n o f t h e i m a g e . T h i s i s a c c o m p l i s h e d by a d j u s t i n g t h e g a i n and o f f s e t o f t h e a m p l i f i e r s f o r e a c h c o l o u r component ( r e d , g r e e n and b l u e ) s u c h t h a t a s i m i l a r l i g h t l e v e l i s o b s e r v e d a t t h e o u t p u t o f e a c h c h a n n e l when o n l y t h e b a c k g r o u n d l i g h t i s m e a s u r e d . L i g h t s o u r c e and f rame g r a b b e r c a l i b r a t i o n i s a l s o e x e c u t e d b e f o r e t he e x p e r i m e n t b e g i n s . I n o r d e r t o u t i l i z e t h e f u l l p h o t o m e t r i c r ange o f t h e d i g i t i z e r (8 b i t s o r 256 g r e y l e v e l s ) , t h e v o l t a g e l e v e l o f t he l i g h t s o u r c e and t he g a i n and o f f s e t o f t h e a n a l o g u e t o d i g i t a l 26 c o n v e r t e r o f t h e f rame g r a b b e r b o a r d a r e a d j u s t e d a c c o r d i n g l y . The l e v e l o f t h e b a c k g r o u n d , w h i c h i s t h e b r i g h t e s t p a r t o f t h e image , i s s e t t o a p p r o x i m a t e l y f i v e p e r c e n t b e l o w t h e maximum t o a v o i d image s a t u r a t i o n . The d a r k e s t s t a i n e d n u c l e u s i n t h e image i s s e t t o a p p r o x i m a t e l y f i v e p e r c e n t above t h e minimum d e t e c t a b l e l e v e l . I n t h e a n a l y s i s , images f r o m e a c h o f t h e r e d , g r e e n , and b l u e c o l o u r s p e c t r u m a r e a c q u i r e d . S i n c e t h e s c e n e i s s t a t i c , an a v e r a g e o f a number o f images f r o m e a c h s p e c t r u m i s t a k e n b e f o r e h a n d . T h i s a v e r a g i n g h a s t h e e f f e c t o f r e d u c i n g t h e random n o i s e i n t r o d u c e d b y t he l i g h t s o u r c e , d e t e c t o r and d i g i t i z e r . S i n c e e a c h d e t e c t e d image I ^ ( x , y ) , c a n be r e p r e s e n t e d as a sum o f a s t a t i o n a r y p a r t S ( x , y ) , and a p a r t c o n t a i n i n g random n o i s e N ^ ( x , y ) , i . e . I ^ x . y ) - S ( x , y ) + N j / x . y ) (1) t he a v e r a g e o f M images i s o f t he f o r m 1 M I ( x . y ) - J Z { S ( x , y ) + N ^ x . y ) } . (2) i - 1 I f t h e n o i s e i s assumed t o be u n c o r r e l a t e d w i t h t h e i m a g e , t h e n i t c a n be shown t h a t t h e power s p e c t r u m o f t he n o i s e (^NN^ ' *" s r e d u c e d by a f a c t o r o f M ( C a s t l e m a n , 1 9 7 9 ; P r a t t , 1 9 7 9 ) , i . e . (3) 27 where ( S j r ^ ) a v i s t h e power s p e c t r u m o f t h e n o i s e i n t h e a v e r a g e d i m a g e . I f t h e n o i s e i s assumed t o h a v e a G a u s s i a n d i s t r i b u t i o n , t h e n t h e s t a n d a r d d e v i a t i o n a, o f t h e random n o i s e i s r e d u c e d b y t h e s q u a r e r o o t o f t h e number o f f r a m e s a v e r a g e d , i . e . a a v " jk ai <*> where <7av i s t he s t a n d a r d d e v i a t i o n o f t h e n o i s e i n the a v e r a g e d image . Due t o t h e q u a n t i z a t i o n l i m i t , t he s t a n d a r d d e v i a t i o n o f t h e a v e r a g e d image i s c h o s e n t o be a t most a h a l f o f a g r e y l e v e l ( f f a v < 0 . 5 ) . A c h i e v i n g t h i s a v e r a g e n o i s e l e v e l i m p l i e s t h a t t h e number o f f r ames r e q u i r e d f o r e a c h samp le ( i . e . t h e number o f f r a m e s t o be a v e r a g e d ) s h o u l d a t l e a s t be f o u r t i m e s t h e v a r i a n c e o f t h e d e t e c t e d random n o i s e , i . e . M > 4 o^2 (5) The s t a n d a r d d e v i a t i o n o f t he g r e y l e v e l o f t h e random n o i s e i n t h e a c q u i r e d s p e c t r a l images r a n g e s f r o m 2 t o 3 . H e n c e , M, t h e number o f f r a m e s a v e r a g e d f o r e a c h image a n a l y z e d , i s c h o s e n t o be 36 ( F i g u r e 8 ) . 4 . 3 Image C a l i b r a t i o n A l t h o u g h random n o i s e c a n be r e d u c e d b y a v e r a g i n g , f i x e d p a t t e r n n o i s e must be c o r r e c t e d by u s i n g o t h e r means. These n o i s e p a t t e r n s a r e 28 32 Pixels H F i g u r e 8 NOISE REDUCTION DUE TO IMAGE AVERAGING The v a r i a t i o n i n b a c k g r o u n d i n t e n s i t y i n t h e o r i g i n a l image and i t s h i s t o g r a m ( t o p ) c a n r e a d i l y be s e e n . The c o l o r m a p p i n g t a b l e i s a d j u s t e d t o h i g h l i g h t s m a l l changes i n i n t e n s i t y l e v e l s . The same image was c a p t u r e d 36 t i m e s and a v e r a g e d (bo t t om) t o r e d u c e the n o i s e v a r i a t i o n s . 29 p r o d u c e d b y v a r i o u s components i n t h e i m a g i n g s y s t e m . U n e q u a l s e n s i t i v i t y b e t w e e n e l e m e n t s o f t h e d e t e c t o r r e s u l t s i n d i f f e r e n t g r a y l e v e l v a l u e s among p i x e l s t h a t have r e c e i v e d t h e same i l l u m i n a t i o n . Some camera c o n t r o l c i r c u i t r i e s g e n e r a t e e v e n l y s p a c e d v e r t i c a l b a r s w i t h s l i g h t l y v a r y i n g i n t e n s i t i e s . S h a d i n g and a b e r r a t i o n e f f e c t s c a u s e d b y t h e o p t i c s a l s o p r o d u c e uneven i l l u m i n a t i o n a t t h e d e t e c t o r r e s u l t i n g i n a b r i g h t e r s p o t n e a r t he c e n t r e o f t h e image w h i c h f a d e s t o t he e d g e s . D e c a l i b r a t i o n i s one method w h i c h i s u s e d t o c o r r e c t t h e f i x e d p a t t e r n n o i s e . E a c h p i x e l i n t he image I ( x , y ) i s t r a n s f o r m e d i n t o a new image C ( x , y ) , u s i n g an e q u a t i o n i n v o l v i n g t h e b r i g h t b a c k g r o u n d image B ( x , y ) , a d a r k image D ( x , y ) , and a c o n s t a n t s c a l i n g f a c t o r k: nt \ i H x . y ) - D(x .v ' ) . , . T h i s f o r m u l a r e p r e s e n t s t he t r a n s m i t t a n c e o f t h e image where e a c h p i x e l i s c o r r e c t e d f o r by t h e v a l u e s o f the c a l i b r a t i o n i m a g e s . A range o f b r i g h t and d a r k images i s u s e d t o l i n e a r l y map t h e d e t e c t e d image t o a s c a l e o f 0 t o k. B e c a u s e d i v i s i o n i s u s e d , t r u n c a t i o n e r r o r i s l i k e l y t o d i s t o r t t h e d i s t r i b u t i o n o f g r a y l e v e l s . I n b r i g h t f i e l d m i c r o s c o p y , t h e d a r k image u s u a l l y h a s a g r a y l e v e l o f 0 and t he b r i g h t image h a s a i n t e n s i t y r a n g i n g f r o m 200 t o 2 2 0 . H e n c e , up t o 10% improvement i s s e e n u s i n g t he d e c a l i b r a t e d i n s t e a d o f t he raw i m a g e . 30 Background subtraction of o p t i c a l d e n s i t i e s can be used instead to achieve a s i m i l a r r e s u l t . This method i s based on the conversion of the transmittance to o p t i c a l density by taking logarithm of the p i x e l values; the dark image i s assumed to be zero i n t h i s case. where the background i s subtracted from the o r i g i n a l o p t i c a l image and a constant value of K i s added to o f f s e t the i n t e n s i t y d i s t r i b u t i o n . Taking logarithms on a d i s c r e t e image w i l l produce gaps i n the d i s t r i b u t i o n of the o p t i c a l density p r o f i l e which adds complications l a t e r i n the analysis of the images. A t h i r d approach to correct images i s to subtract the b r i g h t image and then add an o f f s e t equal to an average value of the b r i g h t image, i . e . This approach was used to analyze white blood c e l l s i n t h i s work (Figure 9 ) . The method adjusts each p i x e l by adding an o f f s e t such that a b r i g h t background image appears to have equal gray l e v e l value for a l l p i x e l s . Most of the image c o n s i s t i n g of background p i x e l s i s c o r r e c t l y adjusted f o r . The advantage of t h i s method i s that no truncation error i s introduced since only subtraction of integers i s involved. Although the approach i s not as good as the f i r s t two methods, i t does serve as a good approximation f o r image correction. The error i n the c o r r e c t i o n log C(x,y) - log I(x,y) - log B(x,y) + K (7) C(x,y) = I(x,y) - B(x,y) + K. (8) 50K + 40K • C 30K-I u 3 cr g 20K--h 10K--0 4—V + 230 240 250 Intensity 256 Pixels +\ F i g u r e 9 NOISE REDUCTION USING BACKGROUND SUBRACTION The a b e r r a t i o n and s h a d i n g e f f e c t s i n t h e o r i g i n a l image and i t s h i s t o g r a m ( t o p ) c a n r e a d i l y be s e e n . The c o l o r m a p p i n g t a b l e i s a d j u s t e d t o h i g h l i g h t s m a l l changes i n t h e i n t e n s i t y l e v e l s . B a c k g r o u n d s u b t r a c t i o n ( t h e t h i r d method as d e s c r i b e d i n t he t e x t ) was t h e n a p p l i e d t o t h e image (bo t t om) t o remove t h i s s h a d i n g e f f e c t . 32 does i n c r e a s e i n t h e d a r k e r r e g i o n s where t h e s t a i n e d c e l l s l i e , b u t t he change i n t h e e r r o r i s g r a d u a l and i n s i g n i f i c a n t i n a f f e c t i n g t h e s e g m e n t a t i o n a l g o r i t h m s . 4 . 4 R e c o g n i t i o n o f N u c l e a t e d C e l l The f i r s t s t e p i n t h e a n a l y s i s i s t o d e t e r m i n e i f t h e r e a r e any n u c l e a t e d c e l l s o f i n t e r e s t i n t he image . S i n c e c o l o u r i s u s e d f o r v i s u a l d e t e c t i o n o f n u c l e a t e d c e l l s , r e d , g r e e n , and b l u e s p e c t r a l i n f o r m a t i o n was u s e d . To i l l u s t r a t e t h i s p o i n t , two c o l o u r h i s t o g r a m s w i t h c o l o u r i n f o r m a t i o n on e a c h o f i t s two a x i s , and t he f r e q u e n c y o f o c c u r r e n c e o f a p a r t i c u l a r c o l o u r p a i r on t h e t h i r d a x i s was g e n e r a t e d as shown i n F i g u r e 1 0 . I t c a n be s e e n t h a t no s i n g l e t h r e s h o l d c a n be u s e d i n any o f t h e i n d i v i d u a l c o l o u r h i s t o g r a m s t o s e p a r a t e t he n u c l e a t e d c e l l s f r o m t h e r e s t o f t he i m a g e . Howeve r , t h e r e g i o n s o f t he n u c l e u s and t h e c y t o p l a s m o f t h e n u c l e a t e d c e l l s , , t he r e d b l o o d c e l l s , and t h e b a c k g r o u n d f o r m c l u s t e r s i n t h e two c o l o u r h i s t o g r a m s . Wermser , Haussmann and L i e d k e (1984) p r o p o s e d a method f o r a n a l y s i s o f a one d i m e n s i o n a l h i s t o g r a m on Pappenhe im s t a i n e d p e r i p h e r a l b l o o d smears i n s t e a d o f t h e more t i m e c o n s u m i n g m u l t i d i m e n s i o n a l c l u s t e r a n a l y s i s . I n t h i s a p p r o a c h , c h a r a c t e r i s t i c f e a t u r e X ( x , y ) , i s g e n e r a t e d f r o m a l i n e a r c o m b i n a t i o n o f t h e g r e e n G ( x , y ) , and b l u e B ( x , y ) , i m a g e s : X ( x , y ) = a G ( x , y ) + b B ( x , y ) (9) 33 CLUSTER PLOTS OF SPECTRAL IMAGES OF BLOOD CELLS A p h o t o g r a p h o f t h e i n t e n s i t y v a r i a t i o n s o f e a c h o f t h e s p e c t r a l images ( l e f t ) : r e d , g r e e n , and b l u e ( f r o m top t o b o t t o m ) , and c l u s t e r p l o t s o f e a c h o f t h e two c o l o u r s ( r i g h t ) : r e d - b l u e , r e d - g r e e n , and g r e e n - b l u e ( f r o m t o p t o b o t t o m ) , a r e shown. C l u s t e r i n g o f t he (A) b a c k g r o u n d , (B) r e d b l o o d c e l l s , (C) c y t o p l a s m , and (D) n u c l e u s a r e a s c a n be s e e n . 34 where a and b are constants using values of a=0.390 and b-0.546. A histogram of the new image i s generated and a simple threshold at the v a l l e y of the histogram of t h i s c h a r a c t e r i s t i c feature i s used to separate the red blood c e l l s from the cytoplasm of nucleated c e l l s . A s i m i l a r approach can be employed with the Wright's stained c e l l s used i n t h i s work. The c h a r a c t e r i s t i c feature uses the red, R(x,y), and blue, B(x,y), images i n a formula: X(x,y) = R(x,y) - B(x,y) +128 (10) This method involves simple subtraction of the images. No f l o a t i n g point arithmetic i s involved which would introduce truncation errors. The constant value of 128 i s added to s h i f t the o r i g i n such that values from -128 to 127 are mapped to the values 0 to 255. To extract the nucleated c e l l s i n the image, thresholding on the histogram of the smoothed subtracted image i s performed. The subtracted image i s f i r s t averaged to remove speckled noise at the edges of the red blood c e l l s . This f i l t e r consists of convoluting the image with a 3x3 kernel of weights equal to l ' s and d i v i d i n g the r e s u l t by a fac t o r of 9. A histogram of t h i s f i l t e r e d image i s produced (Figure 11). The threshold T, near the v a l l e y i s found by s e l e c t i n g the f i r s t point to the l e f t of the peak where p i x e l s belonging to the red blood c e l l s and background are represented i n the histogram H(T), and which has a gradient l e s s than 2% of the maximum peak value P m a x , i . e . 35 Intensity (Grey Levels) Figure 11 SEGMENTATION OF NUCLEATED CELLS The p h o t o g r a p h o f t h e b l u e image s u b t r a c t e d f r o m t h e r e d image i s shown a t t h e t o p . B e c a u s e o f t h e n o i s e i n t he i m a g e , a f i l t e r e d v e r s i o n o f the image h i s t o g r a m i s u s e d t o d e t e r m i n e t h e l o c a t i o n o f t h e t h r e s h o l d ( b o t t o m ) . 36 H(T ) - H ( T - l ) < 2% P m a x (11) A l l p i x e l s b e l o w t h i s t h r e s h o l d a r e s e t t o a g r a y l e v e l o f 255 ( t h e c e l l mask) and a l l o t h e r p i x e l s a r e s e t t o 0 . The 2% P m a x g r a d i e n t t h r e s h o l d i s s e l e c t e d e x p e r i m e n t a l l y t o o p t i m a l l y encompass n o t o n l y t h e n u c l e a t e d c e l l s b u t a l s o s e v e r a l p i x e l s b e l o n g i n g t o t h e r e d b l o o d c e l l s . I f a h i g h e r g r a d i e n t t h r e s h o l d i s s e l e c t e d ( g r e a t e r t h a n 2% P m a x ) , more r e d b l o o d c e l l s w i l l be i n c l u d e d i n t he c e l l mask. I f a l o w e r g r a d i e n t v a l u e i s c h o s e n , some o f c y t o p l a s m o f t h e n u c l e a t e d c e l l s w i l l be l o s t t o t h e b a c k g r o u n d r e g i o n . A 3x3 m e d i a n f i l t e r i s a p p l i e d on t he c e l l mask t o f i l l t h e h o l e s i n t h e w h i t e b l o o d c e l l s and t o remove t h e unwan ted p o i n t s i n t h e r e d b l o o d c e l l s and b a c k g r o u n d . T h i s f i l t e r a c t s as a l o w p a s s f i l t e r r e m o v i n g any h i g h s p a t i a l f r e q u e n c i e s f r o m t h e i m a g e . B e c a u s e a b i n a r y image i s u s e d as t h e i n p u t , t h e f i l t e r c a n be i m p l e m e n t e d as a l o c a l 3x3 a v e r a g e o p e r a t i o n on e a c h p i x e l and t h e n t he r e s u l t i s t h r e s h o l d e d a t t h e 128 l e v e l t o g e n e r a t e t h e new b i n a r y mask. A s i n g l e d i l a t i o n f o l l o w e d b y two e r o s i o n s u s i n g a 3x3 window a r e a p p l i e d t o t h i s b i n a r y image t o f i l l t h e l a r g e r h o l e s and smooth t he c o n t o u r o f t h e n u c l e a t e d c e l l mask ( F i g u r e 1 2 ) . I n t h e d i l a t i o n p r o c e s s , a p i x e l i s i n c l u d e d i n t he new c e l l mask, i f any o f i t s e i g h t a d j a c e n t n e i g h b o u r s b e l o n g i n t he c e l l mask. I n t h e e r o s i o n p r o c e s s , a p i x e l i s removed f r o m the c e l l mask i f any o f i t s e i g h t a d j a c e n t n e i g h b o u r s does n o t b e l o n g t o t h e mask. 37 F i g u r e 12 SMOOTHING THE NUCLEATED CELL MASK The p r o c e s s o f t h e o p e r a t i o n s u s e d t o smooth t h e n u c l e a t e d c e l l mask i s shown. The o r i g i n a l n u c l e a t e d c e l l mask i s shown i n t h e t o p l e f t . A l ow p a s s f i l t e r i s f i r s t a p p l i e d t o the mask ( t o p r i g h t ) , f o l l o w e d by a d i l a t i o n ( bo t t om l e f t ) and two e r o s i o n s ( b o t t o m r i g h t ) . J a g g e d edges a re smoo thed , h o l e s a r e f i l l e d , and s m a l l f r a g m e n t s a r e removed by t hese p r o c e s s e s . 38 4 . 5 B o u n d a r y D e t e c t i o n o f S i n g l e C e l l s Some o f t h e n u c l e a t e d c e l l s e x t r a c t e d f r o m t h e mask may be u s e f u l f o r f u r t h e r a n a l y s i s . B e c a u s e o f t he c o m p l e x i t y and t h e h i g h e r r o r r a t e i n s e g m e n t a t i o n , c e r t a i n a r r a n g e m e n t s o f c e l l s c a n n o t be a n a l y z e d . These i n c l u d e o v e r l a p p i n g n u c l e a t e d c e l l s o r c e l l s w h i c h a r e t oo c l o s e t o g e t h e r t h a t e v e n a human o b s e r v e r w o u l d have a h a r d t i m e s e g m e n t i n g them p r o p e r l y . H e n c e , o n l y n u c l e a t e d c e l l s w h i c h a r e s t a n d i n g a l o n e o r a r e j u s t t o u c h i n g were u s e d f o r f u r t h e r a n a l y s i s . To e x t r a c t t h e s e c e l l s , t he n u c l e a t e d c e l l mask i s s e a r c h e d u n t i l t he b o u n d a r y o f a c e l l i s e n c o u n t e r e d . A b o u n d a r y c h a i n code s t a r t i n g a t t h i s p o i n t i s t h e n g e n e r a t e d . The c h a i n code i s a b o u n d a r y n u m b e r i n g scheme t h a t l a b e l s e a c h b o u n d a r y p o i n t w i t h t h e d i r e c t i o n code ( F i g u r e 13) o f one o f i t s e i g h t p o s s i b l e n e i g h b o u r s as t h e n e x t b o u n d a r y p o i n t . H e n c e , a c o n s e c u t i v e l i s t o f t h e s e p o i n t s d e f i n e s t h e b o u n d a r y o f the o b j e c t . 3 2 1 4 c e n t r e 0 5 6 7 F i g u r e 13 BOUNDARY DIRECTION CODES. T h i s c o d i n g scheme i s u s e d t o l a b e l t h e b o u n d a r y p o i n t s o f t he n e x t p o i n t r e l a t i v e t o t h e c e n t r e l o c a t i o n i n t h e b o u n d a r y c h a i n c o d e . 39 A n a l g o r i t h m h a s b e e n d e v e l o p e d t o s e p a r a t e t o u c h i n g c e l l s b a s e d on t h i s b o u n d a r y i n f o r m a t i o n . The l o c a t i o n o f t h e b o u n d a r y p i x e l s c a n be c a l c u l a t e d f r o m t h e b o u n d a r y c h a i n c o d e . T h i s i n f o r m a t i o n i s u s e d t o f i n d t h e a n g l e o f t h e t a n g e n t l i n e t o e a c h p o i n t on t he o b j e c t b o u n d a r y . The l e a s t s q u a r e f i t i s u s e d t o d e t e r m i n e t h e s l o p e o f t h e t a n g e n t l i n e b a s e d on t h e n i n e p o i n t s c e n t e r e d a t t he p o i n t o f i n t e r e s t , i . e . 8 d x ( i ) = S [ x ( j + i - 4 ) - x ( i - 4 ) ] (12) j - o 8 d y ( i ) - S [ y ( j + i - 4 ) - y ( i - 4 ) ] (13) j - o where d x ( i ) and d y ( i ) a r e t h e t o t a l v a r i a t i o n s o v e r t he n i n e p o i n t s i n t he x and y d i r e c t i o n s r e s p e c t i v e l y . The a n g l e o f t h i s l i n e 9 ( i ) , r e l a t i v e t o t h e x - a x i s i s t h e n c a l c u l a t e d , i . e . Q / • \ - d v ( i ) . . . . 9 ( i ) - a r c t a n (14) By t a k i n g t h e d i f f e r e n c e o f a n g l e s d 6 ( i ) , be tween p o i n t s w h i c h a r e f o u r b o u n d a r y p o i n t s away, i . e . d 9 ( i ) - 9 ( i - 2 ) 9 ( i + 2 ) (15) 40 a n o t i c e a b l e peak i s s e e n a t t he c o r r e s p o n d i n g l o c a t i o n o f where t he two c e l l s t o u c h ( F i g u r e 1 4 ) . A v a l u e o f 2n i s added o r s u b t r a c t e d f r om t h e d i f f e r e n c e a n g l e t o e n s u r e t h a t t h e r e s u l t i s w i t h i n -n and « . A l i n e segment i s p r o d u c e d b y j o i n i n g t h e p e a k s t o s e p a r a t e t h e t o u c h i n g c e l l s . The v a l u e s o f t h i s l i n e a r e s e t t o t he b a c k g r o u n d mask l e v e l o f z e r o , t h u s s e p a r a t i n g t h e two c e l l s . A s m o o t h i n g o p e r a t i o n i s a p p l i e d t o e a c h o f t h e s e p a r a t e d o b j e c t t o smooth any c o r n e r s i n t r o d u c e d by t he s e p a r a t i o n o f t he t o u c h i n g c e l l s . A f t e r t he t o u c h i n g c e l l s a r e s e p a r a t e d , a l l o b j e c t s ( c e l l s ) w h i c h a r e w i t h i n a p r e s c r i b e d s i z e a r e u s e d f o r f u r t h e r a n a l y s i s . T h i s e l i m i n a t e s t h e u n n e c e s s a r y n e e d t o a n a l y z e o b j e c t s w h i c h a r e t o o s m a l l o r t o o l a r g e t o t r u l y r e p r e s e n t a c e l l . 4 . 6 N u c l e u s and C y t o p l a s m S e g m e n t a t i o n The n e x t s t e p i n t h e s e g m e n t a t i o n p r o c e s s i s t o d e t e r m i n e t he d i f f e r e n t r e g i o n s o f a n u c l e a t e d c e l l : t he n u c l e u s and t h e c y t o p l a s m . The mask o f e a c h s i n g l e c e l l i s o v e r l a i d on t he g r e e n s p e c t r a l image u s i n g t h e l o g i c a l AND f u n c t i o n o f t h e mask w i t h t h e image . A h i s t o g r a m o f t h i s masked image i s g e n e r a t e d . T h i s h i s t o g r a m i s g e n e r a l l y v e r y n o i s y and h a s more t h a n two d i s t i n c t p e a k s ( F i g u r e 1 5 ) . H e n c e , an a d d i t i o n a l o p e r a t i o n u t i l i z i n g t he edge i n f o r m a t i o n i s p e r f o r m e d t o h e l p d e f i n e e a c h r e g i o n more r e a d i l y and t o smooth t h e i n t e n s i t y l e v e l v a r i a t i o n s i n e a c h r e g i o n . 41 T 1 1 1 1 1 1 i 1 »-l 0 20 40 60 80 100 120 140 160 180 Boundary Point Number F i g u r e 14 SEPARATION OF TOUCHING CELLS The o u t l i n e o f a s i n g l e and two t o u c h i n g c e l l s ( t o p ) a r e shown. The p l o t s o f t he d i f f e r e n c e o f t h e a n g l e s o f t h e t a n g e n t a l o n g t h e b o u n d a r y o f t h e s i n g l e c e l l and the two t o u c h i n g c e l l s ( bo t t om) a r e shown. The two p e a k s i n t he a n g l e d i f f e r e n c e p l o t c o r r e s p o n d t o t h e i n d e n t a t i o n s f o u n d a t t he b o u n d a r y o f t he two t o u c h i n g c e l l s . 42 0 40 80 120 160 200 Intensity F i g u r e 15 HISTOGRAM OF THE SEGMENTED NUCLEATED CELLS The o r i g i n a l image i n t he g r e e n s p e c t r u m ( t o p l e f t ) and t h e n u c l e a t e d c e l l mask ( t op l e f t ) a r e shown. The mask i s o v e r l a i d on t h e o r i g i n a l image ( b o t t o m r i g h t ) t o show o n l y t he n u c l e a t e d c e l l . The h i s t o g r a m o f t he g r e e n image i s g e n e r a t e d (bo t t om r i g h t ) . 43 T h i s a d d i t i o n a l o p e r a t i o n i s t h e c o n d i t i o n a l mean f i l t e r . I n t h i s f i l t e r , t h e samp le mean M ( P ) , and t he samp le v a r i a n c e V a r ( P ) , o f t h e g r a y l e v e l s i n a l o c a l 3x3 window a t e a c h p i x e l P ( x , y ) , a r e c a l c u l a t e d . M ( P ( x , y ) ) 1 S i—1 1 S =-1 P ( x + i , y+ j ) (16) V a r ( P ( x , y ) ) = J S S ( P ( x + i , y + j ) - M ( P ( x , y ) ) } 2 (17) i - 1 j - 1 I f t h e v a r i a n c e i s b e l o w a p r e - d e f i n e d " c o n d i t i o n a l " l i m i t , t h e p i x e l v a l u e i s r e p l a c e d b y t h e mean v a l u e . O t h e r w i s e , t h e v a l u e o f t he p i x e l i s e x a m i n e d and a d j u s t e d . I f t he v a l u e i s g r e a t e r t h a n o r e q u a l t o t h e mean t h e n i t i s r e p l a c e d b y t h e sum o f t h e mean and s t a n d a r d d e v i a t i o n . O t h e r w i s e , i t i s r e p l a c e d b y t he d i f f e r e n c e o f t h e mean and s t a n d a r d d e v i a t i o n . The h i s t o g r a m o f t h e r e s u l t i n g image i s u s e d t o d e t e r m i n e t h e b o u n d a r y b e t w e e n t h e n u c l e u s and t h e c y t o p l a s m ( F i g u r e 1 6 ) . T h i s h i s t o g r a m H ( T ) , i s smoo thed by a 9x1 m e d i a n f i l t e r t o remove n o i s y s p i k e s . The p o i n t T , t o t h e r i g h t o f t h e f i r s t d a r k peak where t h e g r a d i e n t i s l e s s t h a n 5% o f t h e maximum peak P m a x , i s c h o s e n as t h e t h r e s h o l d l e v e l , i . e . H(T) - H(T+1) < 5% P (18) F i g u r e 16 PROCESS TO SEGMENT NUCLEUS AND CYTOPLASM A f i l t e r i s a p p l i e d t o t he masked image ( t o p l e f t ) and i t s h i s t o g r a m i s g e n e r a t e d ( t op r i g h t ) . A t h r e s h o l d i s d e t e r m i n e d on t h e h i s t o g r a m and the r e s u l t i n g n u c l e u s mask i s f o rmed (bo t t om l e f t ) . T h i s mask i s t h e n smoo thed ( b o t t o m r i g h t ) . 45 The gradient threshold of 5% P m a x i s experimentally chosen to be the optimal f o r generating the cytoplasm and nuclear mask. I f the threshold i s greater, the less dense ( l i g h t e r stained parts) of the nucleus w i l l be c l a s s i f i e d as cytoplasm. I f the threshold i s lower, the more dense (darker stained parts) of the cytoplasm w i l l be included i n the cytoplasm mask. A median f i l t e r and d i l a t i o n and erosion operations are performed on the r e s u l t i n g mask to f i l l the holes and smooth the boundary of the nuclear mask. 4.7 Simple Feature Extraction Features can be extracted from the regions once t h e i r boundaries are known. Several features are implemented to help v e r i f y the segmentation algorithm and to evaluate the f e a s i b i l i t y of c l a s s i f y i n g d i f f e r e n t types of blood c e l l s . These features include s i z e , i n t e n s i t y , and shape measurements as well as r a t i o s of these measurements. Size features give an i n d i c a t i o n of how large each region of the c e l l i s . These measurements include the area and perimeter of the c e l l , the cytoplasm, and the nucleus. The area measurement i s obtained by determining the number of points i n the s p e c i f i c region ( c e l l , nucleus or cytoplasm) as defined by i t s image mask M(x,y), i n a 64 p i x e l by 64 p i x e l matrix, i . e . 63 63 Area = Z 2 M(x,y) x=0 y=0 (19) 46 where M ( x , y ) i s t h e c e l l mask and has a v a l u e o f 1 i n t h e o b j e c t and 0 e l s e w h e r e . The p e r i m e t e r measurement i s o b t a i n e d u s i n g t h e i n f o r m a t i o n f rom the c h a i n c o d e . The c h a i n c o d e , d e s c r i b e d i n C h a p t e r 4 . 5 , c o n t a i n s o d d , e v e n and c o r n e r e l e m e n t s o f t h e e d g e . A w e i g h t e d sum o f t h e number o f t h e s e e l e m e n t s i s u s e d t o d e t e r m i n e t he p e r i m e t e r . T h i s method w i l l g i v e a more a c c u r a t e p e r i m e t e r v a l u e f o r c i r c u l a r l y s h a p e d o b j e c t s s i n c e i t c o m p e n s a t e s f o r edges w h i c h do n o t a l i g n w i t h t h e s q u a r e image g r i d . The f o r m u l a d e v e l o p e d b y Young (1988) i s u s e d s i n c e i t i s o p t i m i z e d f o r t h e p e r i m e t e r o f c i r c u l a r o b j e c t s , i . e . P e r i m e t e r = 1 .406 N o d d + 0 . 9 8 0 N e v e n - 0 . 0 9 1 N c o r n e r + 272 (20) The r a t i o o f a r e a s o f t h e n u c l e u s t o t h e c y t o p l a s m i s u s e d t o g i v e an i n d i c a t i o n o f t h e p r o p o r t i o n o f t he d i f f e r e n t r e g i o n s i n t h e c e l l . I n t e n s i t y f e a t u r e s g i v e an i n d i c a t i o n o f t h e amount o f s t a i n t h a t i s a s s o c i a t e d w i t h t h e n u c l e u s o f t h e c e l l . The measurements u s e d a r e t h e samp le mean i n t e n s i t y I m e a n » and t he samp le v a r i a n c e o f i n t e n s i t y I v a r i n e a c h o f t h e t h r e e c o l o u r s p e c t r u m s ( r e d , g r e e n , and b l u e ) , i . e . I = " " I ( X - I } M ( X | Y ) (21) mean A r e a x=0 y=0 63 63 [ I ( x , y ) - I m e a n ] 2 M ( x , y ) I „ Q T . - Z S : (22) v a r A r e a x=0 y=0 47 Shape features give an i n d i c a t i o n of how the nucleus of the c e l l looks l i k e . One such measurement i s c i r c u l a r i t y , i . e . o „. , . ^  Perimeter C i r c u l a r i t y - ^ ^ (23) Chapter 5 Discussion and Results 48 5.1 Data C o l l e c t i o n In order to t e s t the blood c e l l analysis algorithm, approximately 1000 c e l l s from 10 s l i d e s of blood smears were used. These s l i d e s were obtained from the Children's Hospital of B r i t i s h Columbia i n Vancouver and contained t y p i c a l v a r i a t i o n s which can be expected i n blood smear preparations. The c l a s s i f i c a t i o n of these s l i d e s was known since they had been prepared at l e a s t one year ago and the h i s t o r y and progress of each patient are known to-date. To c o l l e c t the c e l l s , randomly chosen areas on the s l i d e were brought to the microscope f i e l d of view, using the motorized x,y stage of the device. Each f i e l d was manually focussed to obtain the greatest contrast i n the image as seen on the monitor. Spectral images were then acquired and the system was programmed to automatically f i n d the nucleated c e l l s and to perform the segmentation. The r e s u l t i n g boundaries were o v e r l a i d on the c e l l images and displayed on the monitor for v i s u a l inspection. The spe c t r a l images (each 64 pi x e l s by 64 p i x e l s ) , the nucleus and cytoplasm masks, and. the l o c a t i o n of the c e l l on the s l i d e were stored f o r l a t e r observation. At the end of the search, each of the detected c e l l s was manually c l a s s i f i e d by a pathologist into 19 groups (Appendix A). The c e l l c l a s s i f i c a t i o n was 49 u s e d i n c o n j u n c t i o n w i t h t h e v a l u e s o f t h e c a l c u l a t e d f e a t u r e s t o d e t e r m i n e i f a c o r r e l a t i o n e x i s t s . S l i d e s 1 , 3 , 4 , 5 , 6 , and 8 came f r o m p a t i e n t s w i t h a c u t e l y m p h o b l a s t i c l e u k e m i a ( A L L ) and c o n t a i n a l a r g e number o f l y m p h o b l a s t c e l l s amongst t h e n u c l e a t e d c e l l p o p u l a t i o n . A l l t h e s e s l i d e s h a v e l y m p h o b l a s t c e l l s o f t h e c l a s s i f i c a t i o n o f L I t y p e w i t h t he e x c e p t i o n o f s l i d e 5 w h i c h has b o t h t h e L I and L2 s u b - c l a s s i f i c a t i o n t y p e s . S l i d e s 2 and 7 were f r om p a t i e n t s w i t h a c u t e mye logenous l e u k e m i a (AML) and t h e y c o n t a i n a l a r g e p o p u l a t i o n o f m y e l o b l a s t c e l l s . S l i d e s 9 and 10 were f r o m p a t i e n t s who h a d b e e n t r e a t e d f o r A L L and hence t h e y c o n t a i n a m i x t u r e o f t he 15 d i f f e r e n t c l a s s e s o f n o r m a l n u c l e a t e d c e l l s as w e l l as some a b n o r m a l c e l l s . 5 . 2 D e t e c t i o n A c c u r a c i e s A n i m p o r t a n t a s p e c t o f b l o o d c e l l a n a l y z e r s i s t h e i r a b i l i t y t o c o r r e c t l y d e t e c t a l l n u c l e a t e d c e l l s i n a g i v e n f i e l d and e l i m i n a t e a l l o t h e r d e b r i s . Any n u c l e a t e d c e l l s w h i c h a r e l e f t u n d e t e c t e d , e s p e c i a l l y t h o s e o f a s p e c i f i c c l a s s , may g e n e r a t e r e s u l t s w h i c h i n d i c a t e t h a t t he s l i d e i s n o r m a l where i n f a c t i t i s n o t . D e b r i s w h i c h a r e n o t e l i m i n a t e d f r o m t h e a n a l y s i s may p r o d u c e r e s u l t s t h a t i n d i c a t e a b n o r m a l i t y i n t h e s l i d e . A l t h o u g h t h i s i s n o t as s e r i o u s an e r r o r as n o t d e t e c t i n g c e l l s ( f a l s e n e g a t i v e s ) , t h e s e s l i d e s w i l l have t o be m a n u a l l y e x a m i n e d t o v e r i f y i t s n o r m a l i t y . 50 A l l nucleated c e l l s were c o r r e c t l y detected i n the selected frames. Most debris have equal i n t e n s i t y i n a l l colour spectra and hence were e a s i l y eliminated by the subtraction of the blue image from the red. Although there are many regions i n the image which belong to parts of red blood c e l l s , p l a t e l e t s , and other debris, these were generally very small and were eliminated by the erosion process and the s i z e c r i t e r i a imposed on each i s o l a t e d object. Objects which are too large (such as clumps of c e l l s ) were also eliminated by the siz e c r i t e r i o n . C e l l s which were touching the borders of the image were not included i n the data since there was not enough information to c l a s s i f y a f r a c t i o n of a c e l l . Of the 1078 detected nucleated c e l l s , 781 were i n d i v i d u a l , s i n g l e c e l l s and 297 were two or three c e l l s that were j u s t touching each another. Of these touching c e l l s , 271 were c o r r e c t l y divided into i n d i v i d u a l c e l l s , 18 had minor errors i n the p o s i t i o n of the boundary, and 8 experienced major errors where the l o c a t i o n of the boundary was misplaced (Figure 17). Minor errors i n separating touching c e l l s i s l a r g e l y due to the boundary smoothing operation applied to the nucleated c e l l mask. This smoothing operation i s performed to c o l l e c t and merge any scattered pieces which belongs to the cytoplasm a f t e r the thresholding operation. As a r e s u l t , the l o c a t i o n where two c e l l s touch i s blurred. Hence, the touching c e l l separating algorithm can choose points which are upto 3 p i x e l s away from the actual boundary p o s i t i o n . This error can be corrected by 51 Figure 17 MINOR AND MAJOR ERRORS IN SEPARATING TOUCHING CELLS The minor (top) and major (bottom) errors i n separating touching c e l l s i s shown. The separating l i n e produced by the algorithm i s shown i n black and the actual boundary l o c a t i o n i s shown i n white. This l i n e i s s l i g h t l y s h i f t e d i n the case of the minor errors. Major errors includes those which are not separated or i n c o r r e c t l y separated. 52 i n t r o d u c i n g an a l g o r i t h m w h i c h w i l l s e a r c h i n t h e n e i g h b o u r h o o d o f t he c o a r s e l y c h o s e n c e l l s e p a r a t i o n p o i n t , u s i n g t h e i n t e n s i t i e s o f t he image and o t h e r c r i t e r i a , t o l o c a t e t h e e x a c t d i v i d i n g p o i n t . M a j o r e r r o r s a r e due t o i r r e g u l a r l y s h a p e d ( n o t e l l i p t i c ) c e l l s o r c e l l s w h i c h a r e n e a r p l a t e l e t s o r f r a g m e n t e d p i e c e s o f c y t o p l a s m . The a l g o r i t h m w i l l e i t h e r s p l i t t h e s i n g l e c e l l i n t o p i e c e s o r i n c l u d e f r a g m e n t s w h i c h do n o t b e l o n g t o t he c e l l i n t o t h e n u c l e a t e d c e l l mask. Some o f t h e s e e r r o r s c a n be c o r r e c t e d f o r b y a n a l y z i n g t he f e a t u r e s o f t h e s e o b j e c t s . 5 . 3 S e g m e n t a t i o n A c c u r a c i e s A n o t h e r i m p o r t a n t c r i t e r i o n i n a n a l y z i n g b l o o d c e l l images i s t he a c c u r a c y i n d e f i n i n g t h e r e g i o n s o f t he c e l l . The c o r r e c t n e s s o f s e g m e n t a t i o n i s c r u c i a l s i n c e t h e r e s t o f t he a n a l y s i s i s b a s e d on t h e d e f i n e d r e g i o n s . The r e s u l t s o f t h e c e l l s a n a l y z e d a r e t a b u l a t e d i n T a b l e s I t o V . M i n o r c y t o p l a s m e r r o r s ( F i g u r e 18) a r e t h o s e e r r o r s where s m a l l f r a g m e n t s o f t h e c y t o p l a s m o f t h e c e l l a r e n o t i n c l u d e d i n t h e r e g i o n o r t o o much o f o t h e r a r e a s , s u c h as c y t o p l a s m o f a n o t h e r c e l l and b a c k g r o u n d , a r e i n c l u d e d i n t h e d e f i n e d c y t o p l a s m i c r e g i o n . M i n o r n u c l e u s e r r o r s ( F i g u r e 19) a r e t h o s e e r r o r s where p a r t s o f t h e c y t o p l a s m a r e i n c l u d e d o r p a r t s o f t h e n u c l e u s a r e n o t i n c l u d e d i n t h e n u c l e a r r e g i o n . The m a j o r r e a s o n f o r t h e l a r g e number o f m i n o r n u c l e u s e r r o r s i s t h e s m o o t h i n g p r o c e s s w h i c h smooths o u t s h a r p c o n c a v i t i e s p r e s e n t i n c e r t a i n t y p e s o f w h i t e b l o o d c e l l s . 5 3 Major cytoplasm errors (Figure 20) a r i s e when c e l l s are i r r e g u l a r l y shaped or when the cytoplasm of some nucleated c e l l s possess a colour very s i m i l a r to that of the red blood c e l l s and thus i s eliminated from the c e l l mask. Major nucleus errors (Figure 21) are contributed to the uneven s t a i n uptake i n the c e l l s . The cytoplasm of some c e l l s incorporates too much s t a i n r e s u l t i n g i n a larger segmented nuclear region. Some n u c l e i have very dark stained regions and thus the algorithm assigns the l i g h t e r parts of the nucleus to the cytoplasm area. The accuracy of the segmentation also depends on how well the c e l l s are focussed. The focussing range i s i n the order of f i v e microns at the chosen microscope setup. As the focus i s changed, the t r a n s i t i o n of i n t e n s i t y l e v e l s at the nucleus and cytoplasm boundary i s not abrupt enough and may r e s u l t i n errors i n defining the nucleus region. The cytoplasm of the nucleated c e l l s can also blend into the re s t of the background causing cytoplasm segmentation errors. As shown i n the Tables I to V, the error rates i n segmentation vary from s l i d e to s l i d e . This i s due to the type of c e l l s on the s l i d e as well as the way the s l i d e was prepared. In s l i d e s 9 and 10 which contain a mixture of the c e l l types i n the blood, more cytoplasm r e l a t e d errors are present because there i s a greater p r o b a b i l i t y of f i n d i n g c e l l s which have a cytoplasm colour s i m i l a r to the red blood c e l l s compared to the other eight s l i d e s . The percentage of correct segmentation ranges 54 Figure 18 MINOR CYTOPLASM ERRORS Examples of minor cytoplasm errors are shown. The algorithms e i t h e r include small fragments of the background or other c e l l s into the cytoplasm or exclude parts of i t s own cytoplasm. 55 Examples of minor nucleus errors are shown. The algorithms include parts of the cyotplasm into the nucleus region. Examples of major cytoplasm errors are shown. The algorithms exclude a major portion the cytoplasm of the c e l l from the cytoplasm region. 5 7 Figure 21 MAJOR NUCLEUS ERRORS Examples of major nucleus errors are shown. The algorithms exclude a major po r t i o n of the nucleus. 58 T a b l e I SEGMENTATION ERRORS IN NON-TOUCHING NUCLEATED C E L L S . S l i d e C o r r e c t M i n o r M i n o r M a j o r M a j o r M a j o r T o t a l Number S e g . N u c l e u s C y t o . N u c l e u s C y t o . N & C 1 82 7 2 5 2 2 100 2 93 10 0 0 0 0 103 3 63 2 2 0 2 0 69 4 72 6 0 0 0 0 78 5 68 2 3 11 0 0 84 6 67 2 1 0 0 0 70 7 58 7 1 8 0 0 74 8 59 2 3 2 0 0 66 9 56 1 1 2 5 1 66 10 58 0 4 2 7 0 71 o t a l 676 39 17 30 16 3 781 T a b l e I I PERCENTAGE ERRORS IN NON-TOUCHING NUCLEATED C E L L S . ( T a b l e I r e p r e s e n t e d as p e r c e n t a g e s ) S l i d e C o r r e c t M i n o r M i n o r C o r r e c t Maj o r M a j o r Maj o r Number S e g . N u c l e u s C y t o . & M i n o r N u c l e u s C y t o . N & C 1 8 2 . 0 7 . 0 2 . 0 9 1 . 0 5 . 0 2 . 0 2 . 0 2 9 0 . 3 9 .7 0 1 0 0 . 0 0 0 0 3 9 1 . 3 2 . 9 2 . 9 9 7 . 1 0 2 . 9 0 4 9 2 . 3 7 .7 0 1 0 0 . 0 0 0 0 5 8 1 . 0 2 . 4 3 . 6 8 7 . 0 1 3 . 1 0 0 6 9 5 . 7 2 . 8 1 .4 1 0 0 . 0 0 0 0 7 7 8 . 4 9 . 4 1 .4 8 9 . 2 1 0 . 8 0 0 8 8 9 . 4 3 . 0 4 . 6 9 7 . 0 3 . 0 0 0 9 8 4 . 8 1.5 1.5 8 7 . 8 3 . 0 7 .6 1.5 10 8 1 . 7 0 5 .6 8 7 . 3 2 .8 1 0 . 0 0 T o t a l 8 6 . 4 5 . 0 2 . 2 9 3 . 6 3 .8 2 . 0 0 . 4 59 T a b l e I I I SEGMENTATION ERRORS IN TOUCHING NUCLEATED C E L L S . S l i d e C o r r e c t M i n o r M i n o r M a j o r M a j o r Maj o r T o t a l Number S e g . N u c l e u s C y t o . N u c l e u s C y t o . N & C 1 8 0 1 1 0 0 10 2 3 0 0 0 0 0 3 3 35 0 0 1 3 0 39 4 23 0 2 1 0 0 26 5 24 1 0 3 0 0 28 6 29 2 2 0 0 0 33 7 30 5 0 0 0 0 35 8 35 8 3 1 0 0 47 9 31 0 5 1 2 0 39 10 29 0 5 0 3 0 37 T o t a l 247 16 18 8 8 0 297 T a b l e : IV PERCENTAGE ERRORS IN TOUCHING NUCLEATED C E L L S . ( T a b l e I I I r e p r e s e n t e d as p e r c e n t a g e s ) S l i d e C o r r e c t M i n o r M i n o r C o r r e c t M a j o r Maj o r M a j o r Number S e g . N u c l e u s C y t o . & M i n o r N u c l e u s C y t o . N 6c C 1 80.0 0 10.0 90.0 10.0 0 0 2 100.0 0 0 100.0 0 0 0 3 89.7 0 0 89.7 2.6 7.7 0 4 88.5 0 7.7 96.2 3.8 0 0 5 85.7 3.6 0 89.3 10.7 0 0 6 87.9 6.1 6.1 100.0 0 0 0 7 85.7 14.3 0 100..0 0 0 0 8 74.5 17.0 6.4 87.9 2.1 0 0 9 79.5 0 12.8 92.3 2.6 5.1 0 10 78.4 0 13.5 91.9 0 8.1 0 T o t a l 83.2 5.4 6.1 94.6 2.7 2.7 0 60 Table V PERCENTAGE SEGMENTATION ERRORS IN NUCLEATED CELLS. (Tables I and III combined and represented as percentages) Sl i d e Correct Minor Minor Correct Major Major Maj or Number Seg. Nucleus Cyto. & Minor Nucleus Cyto. N & C 1 81.8 6.4 2.7 90.9 5.5 1.8 1.8 2 90.6 9.4 0 100.0 0 0 0 3 90.7 1.9 1.9 94.5 0.9 4.6 0 4 91.3 5.8 1.9 99.0 1.0 0 0 5 82.1 2.7 2.7 87.5 12.5 0 0 6 93.2 3.9 2.9 100.0 0 0 0 7 80.7 11.0 1.0 92.7 7.3 0 0 8 83.2 8.8 5.3 97.3 2.7 0 0 9 82.9 0.9 5.7 89.5 2.9 6.4 0.9 10 80.6 0 8.3 88.9 1.9 9.2 0 Tota l 85.4 5.1 3.2 94.0 3.5 2.2 0.3 61 from 80.6% to 93.2%. The range of percentages f o r minor errors i n the nucleus i s 0% to 11.0%, minor error i n the cytoplasm i s 0% to 8.3%, major errors i n the nucleus i s 0% to 12.5%, and major errors i n the cytoplasm i s 0% to 1.8%. 5.4 Feature C a l c u l a t i o n Accuracies Features are c a l c u l a t e d from the regions defined by the segmentation process. Even i f there were no errors i n the segmentation, there w i l l be errors introduced i n t e s s e l l a t i n g the image to a square-pixel g r i d , and errors introduced i n the method used to c a l c u l a t e a feature. To i l l u s t r a t e these errors, a t e s t image c o n s i s t i n g of a c i r c l e i s t e s s e l l a t e d at d i f f e r e n t resolutions and the area feature i s c a l c u l a t e d . The c i r c l e image i s generated by assigning a l l points i n the g r i d which s a t i s f i e s the equation to a value of 1 and a l l other p i x e l s to a value of zero, i . e . 2 2 (x - x c) + (y - y c ) < square radius of c i r c l e (24) where x c and y c are the coordinates of the centre of the c i r c l e and x and y are the l o c a t i o n i n the g r i d . The area feature i s c a l c u l a t e d by counting the number of l ' s i n the image. Since the p o s i t i o n of a c e l l can l i e anywhere i n the image, the p o s i t i o n of the c i r c l e i s allowed to randomly vary within a p i x e l spacing. A p l o t of the percentage error from the actual value i s shown 10 100 Diameter of Circle, D (pixels) F i g u r e 22 FEATURE CALCULATION ACCURACIES The p e r c e n t a g e e r r o r s i n c a l c u l a t i n g t he a r e a o f a c i r c l e u s i n g d i f f e r e n t number o f p i x e l s t o r e p r e s e n t t h e c i r c l e i s shown. I t c a n be s e e n t h a t t he e r r o r d e c r e a s e s as more p i x e l s a r e u s e d t o r e p r e s e n t the c i r c l e . 63 i n F i g u r e 2 2 . I t c a n be s e e n t h a t i f t h e d i a m e t e r o f t h e c i r c l e spans more t h a n t e n p i x e l s i n w i d t h , an e r r o r o f l e s s t h a n two p e r c e n t i n t he a r e a c a l c u l a t i o n c a n be o b t a i n e d . A l t h o u g h t h e r e a r e e r r o r s i n t h e c a l c u l a t i o n o f f e a t u r e s , as l o n g as t h e i r d i s t r i b u t i o n f o r a c e r t a i n c l a s s o f c e l l does n o t c o n f l i c t w i t h o t h e r c l a s s e s , t h e e r r o r w i l l n o t be s i g n i f i c a n t i n c l a s s i f y i n g t h e c e l l t y p e . 5 . 5 C e l l C l a s s i f i c a t i o n The n u c l e a t e d c e l l s , w h i c h were d e t e c t e d and s e g m e n t e d , were c l a s s i f i e d by an e x p e r i e n c e d p a t h o l o g i s t i n t o a p p r o x i m a t e l y 20 g r o u p s . S i m p l e f e a t u r e s were c a l c u l a t e d f r o m t h e s e c e l l s and a r e u s e d t o d e t e r m i n e the c l a s s i f i c a t i o n o f c e r t a i n t y p e s o f c e l l s . Some c e l l t y p e s w h i c h a r e i d e n t i f i e d b a s e d on t h e i r c o l o u r i n f o r m a t i o n , s u c h . as t he p o l y c h r o m a t o p h i l i c n o r m o b l a s t and b a s o p h i l i c n o r m o b l a s t , c a n be s e p a r a t e d u s i n g t h e mean i n t e n s i t i e s o f t h e c y t o p l a s m i n t h e r e d and g r e e n s p e c t r u m s ( F i g u r e 2 3 ) . O t h e r t y p e s o f c e l l s , s u c h as the l y m p h o b l a s t and m y e l o b l a s t , c a n be s e p a r a t e d u s i n g t h e p e r i m e t e r o f t he n u c l e u s and t h e r a t i o o f t h e n u c l e u s t o c e l l a r e a f e a t u r e s ( F i g u r e 2 4 ) . I t i s e v i d e n t f r o m p l o t s o f t h e c a l c u l a t e d f e a t u r e s ( F i g u r e s 23 and 24) t h a t c l u s t e r s o f c e r t a i n c l a s s e s a r e p r e s e n t . T h i s v e r i f i e s t h a t the methods u s e d t o segment t h e r e g i o n s and t h e c a l c u l a t i o n s o f t h e f e a t u r e v a l u e s f o r t h e s e c l a s s e s a r e s u f f i c i e n t i n d e t e r m i n i n g t h e c o r r e c t c l a s s i f i c a t i o n o f some t y p e s o f n u c l e a t e d c e l l . A l t h o u g h more f e a t u r e s 64 250 2 0 0 " E i— *-» #150-CQ >, 100" Polychromatophilic Normoblast Basophilic Normoblast c a 50--0+-0 —t-50 1 100 150 Mean Intensity in the Green Spectrum 200 250 Figure 23 CLUSTER PLOT OF THE MEAN INTENSIT IES A c l u s t e r p l o t d i s p l a y i n g the mean i n t e n s i t i e s i n the green and blue spectrums of the images of two classes of blood c e l l s : polychromatophilic normoblast and ba s o p h i l i c normoblast. 65 100-7 5 -<u U o •*-> 3 3 z 5 0 -Lymphoblast Myeloblast o _o J 25-50 100 Perimeter of the Nucleus 150 200 F i g u r e 24 CLUSTER PLOT OF THE PERIMETER AND RATIO OF AREAS A c l u s t e r p l o t d i s p l a y i n g t he p e r i m e t e r o f t he n u c l e u s and t h e r a t i o o f the n u c l e u s t o c e l l a r e a o f images o f two c l a s s e s o f m a l i g n a n t b l o o d c e l l s : l y m p h o b l a s t and m y e l o b l a s t . 66 and multi-dimensional c l u s t e r analysis algorithms are required to separate the d i f f e r e n t classes of c e l l s , i t i s unnecessary to add a d d i t i o n a l algorithms to f i n e tune the boundaries of the segmented regions. Chapter 6 Conclusion and Future Suggestions 67 6.1 Overview The algorithms to i ) capture and c a l i b r a t e the image, i i ) detect and segment the c e l l s i n the image, i i i ) generate features from the segmented regions, and iv) c l a s s i f y the c e l l s based on the feature values, have been shown to be useful i n the analysis of nucleated blood c e l l s . Techniques of image averaging and background subtraction are f i r s t employed to improve the q u a l i t y of the input image. The new method of subtracting s p e c t r a l images i s shown to be very useful i n generating bi-modal grey l e v e l histogram of the r e s u l t i n g image where smoothing and threshold detection techniques are subsequentially employed to separate the nucleated c e l l s from the r e s t of the image. The new method of using the difference i n angles along the boundary of the binary image to separate touching c e l l s has allowed more c e l l s to be analyzed from the chosen areas of the s l i d e . The con d i t i o n a l mean f i l t e r has produced images where the nucleus and cytoplasm boundary i s more r e a d i l y defined. F i n a l l y , erosion and d i l a t i o n operations were found to adequately f i l l the holes i n the regions and smooth the region boundaries. Once, the regions are defined, various features can be calcul a t e d . These feature values are then compared to determine i f there are any groupings amongst the d i f f e r e n t classes of c e l l s . 68 It i s evident from the survey of cytometry systems that blood c e l l a nalysis i s important i n medical p r a c t i c e and that a l l of these systems have d i f f i c u l t y i n c l a s s i f y i n g abnormal blood c e l l s . Hence, the C e l l Analyzer Imaging System was used as the t o o l to develop and t e s t the algorithms discussed i n t h i s t h e s i s . The survey of segmentation algorithms employed to segment blood c e l l s confirmed that no p a r t i c u l a r s i n g l e algorithm w i l l work on a l l images. However, combining several d i f f e r e n t algorithms may perform well; the algorithms which work are those which are t a i l o r e d to a p a r t i c u l a r type of images one obtains. 6.2 System Performance The algorithms perform well i n analyzing the types of c e l l s i n a blood smear. Of the 1078 c e l l s chosen, 3% of the 297 touching c e l l s were not properly separated due to the odd c e l l shape. Although the boundary for 6% of these touching c e l l s i s s l i g h t l y misplaced, these c e l l s were s t i l l properly c l a s s i f i e d based on t h e i r feature values. 6% of the 781 single c e l l s were i n c o r r e c t l y segmented and 7% have s l i g h t e r r o r s . Although there are minor errors i n the segmentation, the d i s t r i b u t i o n of features extracted indicates that d i f f e r e n t classes of blood c e l l s can be distinguished. 6.3 Future Plans The algorithms developed i n t h i s thesis permit c l a s s i f i c a t i o n of several types of d i f f e r e n t white blood c e l l s . To discriminate between more 69 c l a s s e s and s u b c l a s s e s o f b l o o d c e l l s , more f e a t u r e s , b a s e d on t he segmen ted a r e a s , w o u l d have t o be d e v e l o p e d and i n c o r p o r a t e d i n t he a l g o r i t h m . F e a t u r e s s u c h as t h e mean and v a r i a n c e o f i n t e n s i t y l e v e l s a t d i f f e r e n t w a v e l e n g t h s and t h e i r r a t i o s may be u s e d . T e x t u r e f e a t u r e s w h i c h g i v e an i n d i c a t i o n o f how i n t e n s i t y l e v e l s a r e v a r y i n g i n a r e g i o n a r e a l s o good f e a t u r e s t o i n c o r p o r a t e . Once a c o l l e c t i o n o f more t h a n 20 f e a t u r e s a r e c a l c u l a t e d f o r e a c h c e l l , a c r i t e r i o n must be d e v e l o p e d t o i n t e r p r e t t h e f e a t u r e v a l u e s and u s e i t t o c l a s s i f y t h e c e l l s on t he s l i d e ( P o o n , J a g g i and P a l c i c , 1 9 8 7 ) . A l i n e a r s t e p w i s e d i s c r i m i n a n t f u n c t i o n a n a l y s i s s h o u l d be p e r f o r m on t h e f e a t u r e v a l u e s t o s e l e c t t h e f e a t u r e s w h i c h w o u l d b e s t s e p a r a t e t h e d i f f e r e n t c l a s s e s o f c e l l s . E x p e r i m e n t a t i o n i n t h e r e m o v a l o f t h o s e f e a t u r e s , w h i c h a r e c o m p u t a t i o n a l l y e x p e n s i v e , f r o m t h e a n a l y s i s s h o u l d be i n v e s t i g a t e d t o d e t e r m i n e i t s e f f e c t s on t h e c l a s s i f i c a t i o n e r r o r s . A l g o r i t h m s s h o u l d a l s o be d e v e l o p e d t o d e t e c t and d i s c a r d c e l l s t h a t a r e n o t segmen ted p r o p e r l y b a s e d on t h e s e f e a t u r e v a l u e s . S i n c e t h e s h a r p n e s s and c o n t r a s t o f t h e images a r e d e p e n d e n t on t he f o c u s , an i m p o r t a n t f e a t u r e t h a t must be d e v e l o p e d i s a u t o f o c u s (Poon e t a l . , 1 9 8 9 ) . A n a u t o f o c u s s i n g s y s t e m w i l l p r o d u c e an o b j e c t i v e and c o n s i s t e n t l e v e l o f f o c u s f r o m one image t o t h e n e x t b a s e d on some p r e -d e t e r m i n e d c r i t e r i a , whe reas a s u b j e c t i v e l e v e l o f f o c u s ( w h i c h m i g h t v a r y f r o m image t o image) w o u l d be c h o s e n i f human i n t e r v e n t i o n i s i n v o l v e d . A c r i t e r i o n , s u c h as t h e sum o f t h e a b s o l u t e i n t e n s i t y g r a d i e n t s i n t h e image c a n be u s e d t o g i v e an i n d i c a t i o n o f t h e f o c u s 70 l e v e l . A h i g h v a l u e f o r t h e sum w i l l i n d i c a t e t h a t t h e r e i s l a r g e v a r i a t i o n s i n i n t e n s i t y ( d e t a i l s ) i n t h e image w h i c h i s c h a r a c t e r i s t i c o f a f o c u s s e d i m a g e . T h u s , t h e s y s t e m w i l l d e t e r m i n e t h e opt imum f o c u s l e v e l b a s e d on t h e f e a t u r e v a l u e s o b t a i n e d a t d i f f e r e n t f o c u s l e v e l s , and i n s t r u c t t h e s t e p p i n g m o t o r s t o a d j u s t t h e f o c u s b e f o r e t he c e l l d e t e c t i o n and s e g m e n t a t i o n a l g o r i t h m s a r e i n i t i a t e d . U s i n g t h e s e a d d i t i o n a l a l g o r i t h m s , a f u l l y a u t o m a t e d b l o o d c e l l a n a l y z i n g s y s t e m c a n be d e v e l o p e d t o c l a s s i f y t h e b l o o d c e l l s on the s l i d e . The s y s t e m s h o u l d i n c l u d e a m o t o r i z e d x , y s t a g e t o s c a n the s l i d e and a mechan ism and a l g o r i t h m t o a u t o m a t i c a l l y f o c u s t h e c e l l s on t h e s l i d e . A n a u t o m a t i c s l i d e l o a d e r s h o u l d a l s o be i n c o r p o r a t e d t o p l a c e t h e s l i d e on t he m o t o r i z e d s t a g e and t o p l a c e t h o s e s l i d e s w h i c h a r e s u s p e c t e d o f b e i n g a b n o r m a l i n a d i f f e r e n t p l a c e f o r f u t u r e manua l o b s e r v a t i o n and v e r i f i c a t i o n o f t he mach ine c l a s s i f i c a t i o n . T h i s s y s t e m w o u l d a l l o w h o s p i t a l s t o s c a n a l a r g e number o f s l i d e s q u i c k l y w i t h l i t t l e human i n t e r a c t i o n , as w e l l as g e n e r a t i n g a s t a n d a r d i n c l a s s i f y i n g a b n o r m a l s l i d e s . 6 . 4 Summary o f A u t h o r ' s C o n t r i b u t i o n s The C e l l A n a l y z e r I m a g i n g S y s t e m h a s b e e n b u i l t t o a n a l y z e s t a i n e d c e l l s , u n d e r t h e s u p e r v i s i o n o f B r a n k o P a l c i c and B runo J a g g i . The a u t h o r o f t h i s t h e s i s h a s p l a y e d a m a j o r r o l e i n t he d e v e l o p m e n t o f t h i s s y s t e m , i n c l u d i n g h a r d w a r e , d e v i c e d r i v e r s , and s o f t w a r e ( J a g g i , Poon and P a l c i c , 1 9 8 6 ; P o o n , J a g g i and P a l c i c , 1987 ; P a l c i c e t a l . , 1988 ; Jaggi et a l . , 1988; Poon et a l . , 1989; Poulin et a l . , 1989; Spadinger, Poon and P a l c i c , 1989a; and Spadinger, Poon and P a l c i c , 1989b). The development of the system was designed to allow work to be performed as presented i n t h i s t h e s i s . Using t h i s system, a procedure containing algorithms f o r automatic segmentation of nucleated blood c e l l s was developed by the author. This included the methods of image a c q u i s i t i o n , corrections, and subtraction, and binary mask processing which were modified and adapted into the c e l l a nalysis procedure. New methods, using the differ e n c e angles to separate touching c e l l s and the co n d i t i o n a l mean f i l t e r to reduce i n t e n s i t y v a r i a t i o n s while preserving edges, were introduced and also incorporated into the procedure. A te s t data set of over 1000 c e l l s was used to evaluate the segmentation algorithms. The main con t r i b u t i o n to new knowledge i n t h i s f i e l d , apart from introducing several new algorithms, i s to combine the hardware and algorithms into a working system which allows s c i e n t i s t s i n the medical f i e l d r e l a t e d to leukemia diseases to study t h i s disease i n much greater d e t a i l than was possible before. 72 Chapter 7 Bibliography Abraayr W . , M a n n w e i l e r E . , O e s t e r l e D . , and Demi E . (1987) S e g m e n t a t i o n o f s c e n e s i n t i s s u e s e c t i o n s . 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(1987) S t r a t e g i e s f o r a u t o m a t e d p l a c e m e n t o f c e l l s f o r m i c r o s c o p y . A p p l i e d O p t i c s , 2 6 ( 1 0 ) : 3 3 0 8 - 3 3 1 4 Umesh R . M . (1988) A t e c h n i q u e f o r c l u s t e r f o r m a t i o n . P a t t e r n R e c o g n i t i o n , 2 1 ( 4 ) : 3 9 3 - 4 0 0 W a l t o n W.H. (1952) A u t o m a t i c C o u n t i n g o f M i c r o s c o p e P a r t i c l e s , N a t u r e , 169 Wermser D . , Haussmann G . , and L i e d k e C . E . (1984) S e g m e n t a t i o n o f b l o o d smears b y H i e r a r c h i a l t h r e s h o l d i n g . Computer V i s i o n , G r a p h i c s , and Image P r o c e s s i n g , 2 5 : 1 5 1 - 1 6 8 Young I . T . (1972) The c l a s s i f i c a t i o n o f w h i t e b l o o d c e l l s . IEEE T r a n s a c t i o n s on B i o m e d i c a l E n g i n e e r i n g , 1 9 ( 4 ) : 2 9 1 - 2 9 8 Young I . T . , and P a s k o w i t z I . L . (1975) L o c a l i z t i o n o f c e l l u l a r s t r u c t u r e s . I E E E T r a n s a c t i o n s on B i o m e d i c a l E n g i n e e r i n g , 2 2 ( 1 ) : 3 5 - 4 0 Young I . T . (1988) S a m p l i n g D e n s i t y and Q u a n t i t a t i v e M i c r o s c o p y , A n a l y t i c a l and Q u a n t i t a t i v e C y t o l o g y and H i s t o l o g y , 4 : 2 6 9 - 2 7 5 Young J . Z . and R o b e r t s F . (1951) A F l y i n g S p o t M i c r o s c o p e , N a t u r e , 1 6 7 : 2 3 1 78 Young T.Y., and Fu K.S., Eds. (1986) Handbook of Pattern Recognition  and Image Processing. Academic Press, London Zahniser D.J., Brenner J.F., and Se l l e s W.D. (1986) Spectral bandwidth i n automated leukocyte c l a s s i f i c a t i o n . Cytometry, 7:518-521 Zucker-Franklin D. , Greaves M.F., Grossi C.E., and MArmont A.M. (1988) Atl a s of Blood C e l l s : Function and Pathology. Second E d i t i o n , Edi. Ermes, Milan. Appendix A 79 The nucleated blood c e l l s are c l a s s i f i e d into the groups with the following codes. Objects with codes '0' to '9' and 'a' to 'c' belong to the normal blood c e l l s and objects with codes ' f , 'g', and 'h' belong to the malignant blood c e l l s . Code C e l l C l a s s i f i c a t i o n dead c e l l + ignore (too d i f f i c u l t to c l a s s i f y ) 0 neutophil 1 band 2 metamyelocyte 3 myelocyte 4 promyelocyte 5 b l a s t 6 orthochronic normoblast 7 polychromatophilic normoblast 8 b a s o p h i l i c normoblast 9 pronormoblast a lymphocyte b monocyte c plasma c e l l d megakaryocyte e macrophage f lymphoblast LI g lymphoblast L2 h myeloblast Refer to Zucker-Franklin et a l . (1988) and Begemann and Rastetter (1979) for examples and a d e s c r i p t i o n of these d i f f e r e n t c e l l types. P o o n , S . S . S . PUBLICATIONS: 1. J a g g i , B . , P o o n , S . and P a l c i c , B. : I m p l e m e n t a t i o n and e v a l u a t i o n o f t h e DMIPS C e l l A n a l y s e r . I E E E P r o c e e d i n g s , E n g i n e e r i n g i n M e d i c i n e and B i o l o g y 3 : 9 0 6 - 9 1 1 , 1986 . 2 . J a g g i , B . , P o o n , S . and P a l c i c , B . : O p t i c a l Memory D i s k s i n Image D a t a b a s e Management f o r C y t o m e t r y . A p p l i e d O p t i c s p u b l . by O p t i c a l S o c i e t y o f A m e r i c a 2 6 : 3 3 2 5 - 3 3 2 9 , 1987 . 3 . P o o n , S . S . S . , J a g g i , B . and P a l c i c , B . : C e l l r e c o g n i t i o n a l g o r i t h m s f o r t h e C e l l A n a l y s e r . I E E E P r o c . E n g . B i o l . 3 : 1 4 5 4 - 1 4 5 6 , 1987 . 4 . P a l c i c , B . , P o o n , S . S . S . , T h u r s t o n , G. and J a g g i , B . : Time l a p s e r e c o r d s o f c e l l s i n v i t r o u s i n g o p t i c a l memory d i s k and C e l l A n a l y z e r . J . T i s s u e C u l t u r e Methods 1 1 ( 1 ) : 1 9 - 2 2 . 1 9 8 8 . 5 . J a g g i , B . , P o o n , S . S . S . , M a c A u l a y , C . and P a l c i c , B . : I m a g i n g s y s t e m f o r m o r p h o m e t r i c a s s e s s m e n t o f c o n v e n t i o n a l l y and f l u o r e s c e n t l y s t a i n e d c e l l s . C y t o m e t r y 9 : 5 6 6 - 5 7 2 , 1988 . 6 . S p a d i n g e r , I., P o o n , S . S . S . and P a l c i c , B . : A u t o m a t e d d e t e c t i o n and r e c o g n i t i o n o f l i v e c e l l s i n t i s s u e c u l t u r e u s i n g image c y t o m e t r y . C y t o m e t r y . ( I n p r e s s , 1 9 8 9 ) . 7 . P o o n , S . S . S . , J a g g i , B . , S p a d i n g e r , I. and P a l c i c , B . : F o c u s s i n g methods u s e d i n t h e c e l l a n a l y z e r . P r o c . 1 1 t h I E E E E n g . Med . B i o l . S o c , S e a t t l e , WA. , N o v . 8 - 1 2 , 1989 ( A c c e p t e d ) . SUBMITTED PAPERS: 1. P o o n , S . S . S . , J a g g i , B. , S p a d i n g e r , I., P a l c i c , B . : F o c u s s i n g methods u s e d i n t h e C e l l A n a l y z e r . I E E E P r o c . E n g . Med . B i o l . , 1989 ( s u b m i t t e d ) . 2 . P o u l i n , N . , P o o n , S . S . S . , K a n d o l a , G . , P a l c i c , B . : A u t o m a t i c d e t e c t i o n o f me taphase chromosomes. I E E E P r o c . E n g . Med . B i o l . , 1989 ( s u b m i t t e d ) . 3 . S p a d i n g e r , I., P o o n , S . S . S . and P a l c i c , B . : E f f e c t o f f o c u s on c e l l d e t e c t i o n and r e c o g n i t i o n b y t he c e l l a n a l y z e r . C y t o m e t r y 1989 ( s u b m i t t e d ) . REPORTS AND ABSTRACTS: 1. J a g g i , B . , P o o n , S . and P a l c i c , B. : I m p l e m e n t a t i o n and e v a l u a t i o n o f t he DMIPS C e l l A n a l y s e r . I E E E S o c . C o n f e r e n c e , D a l l a s - F o r t W o r t h , T e x a s , November 7 - 1 0 , 1986 . 2 . P a l c i c , B . , P o o n , S . S . S . and J a g g i , B . : The d e v e l o p m e n t o f t he C e l l A n a l y z e r f o r u s e i n a n a l y t i c a l c y t o l o g y . 1 s t I n t ' l . C o n f e r e n c e on A r t i f i c i a l I n t e l l i g e n c e S y s t e m s , Los A n g e l e s , F e b r u a r y 1 - 3 , 1987 . P o o n , S . S . S . 3 . P o o n , S . S . S . , J a g g i , B . , A n d e r s o n , G . and P a l c i c , B . : Image d a t a b a s e s y s t e m u s e d f o r c o m p u t e r - a i d e d t e a c h i n g i n c y t o l o g y . 1 s t I n t ' l . C o n f e r e n c e on A r t i f i c i a l I n t e l l i g e n c e S y s t e m s , L o s A n g e l e s , F e b r u a r y 1-3 , 1 9 8 7 . 4 . J a g g i , B . , M a s s i n g , B . , M a c A u l a y , C , P o o n , S . S . S . and P a l c i c , B . : Q u a n t i t a t i v e m o r p h o m e t r i c a s s e s s m e n t o f l e u k e m i c c e l l s . 1 s t I n t ' l . C o n f e r e n c e on A r t i f i c i a l I n t e l l i g e n c e S y s t e m s , L o s A n g e l e s , F e b r u a r y 1-3 , 1987 . 5 . J a g g i , B . , P o o n , S . S . S . , P o n t i f e x , B . D . , D e e n , M . J . and P a l c i c , B. : D e s i g n and d e v e l o p m e n t o f a s o l i d s t a t e m i c r o s c o p e f o r image c y t o m e t r y . 4 t h I n t ' l . C o n g , o f C e l l B i o l . , M o n t r e a l , A u g . 1 4 - 1 9 , 1 9 8 8 . 6 . P o o n , S . S . S . , J a g g i , B . and P a l c i c , B . : C o l o u r s e g m e n t a t i o n o f b l o o d s m e a r s . 4 t h I n t ' l . C o n g , o f C e l l B i o l . , M o n t r e a l , A u g . 1 4 - 1 9 , 1988 . 7. P a l c i c , B . , Poon S . S . S . and J a g g i , B . : R e c o g n i t i o n and a n a l y s i s o f l i v e u n s t a i n e d c e l l s . D i g i t a l I m a g i n g T e c h n o l o g y . f o r O n c o l o g y , T e r r y Fox W o r k s h o p , V a n c o u v e r , B . C . , O c t o b e r 1 9 - 2 2 , 1988 . 8 . P o o n , S . S . S . , W a r d , R . K . , B e d d o e s , M . P . and P a l c i c , B . : D e t e c t i o n o f l e u k o c y t e s i n W r i g h t ' s s t a i n e d c e l l s . D i g i t a l I m a g i n g T e c h n o l o g y f o r O n c o l o g y , T e r r y Fox W o r k s h o p , V a n c o u v e r , B . C . , O c t o b e r 1 9 - 2 2 , 1988 . 9 . J a g g i , B . , P o o n , S . S . S . , P o n t i f e x , B . , F e n g l e r , J . P . and P a l c i c , B . : The d e v e l o p m e n t o f a q u a n t i t a t i v e m i c r o s c o p e f o r image c y t o m e t r y . 1 s t M t g . E u r . S o c . f o r A n a l y t . C e l l . P a t h . , S c h l o s s E l m a u , FRG, November 1 2 -1 7 , 1 9 8 9 . 1 0 . P o o n , S . S . S . , W a r d , R . K . , B e d d o e s , M . P . and P a l c i c , B . : D e t e c t i o n and s e g m e n t a t i o n o f n u c l e a t e d c e l l s i n b l o o d s m e a r s . 1 s t M t g . E u r . S o c . f o r A n a l y t . C e l l . P a t h . , S c h l o s s E l m a u , FRG, November 1 2 - 1 7 , 1989 . SCHOLARSHIPS AND AWARDS: 1985 U n i v e r s i t y S c h o l a r s h i p , U . B . C . 1984 U n i v e r s i t y S c h o l a r s h i p , U . B . C . 1983 M a c k e n z i e Swan M e m o r i a l S c h o l a r s h i p 1980 B . C . Government S c h o l a r s h i p G o l d L i o n Award (honou r s t u d e n t i n g r a d e s 1 0 - 1 2 ) Book P r o f i c i e n c y Awards ( t o p s t u d e n t i n E l e c t r o n i c s and S c i e n c e ) 

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