5 Spectroscopic Determination of the Low-Frequency Dynamics of Biological Polymers and Cells Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 12, 2018 at 18:14:18 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
L. GENZEL, F. KREMER, L . SANTO, and S. C. SHEN Max-Planck-Institut für Festkörperforschung, 700 Stuttgart 80, West Germany
Raman S c a t t e r i n g Low Frequency S c a t t e r i n g ( 200 cm""*") . Here the technique i s f u l l y developed but sometimes not f u l l y used. A change of the l a s e r wavelength or the a d d i t i o n a l o b s e r v a t i o n of the a n t i - S t o k e s spectrum i s necessary t o check whether luminescence of the sample or plasma-lines of the l a s e r are present. I f the b i o l o g i c a l sample has a b s o r p t i o n bands i n the v i s i b l e , use can be made of resonance Raman s c a t t e r i n g . An example f o r t h i s p o s s i b i l i t y i s the reported i n v e s t i g a t i o n of s i n g l e - c e l l u l a r algae (13) where the c h l o r o p l a s t s y i e l d some broad a b s o r p t i o n bands i n the v i s i b l e due t o c a r o t e n o i d - and c h l o r o p h y l l - m o l e c u l e s . Dependent on which p a r t s of these bands the e x c i t i n g l a s e r i s i r r a d i a t i n g , one gets a strong enhancement of the Raman l i n e s of the corresponding mol e c u l e s o n l y . I n t h i s way an assignment of the Raman l i n e s may a l s o be p o s s i b l e i f the assignment i n the v i s i b l e spectrum i s known. I t i s worth noting i n t h i s connection that Raman s c a t t e r ing on l i v i n g c e l l s needs s p e c i a l c a u t i o n because the s t r o n g , f o cussed l a s e r beam w i l l not be harmless t o the c e l l s , due t o b l e a c h i n g of dyes, f o r i n s t a n c e . I t i s recommended, t h e r e f o r e , to
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
5.
GENZEL ET AL.
Biological Polymers and Cells
85
use a f a s t flow system (11) which exposes the c e l l s f o r only a few m i l l i s e c o n d s to the l a s e r r a d i a t i o n . B r i l l o u i n Scattering B r i l l o u i n s c a t t e r i n g i s a w e l l developed s p e c t r o s c o p i c t o o l t o day (14), e s p e c i a l l y f o r s o l i d s t a t e p h y s i c s i n the very low f r e quency r e g i o n (0 at room temperature for about 3 h. 1
5
Figure 3. Model calculations of the absorption of dipole oscillators as in Figure 1. The oscillator, however, is now centered at 150 cm' in the far-infrared. For sym bols see Figure 1. 1
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
5.
GENZEL ET AL.
Biological Polymers and Cells
89
h i g h l y r e f l e c t i n g w a l l s , thus p r o v i d i n g Q-values of the order of 10->. Owing to the h i g h degree of coherence of m i l l i m e t e r - w a v e or f a r - i n f r a r e d l a s e r sources (28), a mode s t i r r e r i s necessary i n such case to produce a time-averaged field-homogeneity and f i e l d - i s o t r o p y . A s m a l l f r a c t i o n of the c a v i t y f i e l d i s coupled out by a horn and measured by a d e t e c t o r . When a sample i s i n s e r t e d i n t o the c a v i t y , an energy l o s s i s measured which i s caused only by the a b s o r p t i o n , but not by the s c a t t e r i n g , from the sample. The dimensions of t h i s broad-band resonator must, of course, bear a c e r t a i n r e l a t i o n to the wavelength used. I f the resonat o r i s too l a r g e , too l a r g e a sample i s necessary to o b t a i n a measurable energy l o s s . I f the r e s o n a t o r i s too s m a l l , on the other hand, not enough modes are a v a i l a b l e and the sample would s t r o n g l y i n f l u e n c e the mode p a t t e r n and hence a l s o the output c o u p l i n g . A c e r t a i n disadvantage of the untuned c a v i t y t e c h n i que i s the d i f f i c u l t y of e a s i l y c o n v e r t i n g the measured energy l o s s , due to a sample, to the a b s o r p t i o n c o e f f i c i e n t . T h i s i s p o s s i b l e , however,for simple sample geometries, l i k e s l a b s or s m a l l spheres. One of our f i r s t experiments w i t h the o v e r s i z e d - c a v i t y t e c h nique was the i n v e s t i g a t i o n of the temperature dependence of the millimeter-wave a b s o r p t i o n of c e l l s l i k e yeast (saccharomyces c e r e v i s i a e ) and monocellular algae ( c h l o r e l l a pyrenoidosa) , i n the temperature range between -200°C and 0°C. For that purpose, a h i g h l y concentrated ( c e n t r i f u g e d ) paste of these c e l l s was produced. The amount of water o u t s i d e the c e l l s was thus s m a l l compared to that of the c e l l water, which amounts to about 80-85% of the c e l l weight. The paste was p l a c e d i n a l i q u i d - n i t r o g e n cooled c r y o s t a t made from fused s i l i c a , which i s h i g h l y t r a n s parent to r a d i a t i o n of the frequency range used (40-90 GHz). I n a r e f e r e n c e experiment, the c e l l s were r e p l a c e d by a corresponding amount of pure i c e , which i n f a c t showed a n e g l i g i b l e energy l o s s u n t i l the temperature was r a i s e d to the m e l t i n g p o i n t . Since water has an a b s o r p t i o n c o e f f i c i e n t which i s more than three orders of magnitude higher than that of i c e , a strong energy l o s s suddenly occurs at 0°C. I n view of the h i g h water content of c e l l s one would expect f o r them a s i m i l a r temperature dependence of a b s o r p t i o n . The samples of algae or y e a s t , however, showed a completely d i f f e r e n t behavior i n warming-up. As seen i n F i g u r e 4, already at -100°C a measurable energy l o s s was found, which c o n t i n u o u s l y increased towards 0°C. The accuracy of these measurements, as w e l l as the above-mentioned d i f f i c u l t y f o r conv e r t i n g the data to a b s o r p t i o n c o e f f i c i e n t s , do not y e t allow one to draw q u a n t i t a t i v e c o n c l u s i o n s , e s p e c i a l l y above 0°C. We remeasured, t h e r e f o r e , the c e l l - p a s t e a b s o r p t i o n by u s i n g , i n t h i s case, a q u a s i - o p t i c a l spectrometer (23). I n order to avoid wavelength-dependent d i f f r a c t i o n of the sample, the m i l limeter-wave frequency was v e r i e d between 40 and 60 GHz, and the average of the apparent t r a n s m i s s i o n was measured. This averag-
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
90
BIOLOGICAL EFFECTS OF NONIONIZING RADIATION
-200
-100 Temperature / *C
Figure 4. Measured energy loss of pastes of yeast cells and of monocellular algae at 53 GHz as a function of temperature. The energy loss of ice is shown for comparison. All curves have been normalized to unity at —196°C. The measurements were performed with the untuned-cavity technique.
100 t
Figure 5. Absorption coefficient of a paste of yeast cells vs. temperature. Two values of the absorption coefficient of ice 0.001 L J 1 i_ are shown for comparison. The measurements were performed with a quasi-opti-100 -50 0 cal spectrometer. Temperature [°C]—*
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
5.
GENZEL ET AL.
91
Biological Polymers and Cells
ing was p o s s i b l e because o f the s m a l l change of the sample abs o r p t i o n i n that range. The r e s u l t f o r yeast c e l l s i s shown i n F i g u r e 5, together w i t h some data f o r the a b s o r p t i o n c o e f f i c i e n t of i c e (20). The same experiment c a r r i e d out at room temperature (~25°C) showed no a p p r e c i a b l e d i f f e r e n c e between the c e l l - p a s t e and a corresponding amount of water. Since the paste a b s o r p t i o n c o e f f i c i e n t , determined a t -1°C, i s about 10 cm" , and s i n c e pure water a t 0°C has an a b s o r p t i o n c o e f f i c i e n t between 50 c m and 60 cm"" (70 GHz), we conclude that a t l e a s t 20% o f the c e l l water behaves i n an anomalous f a s h i o n below 0°C. T h i s estimate i s based on the h i g h l y probable assumption that the c e l l a b s o r p t i o n i s determined e s s e n t i a l l y by the water molecules and only n e g l i g i b l y by the other c e l l c o n s t i t u e n t s . According to t h i s view, the a b s o r p t i o n mechanisms are Debye-like r e l a x a t i o n processes. Owing to the l a r g e inner s u r f a c e of c e l l s (29),a continuous d i s t r i b u t i o n of r e l a x a t i o n times, which depends on the d i s t a n c e of l a y e r s of water molecules from s u r f a c e s and a l s o on temperature, can be assumed. I t should be mentioned that a s i m i l a r behaviour as that shown here has been found f o r water i n the c a p i l l a r i e s of y-alumina (30), i n the same temperature i n t e r v a l and at 58 GHz. For a d e t a i l e d d i s c u s s i o n of the anomalous p r o p e r t i e s of c e l l water determined by v a r i o u s other methods we r e f e r to the l i t e r ature (29) . 1
- 1
1
Abstract The paper deals with the experimental methods of Raman-scattering, Brillouin-scattering, sub-millimeter-wave and millimeter-wave spectroscopy, emphasizing the special problems arising with biomolecules and biological samples. Our discussion is mainly restricted to spectroscopy for the frequency region below 200 cm where, in general, large parts of a biopolymer contribute to the eigenvector of a given vibrational mode. Various difficulties of such low-frequency spectroscopy are discussed, among which the presence of liquid water is perhaps the most serious. It is found that normal water has one to three orders of magnitude higher absorption in the millimeter-wave range than dry biopolymers. Also, the low-frequency Raman scattering is severely limited by the broad scattering of water. Several techniques for reducing these difficulties are discussed. Also described is a new method for measurement of the low-frequency absorption in cases where the sample exists in form of crystallites or fibres and where scattering normally dominates the extinction. -1
Literature Cited 1.
Fanconi, B.; Tomlinson, B; Nafie, L.A.; Small, W.; Peticolas, W.L., J . Chem. Phys. 1969, 51, 3993.
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
92
BIOLOGICAL EFFECTS OF NONIONIZING RADIATION
2.
Small, E.W.; Fanconi, B.; Peticolas, W.L.; J . Chem. Phys. 1970, 52, 4369.
3.
Fanconi, B. Biopolymers 1973, 12, 2759.
4.
Brown, K.G.; Erfurth, S.C.; Small, E.W.; Peticolas, W.L. Proc. Natl. Acad. Sci. USA 1972, 69, 1467.
5.
Genzel, L . ; Keilmann, F . ; Martin, T.P.; Winterling, G.; Yacoby, Y.; FrBhlich, H.; Makinen, M.W. Biopolymers 1976, 15 219.
6.
Peticolas, W.L.; in "Methods in Enzymology" Vol. 61, Part H; Hirs, C.H.W.; Timasheff, S.N., Eds.; Academic Press, New York 1979.
7.
Peticolas, W.L.; Hibler, G.W.; Lippert, J.L.; Peterlin, A; Olf, H. Appl. Phys. Lett. 1971, 18, 87.
8.
Gupta, V.D.; Trevino, S.; Boutin, H. J . Chem. Phys. 1968, 48, 3008.
9.
"Water", Vol. 1, Chapter 5, p. 159; Franks, F . , Ed.; Plenum Press, New York 1972.
10. Keifer, W.; Bernstein, H.J. Appl. Spectrosc. 1971, 25, 500. 11. Drissler, F . ; Hagele, W.; Schmid, D.; Wolf, H.C. Z. Naturforsch. 1977, 32a, 88. 12. Yacoby, Y.; Linz, A. Phys. Rev. 1974, B9, 2723. 13. Drissler, F. Phys. Lett. 1980, 77A, 207. 14.
Sandercock, J . Phys. Rev. Lett. 1972, 28, 237.
15. Maret, G.; Oldenbourg,· R.; Winterling, G.; Dransfeld, K.; Rupprecht, A. Colloid and Polymer Science 1979, 257, 1017. 16.
Buontempo, U,; Careri, G.; Fasella, P.; Ferraro, A. Biopolymers 1971, 10, 2377.
17.
Chirgadze, Y.N.; Ovsepyan, A.M. Biopolymers 1973, 12, 637.
18.
Shotts, W.J.; Sievers, A . J . Biopolymers 1974, 13, 2593.
19. Ataha, M.; Tanaka, S. Biopolymers 1979, 18, 507. 20. Ray, P.S. Appl. Optics 1972, 11, 1836.
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
5.
GENZEL ET AL.
Biological Polymers and Cells
21. Afsar, M.N.; Hasted, J.B. Infrared Physics 1978, 18, 835. 22. Genzel, L . ; Sakai, K. J. Opt. Soc. Am. 1977, 67, 871. 23.
Chandrasekhar, H.R.; Genzel, L. Z. Physick 1979, B35, 211.
24. Lamb Jr., W.E. Phys. Rev. 1946, 70, 308. 25. Gebbie, H.A.; Bohlander, R.A. Appl. Optics 1972, 11, 723. 26. Llewellyn-Jones, D.T.; Knight, R . J . ; Moffat, P.H.; Gebbie, H.A. private communication. 27. Kremer, F . ; Izatt, J.R.; to be published. 28. Yamanaka, M. The Review of Laser Engineering (Japan), 1976, 3, 253. 29.
Clegg, J.S. in "Cell-Associated Water"; Drost-Hansen, W.; Clegge, J . S . ; Eds.; Acadmeic Press, 1979, pp. 363-413.
30. Dransfeld, K.; Frisch, H.L. Wood, E.A. J. Chem. Phys. 1962, 36, 15574. RECEIVED October 31, 1980.
Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
