Biological Effects of Nonionizing Radiation - American Chemical Society

0. ' I. ^. I ι. 0.5. Figure 1. Model calculation of the absorption, centered at 150 GHz, of dipole ... (klystrons, backward-wave oscillators, impatt ...
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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

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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

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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

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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.

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-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]—*

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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

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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

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Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.