Application of photoacoustic spectroscopy in photosynthesis research

217 (1990) 69-84 ElsevierScience PublishersB.V., Amsterdam- Printedin The Netherlands

Journal of Molecular Structure,

69

APPLICATION OF PHOTOACOUSTIC SPECTROSCOPY IN PHOTOSYNTHESIS

C.N. NSOUKPOlkKOSSI

RESEARCH

and R.M. LEBLANC

Centre de recherche en photobiophysique, Universite du QuCbec h Trois-Rivieres, C.P. 500, Trois-Rivikes, Qu&ec, G9A 5H7 (Canada) SUMMARY Photoacoustic spectroscopy is a new analytical technique which allows the detection of light-induced heat production due to non-radiative deactivation of light excitation. This paper gives an overview of the theory of photoacoustics and its application in photosynthesis research to measure energy conversion and storage, and for molecular structure and interaction studies as well as oxygen evolution in photosynthetic systems. INTRODUCI’ION The photoacoustic (PA) effect was discovered over hundred years ago by Alexander Graham Bell (1). Since that time, much research has been done. But it was only during the 1970’s, with the evolution of electronics, that Rosencwaig and Gersho from Bell Laboratories at Murray Hill in New Jersey proposed a theory explaining photoacoustic effect in solids (2). Photoacoustic spectroscopy (PAS) consists of exciting a sample, contained in a hermetically closed cell, with a monochromatic beam which is chopped at audio frequencies.

light

The sample is therefore alternately

subjected to light excitation and thermal deactivation.

This cycling induces periodic

heating and cooling of the gas in contact with the surface of the sample. The resulting pressure oscillations are sensed by a highly sensitive microphone which generates corresponding electric signals at the given wavelengths and the PA signals are amplified and sent to a recorder or to a computer. In case of active photosynthetic systems, 02 evolution also contributes to pressure oscillations. This relatively new spectroscopic methodology has long been utilized to study light absorption by gases.

But only recently has its use been applied in other fields

including biology and biophysics (3-6), physics (7), medicine (8-14), food (15,161 and plant sciences (17-20 for reviews). THEORY OF PHOTOACOUSTICS Photoacoustic effect is based on Boyle’s law which states that the product of pressure (P) and volume (V) of a gas at a given (or constant) temperature is constant

0022-2860/90/$03.50

0 1990 Elsevier Science PublishersB.V.

70

PV =

c

(1)

This constant C has later been equated to the number of moles of gas (n) times R (ideal gas constant) times T (absolute temperature, K) PV = nRT P =m

(2) (3)

V

If we assume that the thermal properties of the material are independent of position and temperature, and consider only heat flow in one direction (parallel to the X-axis), the equation of unidirectional linear heat flow is expressed by:

a*T

1. . 2

ax*

B

-_

=

0

(4)

where T (K) is temperature and is a function of position x and time, t, and J3 (m2 s-l) is the thermal diffusivity of the substance and is a derived quantity mathematically defined as: B =

(5)

+#

where k is the thermal conductivity (J s-1 m-1 K-l), p, the density (kg m-s), C: the specific heat of the substance (J kg-1 K-1). The boundary conditions will determine the adequate solution of equation (4). If the surface temperature,

at x = 0, is a harmonic function of time given by the

relationship T = To cos (ot)

(6)

where w/27t is the frequency of temperature oscillation, then the solution of equation (4) is expressed as: T = To exp [-x (w/2J3)*] . [cos (wt - x (o/2J$)J

- A

(7)

where A is transient disturbance induced by starting the oscillation at time, t = O. As t increases, A decreases down to zero. number of the temperature A=--_T

diffusion

The function

(0/2p)

represents

the wave-

wave of wavelength

27l

(8)

(wlz3)’ From equation

(7) it is easy to see that the amplitude

will diminish

exponentially

exp I- b/2P)f

x1

according

of the temperature

wave

to

(9)

and consequently dimensionless

will decrease rapidly as w or x increases.

quantities,

Equation (7) involves two

wt and ax where (10)

a = (o/2p11. is the thermal diffusion The function Rosencwaig

2rr/a derived

and Gersho

Only this relatively the temperature diffusion

coefficient. from equations

shallow depth of material

wave emanating

coefficient,

(8) and (10) has been

(2) as the active thermal

i.e.,

need be considered

from the surface.

a-1 represents

defined

length of the temperature

as responding

The reciprocal

the thermal

by

wave.

diffusion

to

of the thermal length,

l’p, also

symbolized by l.tLs.

(11) Another

important

parameter

to be defined

in PAS is a, the optical

absorption

coefficient a = 2.3 E X molar concentration where

E

denotes

represents

the molar

(12)

absorptivity

the optical absorption

the incident

light intensity

view, it is important

length,

i.e., a-l

From pratical point of

under which its < pa to avoid optical satu-

of sample) otherwise,

severe deformation

of the

It is worthy to say that by simply changing the frequency of

of the light, we can obtain useful information

the sample. stratified

by a factor of l/e.

to create conditions

PA spectra would occur.

Its reciprocal,

l.t*, or the depth at which, in the material,

has decreased

ration when l.tLa< R((1 = thickness modulation

(dm3 mol-l cm-l).

This depth profile technique

is of especial

from different

interest

depths of

when dealing

with

or thick materials.

To summarize, contained

change

in the temperature

of the gas surrounding

in a closed vessel will result in change

tions are then sensed variations

will

by a very sensitive

be recorded

Furthermore,

these pressure

microphone.

A schematic

through

microphone.

the microphone,

changes diagram

in gas pressure.

should

varia-

Note that, only pressure and

be fast enough

of a photoacoustic

the sample

Pressure

not

pressure

to be sensed

spectrometer

itself. by the

is shown

in

figure 1. Under could

specific

be obtained.

measured

conditions,

a PA spectrum

In contrast

is determined

with

equivalent

absorption

spectroscopy

by the sample’s optical constant,

the sample usually play the central role in the photoacoustic signal is essentially

proportional

ed, the radiationless

conversion

to three parameters, efficiency

to absorption where

the thermal

spectrum the signal

properties

signal (21,22).

of

The PA

i.e., the radiant power absorb-

and the thermal transfer efficiency.

Chopper

Selector PA Cell PA Sign01

Reference Signal

$ Amplifier

I

Recorder

Fig. 1. Schematic

PA signal

determined

i)

of the photoacoustic

=

Radiant power absorbed

the

amplitude

intensity

Consequently,

diagram

x Radiationless efficiency

of PA signal

spectrometer.

conversion

for optimum

x Thermal transfer efficiency signal/noise

ratio

by (17):

high photon flux of modulated photodestruction

light beam with precautions

concerning

of the sample,

ii)

appropriate

iii)

low temperature,

modulation

frequency,

iv)

the nature and amount of gas layer above the sample,

v)

high light absorption

vi)

high quantum yield for radiationless

vii)

low thermal conductivity

viii)

high thermal conductivity

of the sample, de-excitation

of the sample,

of samples with high light absorption, of optically more transparent

samples

(i.e., with

lower light absorption), ix)

large sample surface (to allow a good thermal transfer from sample into the gas phase of the sample compartment).

is

73

MOLECULAR Molecular

STRUCTURE

AND INTERACTIONS

structure

In figure 2, we show PA spectra of a pea (Pisum sativum L.) leaf exhibiting main bands:

one around 300 nm corresponding

lipids, and two other bands for chlorophyll 680 nm.

For curve -1 obtained

modulated

white light (actinic

reactions

centers,

deactivation. increases

the signal

under additional

illumination

light) which saturates is only photothermal

due to 02 evolution (I’D) technique,

of protein and cuticle

at 470 nm (Soret band) and at with a strong

non-

photosynthesis

by closing

resulting

non-radiative

As the actinic light is off, photosynthesis

PA bands are rather broad. deflection

to absorption

absorption

three

from

will operate

all

and the signal

(Fig. 2, curve -2). As can be noticed from these curves, Better resolution

is achieved

known as “mirage effect”.

by using the photothermal

This is evidenced

by figure 3

(curve PD vs curve PA) (23). Depth profile Photoacoustic the sample

signal

essentially

which is inversely

by changing the frequency, the sample. cular

of a sample.

0.00

‘2 305

Fig.

upon the thermal to the frequency

diffusion

length

of modulation.

of

Thus,

PA signals are obtained from different depths or layers of

This depth profiling

structure

depends

proportional

is very useful to gain insight For instance,

PA signal

415 525 WAVELENGTH

from

into internal

mole-

anthocyanins

and

635 (nm)

2. Photoacousiic spectra of a pea leaf 2) without actinic light at 80 Hz.

1) with and

(PO)

400

500

Fig. 3. Photoacoustic (PA) and photothernal deflection (PD) spectraofa pea leaf at 18 Hz.

700

600

WAVELENGTH

( nm)

chlorophylls are found to have different relative intensities at different frequencies, or depths (Fig. 4). Molecular interactions in oriented svstems Light-harvesting

pigments of chloroplast lamellae are functionally organized to

efficiently transfer the absorbed excitation energy to the reaction centers.

Various

pigments and their spectroscopically different “forms” (created by the interaction of molecules membrane.

with environment)

are differently

oriented with respect to thylakoid

Different forms of pigments have overlapping absorption spectra but

various orientations and maybe different energy conversion efficiencies.

Therefore

it might be convenient to investigate them by PA and fluorescence spectroscopies in natural and polarized light. For example, PA, fluorescence and absorption spectra of biliprotein,

namely R-phycoerythrin

(PE) and R-phycocyanin

(PC) in polyvinylal-

cohol (PVA) films (24) have revealed the presence of mixed aggregates for which the thermal deactivation of excitation is more efficient compared with that for PE and PC alone.

The fluorescence

quantum yield has been found to be 0.45 for PC

(considering a value of 0.56 for FE (25)) which is lower than that reported (0.59) earlier by Grabowski and Gantt (26) for PC solution. Changes in the molecular interactions can be induced by having the pigments in solution in a liquid crystal medium, which simulates to some extent natural chlorophyll environment, and applying external electric field. For instance, strong fields of the order of 107 V m-1 have been employed to study the polarized absorption spectra of chlorophyll a (chl aJ and chlorophyll b (chl h) solutions in nematic liquid crystals.

WAVELENGTH WAVELENGTH A

220240

no

470

220

WAVELENGTH

240

470

2.

( nm) 2.0

220

240

4Ta

220

(nm) 220

220

4SO

220

(nm)

240

410

220

WAVELENGTH

-0

(nm)

Fig. 4. Depth profile spectra of sugar maple green leaf showing chlorophyll absorption around 470 nm and 680 nm (A), and a yellowing leaf with anthocyanin band at 525 nm (B).

The following liquid crystals have been used: i) p-methoxybenzylidene line (MBBA) + p-ethoxybenzylidene

p’-butylani-

p’-butylaniline (EBBA) (3:2), ii) MBBA + EBBA

(3:2) and iii) p-pentyl-p-cyanobiphenyl

(PCB). In liquid crystals chl is photochemi-

tally very stable and occurs only in monomeric form because of strong interactions with liquid crystals (27). Upon external electric field, polarization of absorption spectra was only slightly perturbed and position of main bands were not shifted.

The difference between

spectra recorded with and without field was explained as being due predominantly to reorientation of chlorophyll attached to liquid crystals.

These perturbations have

further been confirmed by dielectric measurements of chl in the liquids crystals (28). Chl a and b have been found to perturb the regular arrangement of liquid crystals and thus influence their E’ and E” values, respectively, the real and imaginary parts of dielectric constant. Molecular spectrocopic properties of the biliproteins PE and PC (29) using PAS in natural and polarized light, and of chloroplasts (30) embedded in stretched and unstretched WA films have been examined as already reviewed by Leblanc and N’soukpoCKossi (31). It has also been found (32) that the shapes of chloroplasts and isolated thylakoids have changed as the result of the PVA film stretching.

The

relationship between mechanical deformation and absorption anisotropy was different for whole chloroplasts and isolated thylakoids.

Thermal deactivation of various

chl-protein complexes was calculated from polarized PA and absorption spectra (Tables 1 and 2).

TABLE 1 The factors of mechanical deformation

D and absorption

anisotropy

A.

Sample Congored c=5x1U5 Fl Whole chloroplasts (~2) Broken chloroplasts (~2) Thylakoids (~2) Chloroplasts (~1) Thylakoids (~1)

5.250 1.022 1.179 1.183 1.197 1.331

1.000 1.000 1.027 0.930 0.996 0.980

2.291 1.011 1.071 1.128 1.096 1.165

5.78 3.00 3.00 3.00 4.35 2.15

0.396 0.337 0.357 0.376 0.252 0.542

TABLE 2 Polarized thermal deactivation red band for chloroplasts

(TD = PAS/A, arbitrary

and thylakoids

Sample Chloroplasts

Thylakoids

units) for the main Gaussian

Photoacoustic conversion

Unstretched TD,, TD,

Stretched TD II TDI

640 648 662 668 676 682 687 693

0.091 0.082 0.089 0.076 0.071 0.088 0.100 0.100

0.087 0.077 0.094 0.067 0.074 0.088 0.105 0.108

0.076 0.112 0.084 0.100 0.074 0.125 0.123 0.183

-‘0.142

640

0.078 0.060 0.050 0.066 0.087 0.120 0.100 0.399

0.078 0.043 0.066 0.066 0.130 0.092 0.087 0.199

0.065 0.073 0.068 0.075 0.180 0.100 0.092 1.600

0.150 0.110 0.097 0.100 0.300 0.150 0.330 0.130

and storage

yield.

emission i)

transfer between dissipation

to study Irradiation

the measure

deactivation

stored in chemical

(5).

energy

of a sample

of the conversion

with

of light

In the case of photosyn-

intermediates

activity can thus be estimated

excitation

decreases

by comparison

the thermal

of the thermal

active sample and an inactive one (18).

bacteria

Using photoacoustic Rhodospirillum

used

research.

allows

non-radiative

of a photosynthetically

Photosvnthetic

widely

in photosynthesis

the energy

Photosynthetic

is being

light beam

into heat through

thetic materials,

0.111 0.108 0.106 0.108 0.116 0.154 0.175

and storage methodology

amplitude-modulated energy

of the

Max [NIlI

648 662 668 676 682 687 693

Energv conversion

components

isolated from Pisum sativum L.

spectroscopy,

carotenoids (l&s.)

spectrum

rubrum

we have evaluated

and bacteriochlorophyll and Rhodopseudomomns

of Rhs. rubrum

showed

(331 the efficiency

of energy

in the photosynthetic (Rhp.)

a maximum

sphaeroicles.

bacteria The

in the spirilloxanthin

77

absorption spectral region (450-550 nm).

In Rhp. sphaeroides,

where all pigments

are efficient antenna, the wavelength dependence of the spectrum was absent.

The

ratio of quantum yields in the photochemical reactions of bacteriochlorophyll

and

carotenoids was found to be 2. ii) Plant nhotosvstems The energy conversion has also been studied in subcellular fractions such as photosystem II (PSII) isolated from spinach (34). The energy storage was found to depend upon the intensity (I) of modulated light used. By extrapolating to I = 0, a maximum value of 22% was obtained at 35 Hz, which is similar to what has been obtained in broken barley chloroplasts (i.e., 24%) (35). PA analysis of photosynthetic energy storage in bundle sheath cells of Zen mays has allowed to clarify the role of PSII as being a prevention of overoxidation of PSI (36) and led to the conclusion that PSI is reduced by some exogen electron donor (s) and that PSI1 only partially participates in PSI reduction (37). We further observed from PA measurements that PSII energy storage is mediated by pheophytin (38). Additional experimentation with additives showed two types of effects (39): the first group indicated quenching effects on the energy storage induced by malate, ribulose 5-phosphate (R5P) + NaHC03 + NADP, DCMU [3-(3,4-dichlorophenyl)-Lldimethylurea],

DBMIB (2,5-dibromo-3-methyI-6-isopropyl-p-benzoquinone),

MV (methyl viologen);

and

the second group caused enhancement of energy storage as

evidenced with ascorbate and DTT (dithiothreitol).

In an earlier report (401, it has

been noticed that Rsl?, HC03 and NADP stimulated the photosynthetic carbon fixation in bundle sheaths, increasing the demand for NADPH and ATI?. Consequently, a rapid turnover through electron transport can be expected.

As it has been shown,

under these circumstances, an energy storage signal did not appear. the following:

We then infer

when the electron transport traps of the photosytems are open, heat

dissipation is less favorable.

The reduced electron transport system induced by the

presence of ascorbate or DTI’ (41,421 may provide reductant to PSI and poise cyclic electron flow, which will result in energy storage by means of reduction of interelectron transport carriers. The stimulating effect of ascorbate on the energy storage signal is a sign that an electron donor regulates PSI activity. of ascorbate are found in stromal compartment ranging from 15 to 75 mM (43).

Considerable amounts

of chloroplasts

in concentration

The recovery of energy storage signal, in the

presence of ascorbate after treatment with DCMU, indicates that PSI activity in bundle sheath chloroplasts is highly dependent on some stroma containing reductant(s).

The addition of ascorbate in presence of a higher concentration of DBMIB

does not recover the energy storage signal.

The inhibitory effect of DBMIB shows

that the electron donor is acting via PQ (plastoquinone)

and cytochrome (cyt.) f

reduction and confirms that PQ and cyt. f are tightly associated as electron carriers of

PSI.

As for malic acid, we observed no energy storage signal in bundle sheath

chloroplast in accordance with earlier results indicating that malic acid stimulates photosynthetic carbon assimilation and also increases the demand for NADPH and ATP through donation of CO2 to the reductive pentose-P pathway (44). It is worth noting that these experiments

have been performed

frequency unable to detect fast turnover reactions.

at 75 Hz, a relatively

low

In order to measure energy

storage for fast reactions, higher frequencies of the modulation of light are required. iii) Algal uhotosvstems Cyanobacteria (blue-green algae) are alone able among the bacterial kingdom to perform a plant-type photosynthesis

which involves two consecutive

tions linked to each other by a chain of electron carriers. blue chromoprotein,

phycocyanin,

pigment in cyanobacteria.

replaces

photoreac-

Unlike plants, however, a

chl b_ as a major light-harvesting

This photosynthetic process leads to the photophospho-

rylation of ADP to ATE’ with a final transfer of reducing equivalents to NADP+. A cyclic (non-reducing) photophosphorylation pathway is also available around PSI. Photosynthetic

activities of algae, Anacystis nidulans have been investigated by

PAS. First studies (45) showed that the amplitude of PA signal increases with thickness of the algae layer and reached a plateau when PA signal became saturated. This saturation effect which started from a thickness of 10 pm (46) was believed to originate mostly from the limited optical penetration of the sample and there was distortion of the PA spectrum.

A theoretical model was proposed to explain these results,

and practical means to obviate the limitations of this spectroscopic technique were suggested.

At room temperature as well as low temperature the bands of pigment

holochromes were well resolved and similar to absorption spectrum (Fig. 5) (47). Since the quantum yield of fluorescence in algae pigments at room temperature is negligible (48), the absorbed energy is dissipated between heat production and photochemical DCMU-inhibited

energy storage.

As could be expected, the comparison

between

and active algae indicated (46) that DCMU induced a significant

increase of PA signal between 400 and 700 nm, with a maximum between 550 and 650 nm similar to absorption spectrum.

The coincidence between absorption and

action spectra points out that phycocyanin pigment in linear photosynthesis. methyl-p-phenylenediamine)

is the most efficient light-harvesting

The fact that addition of TMPD (N,N,N’,N’-tetra-

to DCMU-treated

algae causes a relatively

larger

decrease around the red absorption band of chl a (which reflects the reactivation of linear PSI in the presence a poisoned PSII), indicated that chl a acts as the most efficient antenna for photoreaction I. However, phycocyanin still retained about two thirds of the quantum yield of chl a. It follows that phycocyanin can distribute its excitation energy to both types of photoreaction almost

exclusively

to

the

I?SII

antenna

centers, while chl a is assigned

(46).

In order

to

detect cyclic

I.5 *

i ; -

1.0.

i

3 J; 0.5 ( : 0.0

I

I

I

I

550 4io WAVELENGTH

1

I

650 (nm)

Fig. 5. Action spectrum for thermal deactivation in ~nacysAbsorption spectrum from a liquid suspension tis nichlans. of algae ( . ..). PA spectrum from l-urn sample (-) and for an infinitely thin sample (0.0). The PA spectra are equalized with the absorption spectrum at 630 nm.

photophosphorylation,

it is necessary

This is achieved

by using DCMU

phosphorylation

from cyclic electron

across the thylakoid

membranes

here to first inhibit

and next adding NH&l, transport

(49).

linear

photosynthesis.

which

uncouples

by collapsing

Quite surprisingly,

the proton

the final increase of the PA

signal was nearly as large as the one induced by DCMU alone indicating of a much

larger

cyclic activity

between

the modulation

photons

absorbed

than usually

frequency

thought.

and PA signal

at 630 nm perform

Based

intensity,

photochemical

ADP

gradient

work

the presence

on the relationship at least 60% of the

and about half of the

useful energy is stored as stable products (50). iv) Photosvnthetic

activities

in preen whole leaves

The solar energy absorbed by green leaves is transferred the chloroplasts photochemical

where, under optimum work of photosynthesis.

as heat through

radiationless

conditions,

to two reaction centers in

most of this energy is stored for

But part of the absorbed energy is dissipated

deactivation

and as fluorescence.

In P700-enriched

particles it was found (19) that 80% of the absorbed energy is used for photosynthesis whereas acoustic

17% is lost in heat production spectroscopy

measures

and 3% in fluorescence

thermal deactivation

of excitation

emission.

Photo-

energy as well as

80

02 evolved

by photosynthesis

modulated

beam illumination,

one is photothermal 02 evolution.

(thermal

detected

including

deactivation) component

storage

is then suppressed.

cies compared

It would

certainly

light

energy

decreases

When

due to energy

Comparing

modulated

x

APT

cosine).

the actinic

light

the maximum reflects

is off, the thermal under

energy

the quantity

component signal

Thus, by putting

component

the modulated

light only.

actinic light.

is delayed

thermal

relatively

by first adjusting signal

storage measured

in cosine

at high frequency

amplitude,

AQ,

signal,

these

lock-in channels channel

signal is recorded

(sine) when

signal will appear in both channels channel.

Considering

tion, & is the amplitude signal increase

is the same as that at low frequencies

(23) 02-

(14)

normalized

of sine signal

under

modulated

illumination,

in cosine channel

under

modulated

and actinic

of cosine signal under modulated

(with actinic light) to the amplitude

light, at high frequency. 02-evolution

in

that the energy

is given by:

Rs is the amplitude recorded

the

in cosine channel.

(m signal

two

(sine and

the phase angle of the lock-in am-

in the quadrature

actinic light off, 02-evolution component

to thermal

them in different

By doing so, only thermal

to thermal

evolution

without

out by recording

so as to cancel

addition

of

(13)

This is easily achieved

actinic light is on.

thermal

actinic light illumination,

storage for photosynthesis

Since 02-evolution

where

signal could be recorded.

at high frequencies,

100%

signals can be singled

Ao2 =

rate, only photothermal

)

where Th is thermal

plifier

rate of oxygen at these high frequen-

the signal change with APT, the percent of energy storage is obtained:

APT - Th (

absorbed.

upon

PA

to measure

(APT) measured

which the leaf can release

Consequently,

is only photothermal

be interesting

with heat diffusion component

as heat.

This is achieved by using high-frequency

beam (f Z=400 Hz). Because of the low diffusion This thermal

But the energy which

actinic light saturation

component.

the energy storage component.

due to

white light (which saturates

in the active leaf is released

in green leaves upon

energy

and the other one is photobaric

of strong non-modulated

02-evolution

have been stored

signal

energy storage (18, 50-52). Under

PA signal of leaf sample is made of two components:

Upon addition

photosynthesis), should

and photochemical

Oz-evolution was found

beam, k is the ratio of PA

of thermal

signal without

signal could be normalized to be 1.7 for Impatiens

0.6 for bean and 0.2 for sugar maple leaves.

Tc’, the illumina-

against

petersiana,

actinic

APT. The

0.7 for corn,

81

Since

ApT depends

which depends

on thickness

on the modulation

frequency

dependence

limit-value

for AQ

of leaf layer probed frequency,

by plotting the Ao, by extrapolating

by the modulated

it seems important

against square root of frequency

to frequency

zero.

light

to get rid of the

This method

to obtain

seems to be

more reliable but time-consuming. It might be worth noting that considerable lution in chloroplasts probably

because

of inefficient

(50) or dissolution been encountered tion technique

and photosynthetic diffusion

of 02 evolved with 02-evolution

(mirage

effect).

effort has been made to detect 02-evo-

bacteria

by PAS but without

of 02 in these photosynthetic

in the aqueous detection

Therefore,

medium.

success

Similar

problem

in leaves with photothermal

the hypothesis

(50)

preparations

of dissolution

has

deflec-

of evolved

02 in aqueous media seems to be more likely. CONCLUSION This brief

review

photosynthesis suitable

the potential

are not applicable. in photosynthesis

and light-scattering

The most interesting research

such as energy

tion, besides molecular

of photoacoustic

It clearly appears that photoacoustic

for in situ and in viva non-destructive

larly useful for opaque

meters

demonstrates

research.

probably

conversion structure

spectroscopic

application

spectroscopy studies.

It is particu-

media for which conventional

applications

of photoacoustic

lie in the determination and storage,

and interaction

methods

methodology

of physiological

and photosynthetic

in

is quite

oxygen

paraevolu-

studies.

ACKNOWLEDGMENT The

authors

Charlebois

would

like to express

and M. Charland

their gratitude

to K. Veeranjaneyulu,

D.

for having allowed us to use some of their unpublished

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