Mapa de Conocimiento del Area

M f

; w. 1 2 8 .

value i s c a lle d th e e x tin c tio n ! B, Thus E =' k c d = 1

l o g T , -

E = k c d | i s only s t r i c t l y tru e fo r a dye in so lu tio n ! but i t has been shown th a t Feulgen sta in e d n u c le i fo llo w th e

c lo se ly

rela tio n /e n o u g h to perm it th e c a lc u la tio n of r e la tiv e amounts < dye per nucleus in a r b itr a r y u n its from e x tin c tio n d a ta (R is & M r sky 19^-9; P o llis te r & Sw ift 19?0)* The e rro rs in tro d u ced vary but have been shown to be le s s than

There are o th er sources o f e rro rs in cytophotom etry and th e se are d iscu ssed below#

(11) HasDonaa o f alefttro n a u l t l p l l a r tube (T^iotoeoll).

The resp o n se, o r e le c tr ic c u rre n t output o f th e c e l l , should be d ir e c tly p ro p o rtio n a l to th e in te n s ity of in c id e n t illu m in a tio n . In th e p r e s e n t study, th e lin e a r it y o f th e p h o to c e ll response was te s te d by in te rp o sin g n e u tra l d e n sity f i l t e r s in th e lig h t p ath and reading on th e galvanom eter th e output of th e c e l l fo r each f i l t e r . The r e s u lts a re given in Graph I* From th i s i t I s seen th a t th e c e ll used had a lin ea] response to varying li g h t in te n s itie s .

M arshall e t . a l . ( 1 ^ 8 ) have shown th a t th e cathode o f e le c tro n m u ltip lie r tubes i s not uniform ly p h o to se n sitiv e , but i s p r a c tic a lly uniform w ith in an area o f 5^ mm. diam eter round th e exact c e n tre o f th e cathode. W ithin th i s a re a and w ith a uniform ly illu m in a te d f i e l d , th e galvanom eter d e fle c tio n is

X U R E

H- W f^R-rrR

" ^ 9 ^

e s s e n tia lly p ro p o rtio n a l to th e area Illum inated*

The e le c tro n m u ltip lie r c e l l used in th e p resen t study % te s te d fo r area response by tak in g a s e rie s of galvanom eter read in g s a t v ario u s a p e rtu re s of th e diaphragm placed in fro n t o f th e photocell# The galvanom eter readings were p lo tte d

a g a in st th e a p e rtu re diam eters and th e re s u ltin g graph shows t t r e s u lts (Graph 2)*

(111) P u rity of li g h t source. ;

In th e ap p aratu s used in th is stu d y , monochromatic lig h t was obtained by is o la tin g th e greeh lin e of th e spectrum from a

, ■ ' ■ ' :

High P ressu re Mercury Arc lamp by a sp e c ia l W ratten No. 77

f i l t e r . This f i l t e r tra n sm itte d 78^ o f th e in c id e n t ra d ia tio n

a t 550 m.u. The s p e c tra l tran sm issio n of th i s system i s n o t, o f co u rse, pure* but according to Drabkin (19?0), and Moses

(19^2 ), th e sm all lo s s o f s p e c tra l p u rity by using such broad band f i l t e r s i s j u s ti f ie d in o rd er to o b ta in th e n ecessary in te n s ity .

(iv ) S c a tte rin g o f li g h t in th e o p tic a l system^

B io lo g ic a l m a te ria l i s considered to be composed o f , c o llo id a l p a r tic le s , and th e se w ill s c a tte r lig h t w hether th ey

a fe sta in e d or n o t. Por example* i f two s tr u c tu re s , say a nucleus and i t s n u cleo lu s have d iffe re n t r e f r a c tiv e in d ic e s fro

th e surrounding medium, they w ill r e f le c t and r e f r a c t th e lig h t passing through th e se c tio n . This s c a tte rin g can a f f e c t th e e x tin c tio n /

' \

8 # i n c t l 6 n 'l n 't v

(1 ); I f th e lig h t i s s c a tte re d but o f th e measured beain due tp t s tru c tu re p f th e p b je c t* th e $ yalue w ill be to o h igh (p â re f l95Q *^,:R andalbl9?0,>:'sqPtt'19^2V ^?W

tb e p re tic a l; p o in t Pf yiew , th is s c a tte rin g can be o p esid erab ly reduced i f th e ti s s u e i s mounted in a medium of re fra o tly e inde approaching th a t o f th e tis s u e (O m steln 1992). N e u tra i Càna balsam meets th i s requirem ent and was used in th e p re se n t stu d ; N evertheless * th e p re p a ra tio n s made weie te s te d to determ ine t t m agnitM e o f th is s tr u c tu r a l li g h t lo s s . The unhydrPlysed c b n trb ls o r blanks fo r th e Feulgen t e s t s were Used fo r th is t purpose ( P o llis te r i R is 1947; SWift 1950 ; Moses 1952; ând '

o th e rs ). These s lid e s showed U nstained n u c le i and had been preparbd and tre a te d e x a c tly as th e sta ih e d t t slid e s* exp ept th a t they were n o t hydrolysed in acfd^ Measurements of lig h t lo s s on th e se s lid e s showed th a t th e re was no s ig n if ic a n t Ib s s o f li g h t between th e u n stain ed nucleus ahd a c le a r p a rt o f th e s iid e , i . e . th e s c a tte rin g of l i g h t dUe to th e s tru c tu re ; o f ?th« nucleus was in s ig n if ic a n t. ;-C:';'

(2) I f lig h t i s s c a tte re d in to th e measured beam from th e

surrounding tissu e * th e B v a l u e w i ll be too low. This i s a more d i f f i c u l t source o f e rro r to determ ine th an ( l ) above

(R andall 19 5 0 ).. There a re two fa c to rs in th e illu m in a tih g system which a re of prime im portance and must be taken in to c b n sid e ra tip n , : These a re th e siz e o f th e f ie ld illu m in ated * ’

and th e angle: a t w hich,th e li g h t converges on th e p h je c t. . ThS f i r s t i s .cp n trp iled la rg e ly hy th e lamp d th é secpn

hy /the: si#tdgP:di#^

\

vr y::'?

y:;: ; ;

;Çphseqûently* èx tin ctip ri, readings were; c a lc u la te d fo r ; d i f f e re n t aAeytunes o f ,';thése\,diaphragms' ; p^^^ sm a]d':T egiohs/\of-

, s p h e ric a l, : Feulgen /sta ih é d :nUPlSii':, .^ hpse o f th e hopt • were;wse fop th i s purpose sin ce they, were la rg e r th an the\fungai.:pnes::.'ai] were; between 8Î and lOp in diam eter. I t wouid be expected frpa :these .read in g s, th a t ■régions ;'in’;the:, p eh trè ;. pf:b h e niicleuS;v;(the:;;:;: th ic k e s t p a r ti would have h ig h er B / values, than ;;thpse ''t a k e n - frpm th e m arginal a re a s (th e th in n e st p a r ts ) . T his was only tru è* h6wever> when the. a p e rtu re s were small* 0 ohseqU ently ir th e p re se n t study th e lamp diaphragm was : stppped down to 1*5mm. a p e rtu re sp th a t th e a re a illu m in a te d was only a few diam eters

g re a te r th a n th e nucleus being measured. S im ila r r e s u lts hàvï been obtaihed by Sw ift 1950; F o iiis te r & M s i p W i a ^

1952. This procédure elim in ated # o h df th e g la re and soatte:: \.ihg frpm': the: surrounding ,tis:sUes.. ' ;

Hàpfh (1951,1952)* hae questioned th e v a lid ity o f d ata obtained by cytpphptom etric methods on th e grounds th a t th e af« o f the s lid e illu m in a te d i s a number o f diam eters g re a te r th an th e a c tu a l nucleus under t e s t . According to th is author a p e rtu re s such as have ju s t been d escrib ed above, in te r n a l r e fle c tio n s a re s e t up when a la rg e area, bf th e s lid e i s i illu m in a te d and th is, a f f e c ts thp'.:..B,.: v alu e. This in te r n a l vgiàii^S;-