Modulated light techniques in polarized light and interference microscop

Allen, R. D.

English

NASA Technical Reports Server

L 7 
, + & C ' L  -25 
.+I?X-- jil a p a p e r  read. € o r  t h e  I n t e m a t i o n n l  Soc ie ty  f o r  Ce l l  Z i o l o . ~ : ~  
- -  
?j Brown Univers i ty ,  Prbvidence, R . I .  N6 5 13559 v ' * '. 
z 4 c 
$> f $ -  '. September 4 ,  1964 
- 4  
:/lodul.?Sad L igh t  Methods i n  P o l m i x e d  L i R h t  and 
R o b e r t  D. Al len,  Department o f  B i o l o g y ,  P r ince ton  Un ive r s i ty  
.... *:ne light microscope i s  used  t o d s y  p r i n c i p a l l y  as  a magni f ie r  
f o r  observing azd photographins  c - Ils, ;issues, microorganisms, and 
vzr ious  o t h e r  saeil  ob jec t s .  
strives t o  achieve i n  h i s  photomicrographs what has come $0 be c a l l e d  
A s  vver-mxe .knows, a good microscopis t  
good image qua l i ty" .  In  o t h e r  w o r d s ,  he a t tempts  t o  c a p t u r e  and record  11 
as nucn I n f o m a t i o n  about the  specimen as p o s s i b l e .  As i s  we l l  known, 
the  f a c t o r s  which l i m i t  t h e  amount o f  d e t a i l e d  informat ion  i n  nn image 
are  ( 5 )  t he  numerical  a p e r t u r e  o f  t h e  o p t i c a l  system, which determines 
the lateral r e s o l v i n g  power, and ( 2 )  t he  e f f i c i e n c y  o f  t he  con t rns t -  
gene ra t in& Gystem and its s p e c i f i c i t y  f o r  a p a r t i c u l a r  o p t i c a l  p roper ty .  
Contrast i r i  ail microscopic images depends upon well-understood 
l i s b t - m a t t e r  i n t e r a c t i o n s ,  such as absorp t ion ,  r e f r a c t i o n ,  d i f f r % c t i o x ,  
doub le - r e f r ac t ion  ( b i r e f r i n g e n c e ) ,  opt: A r o t a t i o n ,  ' luorescence,  
polarized abso rp t ion  ( d i c h r o i s m )  
now well e s t a b l i s h e d  t h q t  each o f  these light-matter inter?< ; i o n s ,  
L p: __ ~ r i z e d  flue,, .?e?..,c?, I t  i s  
I 19 -_  which I shall l s t e r  r e f e r  t o  o ~ l y  as o p t i c a l  p r o p e r t i e s " ,  -.:y i ; - , -on~ t  
extreinui.-;r u se fu l  ,nformation t o  c e l l  biologists, esp.;-.- -;hose who 
a r e  interestea ir, the func t ion  o f  macromolecules 'Is :: - - I  2nd t i s s i l c s .  
. kln : 3 l a s t  q u a r t e r  o f  a century a number Oi , . ;portant 
s p e c ,  zed :. croscopes have been produced which permit  microsco?is,L 
t o  E:- ce c e r t r a s t  in the  image due t o  one s p e c i f i c  o p t i c a l  p r o ? e r t y .  
n- i ne  2 ~ " :  + c o n t r a s t ,  i n t e r f e r e n c e ,  and f luorescence  microscopes and t h e  
x:crosT=ctroThotometer a r e  familiar e x a m g l e a .  ?c!hrizizig m 6  dark- 
f i e i d  m ~ c r o s c o p e s  are o lde r ,  but perhaps l e s s  familfar  instrments o f  
https://ntrs.nasa.gov/search.jsp?R=19650003958 2020-03-24T04:36:20+00:00Z
- *  In the- case o f  each o f  . t hese  s p e c i n l i z e d  microscopes,  Xeontrnst  
* .  
ii:d t o  a single optical ,  p r o p e r t y  is enhanced, but t'ne c o n t r a s t  never  
jecoillcs h i g h l y  s p e c i f l a  f o r  8 p a r t i c u l a r  o p t i c a l  p roper ty .  As a 
consequonce, p r e c i s e  q u a n t l t n t l v e  microscopic measurements are aircost 
impossible.  For example, i n  microspectrophotometric measurements o f  
Light  n,bcCqtiGn, e n ? o r s  may 3 i ue 'to r e f r a c t i o n ,  l i g h t  
s c a t  t o  r i n g ,  an6 dlchroism. I 2- >.r;- in blr, ' r inqence mensurements 
light s c a t t e r i r  -? Tbsorptlon, ~ n :  re-. - a c t i o n  ~ L J '  ii ryoduce e r r o r s .  
The m u l t l p l i c l - -  of c o n t r a s t  o o u r c e s  A u s  p lagues virtually every k ind  
of p h y s i c a l  r - -,-.oscopIc mer, ,a s::~.;,t. 
Considering t h e  f a c t  t h z t  a o s t  3 :ne in fo rma t ion  VB have about 
t he  f o m  ana f u n c t i o n  o f  c e l l s  ka- e G m  t o  us  through t h e  microscope, 
i t  behooves u s  n o t  t o  throw away neec le s s ly  any p o t e n t i a l l y  u s e f u l  
ana i n t e r p r e t a b l e  information just because o f  t he  p u r e l y  t e c P a i c a i  
problem posed 'by  t h e  m u l t i p l i c i t y  on c o n t m s t  sources. A l i s t  of 
i w o r t n n t  infomatior?,  %ha% c,m b e  obtained f r o i a  physlcal microscopic 
measurements is already impressive:  1% i nc ludes  the dry  mass o f  single 
I 
c o l l a  or t h e i r  i n c l u s i o n s ,  orgnnic m a t t e r  concent ra t ion ,  molar con- 
c e n t r a t i o n s  of s p e c i f i o  t 'ne o r l e n t a t l o c  O f  f i b r i l s ,  
membranes o r  molecules below the  r e s o l a t i o n  limit : : 2ror;cor>e 
ie i i seo ,  changes i n  macrornolecu;;.: eon:-srmntion, t h e  o r i e  -:ion of  
.. 
chen ica l  bonds, t h e  l o c a l i z a t i o r i  and concentratLon o f  f l u  -ester,$ 
stziins, antibokies, e t c .  The rewards would t h e r e f o r e  be .,ignificant if 
. cou ld  .-,< found  f o r  making p r e c i s e  measurements o f  t h e  optical . 
t r - ~ i e s  of microscopic spocimens wi-:hout r e c o  J E  t o  t h e  u s w i  
S ~ . - - T ; : . * L ~ O ~  o f  1o;age c o n t r a s t  Intc its zomponcnt .usi?s. 
I am vsry  pleased  t o  r e p w t  t h a t  considera-&,? grogress has been 
mnde toward t h e  development o f  a univers?.: .i..?surlnp, microscope x h i c h  
iy: e f f e c t  allows t h e  microecoplst i o  encods a s e l e c t e d  , o p t i c a l  
\ 
- -c - -  
_ -  ------c--- - . 
0 -37 * e  
. - prope r ty  f o r  r e g i s t r a t i o n  as an e l e c t r i c a l  siwal, w h i c h a n  be used 
f o r  p o i n t  neasurements,  one- o r  two-dimension21 scans o f  t h e  image, 
ncFtsurernents a t  one o r  more p o i n t s  i n  t h e  image throuGh time, and 
s?ectr ; i l  oca i~s .  
Pr ince ton  Applied Research Corporation wi th  t h e  cooperat ion o f  t h e  
C . z r l  Z e l s s  works i n  W e s t o n  I,. 
* -  
An ins t rument  h n s  bcen produced f o r  u s  by t h e  
ry - +a;-u,o::c a Research Contract  w i t h  
X;;atiozlal Aeronzst-Les zinc ; j t ; c ,  ninir  - \ - . ; L L ( ~ ~  n Ihe Par-Zeiss IY;:cro- 
scope Systero,, 1 5  A*d vi;l be c a l l e d ,  i s  an i n t e g r z t e a  o p t i c a l - e l e c t r o n i c  
s p t e z  em'ool:. -2: x- :-"urely new approacn -GG qunr , t i t a t ive  p h y s i c a l  
x i c roscop ic  iensureEents  wnicn er,,irel;y a v o i d s  t h e  use o f  image con- 
t ras t  i n  the convent ional  sense as a measure o f  m y  o p t i c a l  p rope r ty .  
In s t ead ,  each  o p t i c a l  p r o p e r t y  zzy be s e l e c t i v e l y  ellcodsd as an 
irtezsitg zociulatfon p a t t e r n  throzgk; t he  u s e ,  f o r  each o p t i c a l  p rope r ty ,  
of (an unique arrangement o f  a u x i l l i a r y  o p t i c n l  devices  i n  t h e  microscope, 
Zach o p t i c a l  p r o p e r t y  i s  measured e l e c t r i c a l l y  on a l i n e a r  s c a l e  w i t h  
a ioaxiaun al lowable e r r o r  o f  one .;ercent o f  whatever f u l l - s c a l e  
d e f l e c t i o n  s e n s i t i v i t y  i s  ir, 2 ; s  
t h a t  ( 1 )  measurements may be maQd very  r a p i d l y  ( v i t h  response t imes 
ranging f r o 3  one msec t o  t e n  seconds) ,  ( 2 )  t h e  n o i s e  l e v e l  i s  e x t r e a e i y  
low,  s o  t h a t  v e r y  small e f f e c t s  can be de t ec t ed ,  and (3; <here i s  no 
loss whatever i n  l a t e r a l  reso'.czion made p o s s i b l e  by xc riLcroscope 
l e n s e s .  ,Ti; ; l a s t  statemen: 
ii;?erc 3 r d i z - r y  images a r e  dis-;. --oed by anomlous  d i f f r a c t i o n .  
T1 sdv:mtages o f  t h i s  approach are 
, 
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t rue e v ~ n  in p a l ~ A . ~ ~ d  l i g h t  E I ~ C ~ G Z C G ; ; .  
e pr l inc ip les  bas i c  ti 11 o f  t h e  methods t%,x can be use.; : L - ~ l A  
A7 b f i A ~  A Astra::-,,t a re  b e s t  desoribed f o r  t h e  case o f  %he 
a e t e c z i o n  S -  ; t e a ,  publ ished last year by Allen,  BracZt  ;i yoorc ~~. 
l lm Courna; of Ce l l  3iology". In  t h i o  system, iig;?.c from n. suLtxb;c 
source is 73ssed through a p o l a r i z e r ,  an e l e o t r o - o p t i c  light r n o d u l z t o r  
(ZOLX),  a 2;)oci;lzen i n  an optical system f r e e  from e t r a i n , a n d  nn nnzlyzer, 
I 
i I 
e 
. find. i c  de$ected by a s e n s i t i v e  photomul t ip l ie r .  
- A  - ' .  
0 .  The 3OLX i s  t h e  moa t  e s sen t in l  element i n  t h e  system, s i n c e  
it se,nes n o t  on ly  as the  l i g h t  m o d u l a t o r  f o r  encoding t h e  b i r e f r i n -  
gence Lnfornnt ion f r o m  the specimen as a photocurren t  a m p l i t u d e  
coaul%<ion, ~ L T  r l s o  as an au tc  
si?ff',s o r  
s h i f t c .  o f  . 
deuter2um : - 3  ;nnate whicn acqu i r -  I ils-.: erpstnlline axes; 
becon,c.; S i .  
-;IC conpensator  f o r  measuring phase 
r e t a r d a t i o n s  c y  - L > p l y  .:*c e l e c t r i c a l l y - i n d u c e d  phase 
ctJ--ce SI@. The 3OLX i s  ,. 2-cut c r y s t a l  o f  potsss iun:  d i -  
t h a t  I s ,  i t  
' r k p n t ,  whec n vo;%ige - 3  aDplied t o  i t s  optical s u r f a c e s .  
JceL. J l r e f r i n g e n c e  I s  know.ls t h e  Pockels e f f e c t ,  nnd i t s  va lue  
i n  tir: ap: -:cation i s  i t s  s7;rict  i i n o r t r i t y  and known wavelength dependen 
I-? o p s r a t i o n  of  t h e  sys t em ;s SC3t shown wi th  a ciiagran ( F i g .  2 ) .  
oc;ci;la~zT drj.-v.es the  p 7 - v  
U U ~ A  W L C ~  square-waves of AC, f o r c i n g  i t  t o  
in t roduce  square-waves o f  phase shifts on bo th  s i d e s  o f  zero. 
p h x o  Tetard ing  specimen 1s added t o  t h e  o p t i c a l  system, t h e  
total r e t a r d a t i o n  i s  no longe r  s p m e t s i c d  nbout ze ro ,  s o  t h a t  t h e  
r e c t i f i e d  square-waves of photocurrent  from t h e  p h o t o m u l t i p l i e r  cievelo? 
a r e p e t i t i v e  asymmetry, whicn i n  e f f e c t  i s  a pkc tocur ren t  a q l i t u d e  
nodu la t ion  at t h e  findamental frequency o f  t h e  . . , z i l l a t o : * .  The lock- 
When a 
i n  a m p l i f i e r ,  which isba phnse- . rd  frequency- L d v e  dev,ce ciel-',vinc 
i t s  r e f e r e n c e  signal f rom the  a s c i l i a t o r  d r i v k g  t h e  3:CL'" A*A, resYG2;  .r; 
tc t h i s  photoc-c - x n t  modulation w i t h  2 large DC v o l t z g e ,  ?i? i ich :..- -- e -  
tu: 
t o  L L I : ' ~ .  
?ocXe;s e f f e c t ,  a d  t h e  instrument  i s  designed t o  operxte  w i t h  uxii 'orz 
e r r o r  o f  one p r c e n t .  
- t o  t h e  E C X  as nega t ive  feedback t o  r e t u r n  the t o t a i  r e t a x i n t i o n  
Tnis se rvo  i s  l i n e a r  by v i r t u e  o f  t h e  l i n e z r i t y  o f  t h e  
n- Afie microscope' i s  a Zeiss Photomicroscope I aocE:iea t o  ~ c c o ; z o c s t c  
t h e  n o d z l s ~ ~ r  azsaz~bLy ,  a - , e r t u r q  sliclcs, f i l t s r s ,  a i ~ ~ o r a ,  e t c .  
?.re needed f o r  t h i s  method. The design shown i n  Figure' 3 ~ l l o - ~ : : s  tis to 
- 9  
i i iu rnkkAte-  t ne  spec iaen  wi th  ‘€ul l - f io ld  red unmodulated l i g h t  w h i l e  
4 .  
. .  
d.scam:ng it wi th  a nicrobenm o f  short-wavelength l i g h t  w i th  which 
* mcasurenentn a r e  made. L i g n t  f r o m  a zirconium oxide a r c  i s  first 
f i l t e r e d  f o r  tho des i r ed  s p o c t r n l  r ag ion ,  then  passed through t n o  
rcodul:l%or nssenbly .  
x T c r t u r e  Inaged f i r s t  i n  t h o  plana of t h o  rear diaphragm t h e n  n ~ . - ~ i n  
ir, tine spec iaen  >lane.  
T h e  microbeam i s  f o m e d  by a s l i t  or c i r c i l l n r  
A d i c h r o i c  m i r r o r  xiixes short-wnvelenc$?, 
observer t o  locnGe t h e  exac t  p o s i t i o n  o f  t i le m i c r o b e a m  r e l a t i v e  t o  t h e  
s?ecimer; an.; Ls toc t  any f o r m  changes o r  movements o f  t he  specimen dur ing  
measurcnents,  
blue o r  g reenspot  ( t h e  focused microbeam) i n  n. r e d  f i e l d .  
The observer  s e e s ,  o r  may record  photographica l ly ,  a 
A d i c h r o i c  
beaa s? l i t - t ; e r  i n  the  tube-head sends tne microbenm energy t o  t h e  photo-  
n u l t l 2 l i e r  a d  t h e  r e d  illumination t o  f o m  a n  image o f  the specirren. 
T A ~ b h  * L  t h i s  sys tem,  i t  Is p o s s l b l e  t o  make s p o t  mensureaents i n  
t h e  specinex. a d  record  changes with time; o r ,  the  cpecimen may be 
slowly dram SCI’CG:: t h e  microbeam t o  provide 2 o m  o r  t w o  “mensional 
sczn. 
e l e c t r o c e c h a n i c a l  device which p r o v I d \  
reprobucib le  ”uo very  ne-qrly t h e  rt.;;olu4;ion l i m i t  o f  t he  nicroscope.  
The s 
r d ~ 2 2 . o ~ - ~ h  o f  8 wavelength 6f l i g h t ,  o r  a d is tnnca  which i s  mal:- 
coaparec, t o  In t e ra tomic  d i s t a n c e s .  
s e n s i t i v i t y  has proven t o  be photoa n o i s e ;  that 2 a 
f l u c t u z z i o n s  i n  t h e  s t ream o f  photons s t r i k i n g  the  ~ h o t o m u l t i ~ l l c r .  
.r PZ. Bichara Burger i n  my labor?.-Lory A<-.,  Scsignec : :qle 
i i n o a r  m o t i o n s  which a r e  
l e s t  _;base r e t a r d a t i o n  t h a t  can be measured i s  a p p r o x i m x e l > r  cirLe 
The l i m i t n t i o : :  m the  a b s o l u t p  
1 s t a t i s t i c a l  
n- L f i e  s:gdfic--nce of t h i s  f a c t  i s  t’lat s u b s t a n t i c l  ihprovements i n  
? e r f o - m . m c e  of I n s t r u n e n t s  o f  t h i s  ty?o can be a t t a i n e d  o n l y  by incyezsLng 
* (1.- At*,t? inotr",.mcnt which has rn*ide such s e n s i t i v e  mezsurementn pos-  
. L  
A r n a y ,  AS rnost iiliportnnt a p p l i c a t i o n  o f  t n i s  sjrste'2i i n  c e l l  
? A  . . -  G - O ~ O , . ; ~  wiL 16 in i n t e r f e r e n c e  nicroscopy, where o ? t i c - . l  p a t h  a>;- 
f e r ~ . c e s  CLA be used f o r  weighing c e l l s ,  determining their dry -..:: 
c o n c e i - t r a t i o L ,  volumg, and r e f r a c t i v e  index. The p r e c i s i o n  ~ E C  :cei 
o f  c,, i n t e r f e r o m e t r i c  measurements x-y now Le increased  3y  be; L _  
, w  LL ;OO <Lines. 
-,-. 
.C\ 
s i b i e '  has been I n  o u r  I n b o r n t o r y  f o r  on ly  x short time. 
on l o m  t o  t h e  conp;uAy which clcvcloped it Mnile I a t t e n d  t w o  mec';in~:s. 
I t  I s  now on e x h i b i t  at This  mect inc .  
t s i l e c  L f i f G x a t i o n  on t h e  pe r fo rmnce  o f  t h e  instrument  since you nr*y 
However, it i s  
b 
I s h a l l  n o t  give you any de- 
s e e  it i n  c p e r a t i o n  and exarniL.c, - .e ~reliminary specifications yourselves  
u i some o f  t h e  p o s s i k l e  tries 4. - ~ ~ t e a d  I shoald l i k e  50 
o f  3er.surezents tha t  csn  be c-- ,. it - - 7 -  abem. L Tnese fzll i n t o  
t h r e e  c l a s s e s u  i n  t h e  firs; 2 ,  ( .,.- -(' 1-i ?-=-e t h e  f u c t i o n s  
assocrt r ted t r i t h  9 o l a r i z e d  ana XI;: r fez-ncc  xicrcs2opy. 
quarter-wave ? l a t e  f o l l o w s  the  ECLM, t h e  ins t rument  records  no t  
Sote  t h a t  if a 
- 3  ciirc:rixgence b:t o p t i c s 1  r o t z t i c n .  ,c i s  s t i l l  s e i l s i t i ve  t o  S i r e -  
frinygen:, A. o'ajecxs, b u t  only at c e r t a i n  anKles at which these  o b j e c t s  
i ~ ~ r i i z ~ e  2 s;;;rious r o t t L t i s ; ; .  Y e  k.,--rc, f c ~ m _ d  :'.nt i t  i s  n o s s i b l e  t o  
measure rotation and b i r e f r i n p n c e  indepenaent ly  o f  one znother i f  8 
second EOLX i s  added and two  lock-in - a -p l i f i e r s  a t  d i f f e r e n t  f r equenc ie s  
a r e  t lsed. One channel allows the  e x p e r i n e n t o r  t o  r e m v e  --ny spurious 
ro t ;At io i j .  due t o  t h e  b i r e f r i n p n t  o b j e o T  m d  t h e  o t h e r  al. xi a measure- 
nient o f  t h e  r e s i d u a l  r o t a t i o r -  :-dh 1:. real. This is app-rent ly  t h e  ' 
first syr;tea xi:k yhich  it has been p o s s i b l e  t o  measure o-p t ica l  
rotatLori  i - r ,  .:.= -oscs?ic  ob3ects .  
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Modulated light methods in polarized light and interference microscopy

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