Photochemrrrry and Photobiology Vol. zyxwvutsrq 51, No. 4, pp. 491-496, 1990 zyxwvutsr Printed in Great Britain. All rights reserved 0031-8655190 zyxw $03.M+0.00 Copyright zyxw 0 1990 Pergamon Press plc TECHNICAL NOTE PHOTOTHERMAL DEFLECTION SPECTROSCOPY OF PHOTOSYNTHETIC PIGMENTS in vivo AND in vitro LUCIE LOKKAIN~, MICHEL HAVAUX’.’, AIAIN TESSIEK~ and ROGER M. LEBLANC’.“ ‘Centre de Recherche en Photobiophysique, Universite du Quebec a Trois-Rivieres. 3351 Boulevard des Forges, C.P. 500, Trois-Rivieres (Quebec), Canada zyxwv G9A 5147 and ‘Laboratoire de Bioenergetique. Station de Botanique, Universite de Geneve. OH-1254 Lullier. Geneve. Switzerland. (Received I zyxwvutsrqponmlkjihgfedcba 1 zyxwvutsrqponm August 1989; accepted 10 November 1089) Abstract-This Technical Note describes the design of a photothermal beam deflection apparatus which allows the easy and rapid measurement of thermal dissipation of absorbed light energy in various photosynthetic materials including whole plant leaves. This system is based on the “mirage effect” in which the refractive index gradient induced in a fluid in contact with the sample, irradiated with an intensity-modulated light, causes the periodic deflection of a laser beam parallel to the sample surface. The deflection of the probe laser beam is detected by a position sensor, the output of which is processed by a lock-in amplifier. Photothermal deflection signals can be monitored in vivo in intact leaves placed in various (liquid or gaseous) environments with a wtisfactory signal-to-noise ratio between 100 (in water) and 50 (in air) at low modulation frcqtiencies (ca 30 Hz). It is shown that this new and simple photothermal technique is a very sensitive tool for the meawrement of absorption spectra of photosynthetic pigments both zyxwvut in zyxwvutsrqp vivo (leaves, algae or chloroplasts) and in model systems (Langmuir-Blodgett and solid films of chlorophyll). INTRODUCTION In recent years, the pulsed temperature rise induced in an absorbing material by illumination with inten- sity-modulated light has provided the physical basis for the development of a new class of sensitive photothermally based spectroscopic techniques. Until recently, the more familiar member of this family has been photoacoustic (PA)? spectroscopy where modulated heat emission is measured as pressure waves using a microphone or in some cases a piezeolectric transducer (Rosencwaig, 1980; Bras- lavsky, 1986; Coufal and McClelland, 1988). In a conventional gasimicrophone PA cell, modulated thermal energy dissipation causes the periodic heat- ing of a thin layer of gas adherent to the sample. The resulting periodic expansion and contraction of this gas layer ultimately produces a pressure wave in the bulk gas which propagates in the hermetically closed cell where it is detected by a sensitive micro- phone. After analysis with a phase-sensitive lock-in amplifier to obtain both amplitude and phase, the PA signal provides useful information on the optical and thermal properties of the absorbing sample. Because only absorbed radiation contributes to the signal, PA spectroscopy is particularly suitable for the study of highly light-scattering media, such as most biological samples, as an alternative to conven- ___ ~ _-.. ~~~ ~~~~ ~ *To whom correspondence should be addressed. tilbbreviafions: chl, chlorophyll: L-B, Langmuir-Blodgett; PA photoacoustic: PD, photothermal deflection: SIN, signal-to-noise ratio. tional transmission and diffuse reflectance tech- niques. The PA method has been applied lately to the field of photosynthesis and the recent literature provides numerous examples of photoacoustically measured absorption spectra of photosynthetic sys- tems such as whole leaves (Malkin et a/., 1981; Nagel et a/.. 1987; Nery et al., 1987; Szigeti ef a/., 1989), lichens (O’Hara et al., 1983) or algae (Car- pentier et d., 1983). Boccara and co-workers have proposed another means for carrying out PA experiments using the “mirage effect” (Boccara et a/., 1980a, b; Jackson et al., 1981). In this method, the signal is detected by the change of the index of refraction produced in the fluid (gas or liquid) near the sample by the temperature variation of the sample. The index-of- refraction gradient is sensed by the periodic deflec- tion of a laser beam focalized above the sample. A significant advantage of this photothermal deflec- tion (PD) method is that the spatial gradient of the total temperature rise associated with light absorp- tion, not just the acoustic fraction, is used for detec- tion. Although PD spectroscopy is apparently more sensitive than the conventional microphone detec- tion, it has not yet found wide application to biologi- cal samples. This is probably caused by technical problems linked to practical difficulties in the optical alignment of the sample and the probe laser beam. This paper describes an original beam deflection system specially designed to carry out in vivo PD measurements in intact plant leaves. The PD method is particularly suitable for photosynthetic 49 1