PHOTOACOUSTIC SPECTROSCOPY AND RELATED TECHNIQUES

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Colloque C6, suppl6ment au nO1O, Tome 44, octobre 1983 page C6- 339. PHOTOACOUSTIC SPECTROSCOPY AND RELATED TECHNIQUES APPLIED TO ...
JOURNAL DE PHYSIQUE Colloque C6, suppl6ment au nO1O, Tome 44, octobre 1983

page C6- 339

PHOTOACOUSTIC SPECTROSCOPY AND RELATED TECHNIQUES A P P L I E D TO BIOLOGICAL MATER IALS T.A. Moore

*,+,a

, E.P.

O'Hara

, D.M.

Anjo

, R.

Tom

*

and D. Benin

Department of Chemistry, Arizona S t a t e University, Tempe, AZ 85287, U.S.A. Department of Physics, Arizona S t a t e University, Tempe, AZ 85287, U.S.A. + ~ a b o r a t o i r ede Biophysique, INSERM U. 201, Muse'um National d r H i s t o i r e NatureZZe, 61, Rue Buffon, 75005 Paris, France "De'partement de BioZogie, Service de Biophysique, CEN SacZay, 91 191- Gif-sup-Yvette Cedex, France

Rksumk - Les techniques photoacoustiques peuvent Btre utiliskes pour mesurer certaines propriktks spectroscopiques et calorimktriques des matkriaux biologiques. Des exemples comprenant des mod4lisations du profil de profondeur, des ktudes du profil de profondeur des systhmes vivants, ainsi que l'observation du processus photosynthktique actif sont prksentks ici. Abstract - Photoacoustic techniques can be used to measure certain spectroscopic and calorimetric properties of biological materials. Examples including model studies for depth profiling, depth profiling studies of living systems, and the observation of the active photosynthetic process are presented. Photoacoustic (PA) techniques are becoming increasingly useful for the measurement of spectroscopic properties of biomolecules i n v i v o and i n v i t r o 11-41. Often conventional spectroscopic techniques such as transmission or reflectance are not amenable to biological materials due to opacity, scattering, poorly defined or heterogeneous surface properties, etc. However, in ordertounderstand spectroscopic and photophysical aspects of functioning biological systems it is absolutely necessary to have methods available that can be used'on native biological structures. These may include material such as intact leaves of plants, algae cells, subcellular particles, membrane fragments, tissue samples, etc. In addition to the usual spectroscopic information such as band positions and relative intensities, PA techniques can offer new information that arises uniquely from the combination of spectroscopic and calorimetric phenomena that comprise the PA effect. This new information includes quantum yields, lifetimes, energies of various metastable excited states, and the kinetics of relaxation pathways of photobiological systems. The thermal wave physics and general theoretical treatment of the PA response have been described 15-81 ; it is only necessary here to point out that thermal diffusion lengths in biological materials range from a few um at modulation frequencies % 1 KHz to several hundred um at 0.1 to 1 Hz. To date, the gas microphone has probably been the most widely used detection technique. However, photothermal radiometry /9,10/ mirage effect 1111, and PZT detection are important variations that have provided key information on certain samples. Depth profiling and photosynthetic assay are demonstrated by work on the lichen Acarospora schZeicheri, an organism having chromophores arranged in strata. The surface layer of the lichen contains the yellow-green pigment rhizocarpic acid ; a discrete layer of algal cells is located in the interior of the fungal matrix. A two layer plastic model (Fig. I ) was constructed to mimic the layered chromophore structure of the lichen. Figure 2 presents the PA spectra (gas-microphone) ; the amplitude spectrum (Fig. 2b) was similar to the conventional absorption spectrum essentially the sum of the individual components. However, PAS differs from conventional spectroscopy in that the photoacoustic signal can be described as a vector quantity (Fig. 2a). The amplitude (A) can be decomposed into "predominately interior" and "predominately surface" components. The phase difference between these components is denoted by @.In order to obtain the separable absorption spectra for the two layer model, a rigid axis rotation is performed about the origin 1121. This treatment is similar to the data manipulation performed by Betteridgeet/13/. Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1983655

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Fig. 1 - Model consisting of two layers of pigmented polymethylmethacrylate fused together by a mild heating process. The surface layer contained methylene blue with cm thick. The interior layer conBoptica - 2.3 x lo2 cm-'at 660mandwas 1 x tarned &carotene with Boptical = 8.4 x 10' cm-' at 660 nm and was 6.1 r lo-' cm thick. The sample was thermally thick overall with a thermally thin surface layer.

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Fig. 2 - Phase analysis diagrams and photoacoustic spectra for the two layer model. Phase diagrams ; (a) describes the experimental photoacoustic signal, (c) describes the axis rotation to resolve the surface signal and (e) describes the axis rotation to resolve the interior signal. Photoacoustic spectra ; (b) is a composite spectrum of methylene blue and B-carotene, (d) spectrum of methylene blue obtained by axis rotation and (f) spectrum of 6-carotene obtained by axis rotation.

R o t a t i o n of t h e a x i s t o e l i m i n a t e t h e c o n t r i b u t i o n of t h e i n t e r i o r s i g n a l from t h e p r o j e c t i o n of t h e amplitude on t h e Y-axis (Fig. 2c) r e s o l v e s t h e a b s o r p t i o n of methylene b l u e i n t h e s u r f a c e l a y e r (Fig. 2d). The a n g l e t h e amplitude made w i t h the rotated axis i s referred t o as Conversely, r o t a t i o n of t h a~ x i s t o e l i m i n a t e t h e c o n t r i b u t i o n of t h e s u r f a c e s i g n a l from t h e p r o j e c t i o n of t h e a m p l i t u d e on t h e X-axis ( F i g . 2e) r e s o l v e s t h e a b s o r p t i o n of 6-carotene i n t h e i n t e r i o r l a y e r ( F i g . 2 f ) . The a n g l e t h e amplitude made w i t h t h e r o t a t e d a x i s i s r e f e r r e d t o a s @Aos. 'The e x p e r i m e n t a l phase d i f f e r e n c e ($exp) was c a l c u l a t e d /14,15/ and compared t o t h e t h e o r e t i c a l v a l u e s c a l c u l a t e d u s i n g t h e e x p r e s s i o n s i n M o r i t a 1161. The d a t a t a k e n a t 26 Hz (47" +3) and 100 Hz (57O +4) a r e i n good agreement with t h e t h e o r e t i c a l v a l u e s of 45.0' and 57.5O. The model 1171 v e r i f i e s t h a t t h e i n d i v i d u a l a b s o r p t i o n s of t h e s u r f a c e and i n t e r i o r components a r e r e s o l v e d and t h a t t h e phase data obtained i n the photoacoustic c e l l i s c o r r e c t .

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A p p l i c a t i o n of t h e phase a n a l y s i s a t v a r i o u s f r e q u e n c i e s y i e l d s a pigment d e p t h p r o f i l e of t h e l i c h e n A. schZeicheri ( F i g . 3 ) . The high f r e q u e n c y r e s p o n s e (150 Hz)

Fig. 3 - Pigment d e p t h p r o f i l e and schematic diagram of t h e l i c h e n A. s c h l e i c h e r i . ( a ) Pigment d e p t h p r o f i l e of t h e l i c h e n A. s c h l e i c h e r i . (-) i s associated with a s p e c t r a l component of Khizocarpic a c i d (Asin @ ' p h o t o a c o u s t i c component a t 150 Hz). (---) 1. s a s s o c i a t e d w i t h t h e bulk cytochrome a b s o r p t i o n (Acos @ ' p h o t o a c o u s t i c componc:nt a t 150 Hz). (-.-.-) i s t h e a b s o r p t i o n of t h e c h l o r o p l a s t of t h e a l g a e (Acos $' p h o t o a c o u s t i c component a t 32 Hz). ( b ) schematic c r o s s - s e c t i o n a l view of the lichen. c o r r e s p o n d s t o a thermal d i f f u s i o n l e n g t h of 15pm. The p h o t o a c o u s t i c response i s , t h e r e f o r e , l i m i t e d by t h e thermal d i f f u s i o n l e n g t h t o t h e f u n g a l r e g i o n of t h e lichen. The s t r o n g UV a b s o r p t i o n observed i n t h e s u r f a c e s i g n a l c o r r e s p o n d s t o an a b s o r p t i o n r e g i o n of t h e f u n g a l pigment r h i z o c a r p i c a c i d (peak 275, s h o u l d e r 375 nm i n methanol). T h i s pigment i s observed i n high c o n c e n t r a t i o n i n t h e upper c o r t e x of A. schzeicheri. The i n t e r i o r s i g n a l f o r t h e h i g h f r e q u e n c y r e s p o n s e h a s c h a r a c t e r i s t i c s of a b u l k cytochrome a b s o r p t i o n (peak 440, s h o u l d e r 535 / l a / ) . T h i s spectrum can b e a s s i g n e d t o t h e non-pigmented hyphae i n t h e upper c o r t e x . The low f r e q u e n c y r e s p o n s e (32 Hz) c o r r e s p o n d s t o a thermal d i f f u s i o n l e n g t h of 3 2 ~ . A t t h i s d e p t h w i t h i n t h e sample, t h e i n t e r i o r s p e c t r a c o r r e s p o n d s t o t h e c h a r a c t e r i s t i c a b s o r p t i o n of t h e a l g a l c h l o r o p l a s t . A d e p t h of % 30 p m i s well. w i t h i n the a l g a l layer ; we have observed by l i g h t microscopy t h a t t h e a l g a l l a y e r b e g i n s a t a d e p t h of 20pm.The i n vivo a b s o r p t i o n s p e c t r a of t h e t h r e e s t r a t i f i e d chromophorescorrespond

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w e l l w i t h t h e observed thermal d i f f u s i o n l e n g t h s of t h e t h r e e pigmented l a y e r s /17/. An e a r l y example of t h e r e s o l u t i o n of a n a t u r a l l y o c c u r r i n g s t r a t i f i e d system u s i n g

et.

1 1 9 1 i n which l i g h t a b s o r p t i o n i n t h e n e a r PA t e c h n i q u e s i s t h e work by Adams UV by t h e waxy c u t i c u l a r l a y e r of a green l e a f was c l e a r l y r e s o l v e d from t h e l i g h t

absorbed by the c h l o r o p l a s t s . l ' i g u r e 4 p r e s e n t s the spectrum of the leaf of ablackfoot daisy taken i n o u r s p e c t r o m e t e r ; i n t h i s c a s e only t h e a m p l i t u d e s p e c t r a a r e shown. The upper

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wavelength Fig. 4 - Amplitude s p e c t r a of b l a c k f o o t d a i s y l e a f t a k e n a t low modulation frequency. () i s t h e spectrum of a l e a f from a normal p l a n t grown i n t h e f i e l d ; the spectrum of a c l o n e grown i n a s p e c i a l environment. ( a m - )

spectrum shows b o t h t h e wax a b s o r p t i o n (350-400 nm) and t h e c h l o r o p h y l l . bands i n t h e v i s i b l e . I t i s i n t e r e s t i n g t o n o t e t h e spectrum of t h e i n v i t r o l e a f p r e s e n t e d i n f i g u r e 4. T h i s l e a f i s from a c l o n e of t h e d a i s y p l a n t and i t h a s c l e a r l y f a i l e d t o develop t h e waxy c o a t n e c e s s a r y f o r s u r v i v a l . S t u d i e s a r e underway i n o u r l a b o r a t o r y t o d e t e r m i n e t h e biochemical mechanism f o r t h i s f a i l u r e and t o f i n d a means of enhancing t h e v i a b i l i t y of t h e s e c l o n e s i n t h e f i e l d .

We have measured t h e PA spectrum of l o b s t e r s h e l l a t d i f f e r e n t modulation frequencies and p h a s e s , and have found a n unexpected arrangement of protein-pigment complexes ( c a r o t e n o p r o t e i n s ) i n t h e pigmented l a y e r of t h e s h e l l / 2 0 / . The c a r o t e n o p r o t e i n s having t h c l o n g e s t wavelength a b s o r p t i o n maxima were found i n t h e i n t e r i o r - m o s t r e g i o n o f t h e pigmenred l a y e r . T h i s a n i s o t r o p i c d i s t r i b u t i o n of c a r o t e n o p r o t e i n s a s a f u n c t i o n of d e p t h i s d e s t r o y e d by d e n a t u r i n g t h e p r o t e i n ; c l e a r l y i n d i c a t i . n g t h a t t e r t i a r y and q u a t e r n a r y s t r u c t u r a l f e a t u r e s a r e involved i n t h e pigment-protein interaction. The p h o t o s y n t h e t i c a s s a y c o n s i s t s of o b t a i n i n g t h e PA a m p l i t u d e a t 680 nm a s a f u n c t i o n of modulation frequency i n t h e absence and p r e s e n c e of a n i n t e n s e , c o n t i nuous (DC) background l i g h t 121,221. Without t h e DC l i g h t a f r a c t i o n of t h e energy of t h e modulated l i g h t i s conserved by t h e p h o t o s y n t h e t i c p r o c e s s and t h u s t h e PA s i g n a l ( t h e h e a t e v o l v e d ) i s n o t maximal. The DC l i g h t d r i v e s p h o t o s y n t h e s i s t o s a t u r a t i o n ; t h e weaker modulated l i g h t i s t h e n 100 % c o n v e r t e d t o h e a t and t h e PA s i g n a l changes. l l ~ w e v e r , t h e s i g n of t h e change depends on t h e modulation f r e quency ; i t i s (--) a t low frequency and (+) a t high f r e q u e n c y . T h i s can be understood s i n c e b o t h h e a t and 0, e v o l u t i o n c o n t r i b u t e t o t h e PA s i g n a l i n a g a s microphone c e l l . A t low modulation f r e q u e n c y t h e PA s i g n a l c o n s i s t s i n l a r g e p a r t of modulated 0, e v o l u t i o n . The s t r o n g DC l i g h t d r i v e s t h e 0, e v o l u t i o n t o s a t u r a t i o n Q,

and hence the modulated 0, evolution and concomitant PA signal decrease. At higher modulation frequency, above 200 Hz, the slower 0, evolution process is damped and does not contribute to the PA signal and the expected increase in signal upon applicationof the DC light is observed. Using PTR detection 191 only heat is measuredand the PA signal increases at both low and high frequency. This assay is unequivocal for a living photosynthetic system - leaves poisoned by drugs or that are ina dormant state do not respond to the DC light. Figure 5 presents the photosynthetic assay of a living hydrated lichen sample 4

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Fig. 5 - The % effect at 680 nm vs modulation frequency for the lichen A.schZeicheri. % effect 1 2 1 1 is the percent change in the PA signal upon irradiation with DC light. carried out in a gas-microphone cell ; 0, evolution and energy conversion (storage) are evident. In the case of a dehydrated lichen sample it is known that the algal component is dormant. As expected, there is no effect of the DC light at any modulation frequency on these samples. It must be noted that there is essentially no macroscopically observable change in the sample upon hydration, yet the state of the photosynthetic process can clearly be monitored via PA techniques. Lichen have been used as an early warning system for certain types of environmental polution ; it is in this connection that a sensitive method for assaying the health of the lichen is important. Another application of PA techniques is to studies of the absorption and penetration of light into skin tissue. The information about the type and location of chromophores in skin is of importance in the design andevaluationof sun screening chemicals, in understanding the mechanism of skin photosensitization in diseases of light intolerance, and in research and clinical use of photochemotherapy for psoriasis and certain types of cancer. Figure 6(a-e) presents results from our laboratory using a gas-microphone cell and mouse skin. In 6(a) the surface spectrum is shown of an albino mouse ; this is in contrast to the surface spectrum shown in 6(d) of a normal pigmented mouse in which the absorption of the melanin pigment is clearly shown. 6(b) shows the surface as in 6(a) except that the mouse was fed 6-carotene daily for six weeks ; the absorption of the carotenoid pigment in the 450 nm range is clearly evident. The characteristic bands of hemoglobin at ?. 430 nm (Soret), 550 nm and 580 nm (Q-bands) are shown in the interiorspectrum 6(c). 6(b) and 6(c) are spectra of the same tissue sample and indicate that the major reservoir of B-carotene is in the stratum corneum above the capillary bed which is in the living epidermis (Anjo, D.M. and Moore, T.A. submitted to Photochem. Photobiol.) It is interesting to compare the signal to noise ratio between 6(c) and the interior (hemoglobin) spectrum in 6(e). The reduced signal in 6(e) is due to the screening effect of the melanin which substantially reduces the amount of light present at the level of the capillary bed. Furthermore, comparison of the hemoglobin signal

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wavelength (nm) Fig. 6(a) - Surface spectrum of albino mouse (Swiss-Webster) ear skin taken at 10 Hz modulation frequency.

wavelength (nm) Fig. 6(b) - Surface spectrum of mouse tlssue as in (a) except mouse was fed B-carotene over 8 weeks.

Fig. 6(c) - Interior spectrum of the same tissue sample as in (b). 10 Hz modulation.

Fig. 6(d) - Surface spectrum of ear tissue of mouse with normal pigmentation. 10 Hz modulation.

Fig. 6(e) - Interior spectrum of the same tissue sample as in (d). 10 Hz modulation.

wavelength (nm)

from tissue with and without $-carotene treatment shows that the screening by $-carotene is negligible. This result leads us to search for alternative mechanisms for the protective effect /23/ of carotenoids against light-induced diseases. Recently, we have applied a modified form of the technique developed by Quimby and Yen /24/ for measuring the heat evolved when a molecule in an excited state relaxes /25/. Using this method (fully described in this issue) it is possible to measure the energy gap between two states, e.g. the ground state and an upper, metastable excited state, even though an optical transition between them is not observed. It is expected that this will be a useful way to investigate biological (photosynthetic) and photochemical energy transducing systems. TAM and DB acknowledge the NSF (grant no CHE-802171 8) and the Pllysics and Chemistry Departments, Arizona State University, for support of this work. REFERENCES ROSENCWAIG, A., Adv. Electr. Electron Physics. 3 (1978) 207. BALASUBRAMANIAN, D. and RAO, Ch. M., Photochem. Photobiol. 34 (1981) 749. CAHEN, D., BULTS, G., CARTY, H. and MALKIN, S., J. Biochem. Biophys. Methods 3 (1980) 293. MOORE, T.A., in Photochemical and Photobiological Reviews (ed. K. Smith) Vol. 7 Plenum, N.Y., in press. ROSENCWAIG, A. and GERSHO, A., J. Appl. Phys. 47 (1976) 64. J. Appl. Phys. 2 (1980) -52. THOMAS, R.L. MURPHY, J.C. and AAMODT, L.C., J. Appl. Phys. 48 (1977) 3502. AAMODT, L.C., MURPHY, J.C. and PERKER, J.G., ~ 7 ~ Phys. ~ ~ 48 1(1977) . 927. McDONALD, F.A. and WETSEL, G.C., J. Appl. Phys. (1978) 2 3 n . NORDAL, P.E. and KANSTAD, S.O., Appl. Phys. Lett. 2 (1981) 486 ; Physica Scripta 20 (1979) 659. TOX, R.D., O'HAM, E.P. and BENIN, D., J. Appl. Phys. 2 (1982) 5392. BOCCARA, A.C., FOURNIER, D. and BADOZ, J., Appl. Phys. Lett. 36 (1980) 130 ; BOCCARA, A.C., FOURNIER, D., JACKSON, W. and AMER, N.M., Opt. Lett. 5 (1980) 377. FULLER, G., Analytic Geometry, 4thed. (1973) 108, Addison-Wesley Publishing Co., Reading MA. BETTERIDGE, D., LILLEY, T. and MEYLEK, P . J . , Fresenius Anal. Chem. 296 (1979) 28. - @Aos). Calculation of experimental phase : Qexp = 90° + ($:in The experimental values were obtained by rotating the axis until an uncontaminated spectra was observed. The deviation corresponds to values of the phase in which the uncontaminated spectra were judged indistinguishable. MORITA, M., Japan J. Appl. Phys. 20 (1981) 835. O'HARA, E.P., TOM, R. and MOORE, T.A. Second International Topical Meeting on Photoacoustic Spectroscopy, Technical Digest, June 1981 ; 10th Annual Meeting of of the American Society for Photobiology, June, 1982 ; Photochem. ~hotobiol. (1983) in press. Similar photoacoustic spectra have been obtained for the non-lichen fungus VerticiZZiwn agarlici Z Zwn. ADAMS, M.J. and KIRKBRIGHT, G.F., Analyst 102 (1977) 281 ; ADAMS, M.J., BEADLE, B.C., KING, A.A. and E K B R ~ G H T ,G.F., Analyst 101 (1976) 553. 20. MACKENTHUN, M.L., TOM, R.D. and MOORE, T.A. Nature 279 (1979) 265. 21. LASSER-ROSS, N., MALKIN, S. and CAHEN, D., Biochim. Biophys. Acta 593 (1980) 330. 22. BULTS, G., NORDAL, P.-E. and KAHSTAD, S.O., Biochim. Biophys. Acta 682 (1982) 234. KANSTAD, S.O., CAHEN, D. and MALKIN, S., Biochim. Biophys. Acta 7 2 2 7 9 8 3 ) 182. 23. MATHEWS-ROTH, M.M. in the Science of Photomedicine (ed. Kegan and Parrish) p.409 (1982). Plenum Publishing Corp. ; MATHEWS-ROTH, M.M., J.N.C.1, 69 (1982) 279. 24. QUIMBY, R. and YEN, W., J. Phys. 2 (1980) 1780 ; Opt. Lett. 3 (1978) 181. 25. MOORE, T.A., BENIN, D. and TOM, R., J. Am. Chem. Soc. 104 (1982) 7376.

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