23.
INFRARED MICROSPECTROPHOTOMETRY OF T I S S U E C E L L S
JOSEPH G.
HOFFMAN
S T A T E U H I V ' E R S I T Y O F N E WYORK
. . . . . . . . . . . B.U F.F A. L.O ,.N .E W.Y.O R.K . . . . . . . . .
I n t h e course of experiments designed t o measure t h e r a t e of i n corporation of heavy water (deuterium oxide) i n t o cultured mammalian
e p i t h e l i a l c e l l s it became evident t h a t t h e water i n s i d e a c e l l is not l i k e
f r e e water. A reduction i n t h e i n f r a r e d absorption bands indicated i n t r a c e l l u l a r water i s not i n a f r e e s t a t e b u t i s i n t h e "ice-like'' s t a t e . It
has long been suspected t h a t i n t r a c e l l u l a r water is bound although l i t t l e
d i r e c t information about t h e e x t e n t of binding is a v a i l a b l e . In general,
water c o n s t i t u t e s about 75% of t h e c e l l mass, t h i s water probably being
present i n a l l t h r e e s t a t e s , namely: f r e e , bound, and frozen ( i c e - l i k e )
The following r e p o r t is a preliminary review of t h e q u a n t i t a t i v e measurements of t h e e f f e c t of binding of water i n s i d e c e l l s as revealed i n i n f r a r e d absorption.
.
Figure (1)gives t h e comparison of t h e absorption i n H20 and D20
as measured i n t h e wavelength range of one t o 10 microns. The displacement
of t h e major absorption bands t o t h e r i g h t r e f l e c t s t h e increased mass of
deuterium over hydrogen. The displacement i s t h e b a s i s of our work on using
D20 as t r a c e r s i n c e it permits amounts i n t h e range of one t o t h r e e p e r cent
i n H20 t o be r e a d i l y detected. Reflection o p t i c s a r e used (Figure 2) because they allow o p t i c a l l i g h t range viewing without introducing achromatism.
T h i s work could be done with g l a s s o p t i c s i n t h e range of wavelengths one
t o 4.2 microns. Figure (2) shows t h e o p t i c a l path i n two r e f l e c t i n g l e n s
w i t h an o b j e c t specimen i n place. The specimen may be supported by a
p l a s t i c f i l m such a s Saran Wrap o r J i f f y - S e a l , o r by calcium f l u o r i d e ,
KRS5, g l a s s , o r IR-2.
The absorption s p e c t r a i n i c e corresponding t o t h e data i n Figure
(1) f o r l i q u i d s a r e not complete. Only i n t h e p a s t 2 years have data on
l i q u i d water f o r thicknesses down t o 2 microns become a v a i l a b l e . In gene r a l , it i s known that absorption w i l l be reduced i n i c e a t t h e characteri s t i c bands. This reduction of absorption i s our measure of t h e extent of
binding of water. Perhaps more d a t a w i l l show s h i f t s of some absorption
bands i n i c e . If so, it w i l l be most u s e f u l in evaluating r e s u l t s on i n t r a c e l l u l a r water.
The l i n e a r exponential absorption c o e f f i c i e n t , A, Figure ( 3 ) , of
water a t 2.97 microns i s 1 . 5 p e r micron, and a t 6.3 microns i s 0.28 p e r
micron. A t o t h e r regions such a s 1.25 microns t h e t y p i c a l absorption i s
much l e s s , by a f a c t o r of 1000. This means t h a t a c e l l f l a t t e n e d out t o
2.97
one micron t h i c k would give s t r o n g absorption, reducing a beam a t A
microns down t o 20% of i t s i n i t i a l value. The l / e valve a t t h i s wavelength
i s 0.67 microns. It was found t h a t spinner cultured c e l l s could be cent r i f u g e d f r e e of t h e i r surrounding aqueus medium and placed i n a v a r i a b l e
space c e l l . By means of a micrometer, t h e c e l l thickness was adjusted and
24.
transmission measurements made on a double-beam spectrophotometer. Cultured
c e l l s grown on No. 00 g l a s s s l i d e s were cleared of water and measured under
t h e microscope. Figure (4) i s a photomicrograph of Rep I1 c e l l s s t r e t c h e d
and spread on g l a s s . The extensive spreading and f l a t t e n i n g i s characteri s t i c of cells growing attached t o surfaces, and may permit measurement of
i n f r a r e d transmission i n c e l l p a r t s of t h e order of one micron thickness.
The chief t e c h n i c a l problem i n determining t h e change of absorpt i o n c o e f f i c i e n t of i n t r a c e l l u l a r water i s t o measure t h e thickness of t h e
volume viewed. This i s accomplished by using t h e i n f r a r e d spectrophotometer
d i r e c t l y as an interferometer. The i n f r a r e d measurements a r e corroborated
by t h e interference microscope using t h e mercury l i n e a t 5300 angstroms.
In case a cuvette has very t h i c k IR-2 walls t h e w a l l separation i s measured
by l a s e r beam interferometry.
A Further i n d i c a t i o n of t h e absorption by i n t r a c e l l u l a r water i s
given by a t t e n u a t i o n of t o t a l r e f l e c t i o n . Figure (5) i s a schematic sketch
of t h e arrangement i n which a beam o f r a d i a t i o n is attenuated during r e f l e c t i o n a t an i n t e r f a c e . The c e l l mass i s pressed a g a i n s t t h e plane s u r f a c e of t h e semicylinder. The r a d i a t i o n p e n e t r a t e s u s u a l l y t o a d i s t a n c e
of 1 t o 2 wavelengths which a t 2.97 microns r a d i a t i o n would be about 6
microns depth a t most. By comparing absorption i n pure l i q u i d water w i t h
t h a t i n t h e c e l l mass one f i n d s a reduction i n expected absorption i n t h e
latter.
In Figure (6) a r e p l o t t e d t h e data on l i n e a r exponential absorpt i o n i n a semilog p l o t . The s t e e p e s t curve i s t h a t f o r absorption i n pure
water, curve 1. Assuming t h e t i s s u e c e l l i s 75% water, t h e curve 2 i s
derived. Curve 3 i s f o r t h e same c e l l with t h e absorption c o e f f i c i e n t reduced by one h a l f . The p o i n t s f r o m current measurements f a l l beyond curve
3 i n d i c a t i n g t h a t t h e absorption i n t h e c e l l s (Hela and Rep 11) a t t h e
s t r o n g e s t water band, 2.97 microns, is considerably reduced.
25.
26.
IJ
I-
microscope,
Figure 3
Exponential absorption c o e f f i c i e n t s i n
r e c i p r o c a l microns a t several wavelengths
p e r t i n e n t t o water absorption 25%.
A =
A
=
1/A
4.23
=
A = 2.97p
1.25t.L
X
2.36
10 - 5
X
10%
h = 6.3p
1.5
0.28
0.67p
4.42p
27.
Figure 4.
Mouse fibroblasts.
E a g l e ' s Medium.
American Type C o l l e c t i o n L929.
..
Hoffman, J G
28
29.
FIGURE 6
INFRARED MICROSPECTROPHOTOMETRY
WATER ABSORPTION AT 2 . 9 7 ~
I. PURE WATER A= 15;'
2.CELL IS 7 5 ' ! P U R E WATER
3.CELL IS 75% P U R E WATER B U T
A = 0.7 5 p-'
X MEASUREMENTS AS OF 6/1/66 IN
VARIABLE SPACE C E L L
0 ATR MEASUREMENTS
\
\
0
I
X
x
0
h
2
3
4
M I C R O N S , D E P T H OF WATER
I
I
5
6
DR. HEDRICK: Thank you very much D r . Hoffman f o r t h i s
e x c e l l e n t presentation. D r . Harold Tuma of Kansas S t a t e Univ e r s i t y w i l l l e a d t h e discussion. I n order t h a t t h e questions
and answers may be recorded f o r t h e proceedings t h e recording
engineer has asked that you e i t h e r stand o r r a i s e your hand and
w a i t f o r t h e microphone t o be brought t o you, i d e n t i f y yourself
and then s t a t e t h e question. Harold.
DR. HAROLD TUMA: Thank YOU, Harold. I might mention
t h a t many of you have asked t h e question about Davey McIntosh
and Davey w i l l n o t be able t o be here. Probably t h i s i s t h e
f i r s t conference t h a t he has missed b u t he i s escorting some
f o r e i g n v i s i t o r s around t h e states and r e g r e t s very much t h a t
he i s unable t o a t t e n d . He sends h i s regards t o a l l of you,
however. I w i l l e n t e r t a i n questions f o r e i t h e r of t h e
speakers.
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