INFRARED MICROSPECTROPHOTOMETRY OF T I S S U E C E L L S

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1 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 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 cells grown on No. 00 glass slides were cleared of water and measured under the microscope.

2 24. transmission measurements made on a double-beam spectrophotometer. Cultured cells grown on No. 00 glass slides were cleared of water and measured under the microscope. Figure (4) is a photomicrograph of Rep I1 cells stretched and spread on glass. The extensive spreading and flattening is characteristic of cells growing attached to surfaces, and may permit measurement of infrared transmission in cell parts of the order of one micron thickness. The chief technical problem in determining the change of absorption coefficient of intracellular water is to measure the thickness of the volume viewed. This is accomplished by using the infrared spectrophotometer directly as an interferometer. The infrared measurements are corroborated by the interference microscope using the mercury line at 5300 angstroms. In case a cuvette has very thick IR-2 walls the wall separation is measured by laser beam interferometry. A Further indication of the absorption by intracellular water is given by attenuation of total reflection. Figure (5) is a schematic sketch of the arrangement in which a beam of radiation is attenuated during reflection at an interface. The cell mass is pressed against the plane surface of the semicylinder. The radiation penetrates usually to a distance of 1 to 2 wavelengths which at 2.97 microns radiation would be about 6 microns depth at most. By comparing absorption in pure liquid water with that in the cell mass one finds a reduction in expected absorption in the latter. In Figure (6) are plotted the data on linear exponential absorption in a semilog plot. The steepest curve is that for absorption in pure water, curve 1. Assuming the tissue cell is 75% water, the curve 2 is derived. Curve 3 is for the same cell with the absorption coefficient reduced by one half. The points from current measurements fall beyond curve 3 indicating that the absorption in the cells (Hela and Rep 11) at the strongest water band, 2.97 microns, is considerably reduced.

3 25.

4 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 X 10% h = 6.3p p 4.42p

5 Figure 4. Mouse fibroblasts. American Type Collection L929. Eagle ' s Medium. Hoffman, J. G. 27.

6 28

7 29. FIGURE 6 INFRARED MICROSPECTROPHOTOMETRY WATER ABSORPTION AT ~ 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 = p-' X MEASUREMENTS AS OF 6/1/66 IN VARIABLE SPACE C E L L 0 ATR MEASUREMENTS \ \ 0 I X x 0 h 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. 1 7

Chemistry Instrumental Analysis Lecture 15. Chem 4631

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