Photosynthesis Research 66: 13–31, 2000.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Emerging techniques

New applications of photoacoustics to the study of photosynthesis

Stephen K. Herbert∗, Tao Han & Thomas C. VogelmannDepartment of Botany, University of Wyoming, Laramie, WY 82071, USA; ∗Author for correspondence(e-mail:sherbert@uwyo.edu; fax: +1-307-766-2851)

Received 19 January 2000; accepted in revised form 25 July 2000

Key words: photosynthesis, photoacoustic, optoacoustic, spectroscopy

Abstract

Photoacoustic methods offer unique capabilities for photosynthesis research. Phenomena that are readily observedby photoacoustics include the storage of energy by electron transport, oxygen evolution by leaf tissue at micro-second time resolution, and the conformational changes of photosystems caused by charge separation. Despitethese capabilities, photoacoustic methods have not been widely exploited in photosynthesis research. One factorthat has contributed to their slow adoption is uncertainty in the interpretation of photoacoustic signals. Careful ex-perimentation is resolving this uncertainty, however, and technical refinements of photoacoustic methods continueto be made. This review provides an overview of the application of photoacoustics to the study of photosynthesiswith an emphasis on the resolution of uncertainties in the interpretation of photoacoustic signals. Recent develop-ments in photoacoustic technology are also presented, including a microphotoacoustic spectrometer, gas permeablephotoacoustic cells, the use of photoacoustics to monitor phytoplankton populations, and the use of photoacousticsto study protein dynamics.

Abbreviations: DBMIB – 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone; DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethylurea; ES – energy storage; LED – light emitting diode; PA – photoacoustic, PS I – Photosystem I; PSII – Photosystem II; PL – photochemical loss

Introduction

Photoacoustic spectroscopy first emerged as a tech-nique for photosynthesis research more than 20 yearsago (Callis et al. 1972; Cahen et al. 1978; Malkinand Cahen 1979). It continues to emerge today as un-certainties about the nature of photoacoustic signalsare resolved and methodological improvements ac-crue. Photoacoustic methods offer unique capabilitiesfor photosynthesis research. Oxygen evolution by leaftissue can be measured photoacoustically with a timeresolution that cannot be matched by other methods(Canaani et al. 1988; Mauzerall 1990; Kolbowski et al.1990; Malkin 1998). The storage of energy by electrontransport may be measured by photoacoustics, allow-ing study of cyclic electron transport pathways that areinvisible to gas exchange and fluorescence techniques

(Lasser-Ross et al. 1980; Herbert et al. 1990; Chaand Mauzerall 1992; Havaux 1998; Charlebois andMauzerall 1999; Van Thor et al. 2000). Photoacousticscan also detect light-induced conformational changesin isolated photosystems and other photochemicallyactive proteins (Callis et al. 1972; Arata and Par-son 1981; Delosme et al. 1994; Edens et al. 2000;McClean et al. 1998). Recent innovations and incre-mental improvements in photoacoustic methods haveincreased their usefulness in photosynthesis research.In addition to these technical improvements, uncer-tainties in the interpretation of photoacoustic signalsare being resolved. The intent of this review is to eval-uate current interpretations of the most useful kindsof photoacoustic signals and to present some recentdevelopments in photoacoustics technology as appliedto photosynthesis research.

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The PA effect

Photoacoustic methods have been the subject of anumber of reviews (Malkin and Cahen 1979; Cahenet al. 1980; Malkin et al. 1981; Buschmann et al.1984; Braslavsky 1986; Tam 1986; Peters and Snyder1988; Buschmann 1989; Buschmann and Prehn 1990;Braslavsky and Heibel 1992; Fork and Herbert 1993;Malkin and Canaani 1994; McClean et al. 1998). Asa consequence of these, the fundamentals of pho-toacoustics will be presented very briefly here. Thephotoacoustic (PA) effect is the generation of anacoustic wave when light is absorbed by a sample.In photosynthetic samples, light absorption generatesacoustic waves primarily in two ways: By the con-version of light to heat and by the photochemicalrelease or uptake of gas (Bults et al. 1981, 1982).Conversion of light to heat generates a photothermalsignal, which is an acoustic wave caused by thermalexpansion of the sample and its adjacent medium.Photothermal signals are detected in gaseous mediawith a microphone (e.g. Bults et al. 1982; Canaaniet al. 1988) or in liquid media using a piezoelectricdetector (e.g. Jabben and Schaffner 1985; Delosme etal. 1994). Photochemical release or consumption ofgas generates a photobaric signal, which occurs onlyin gaseous media and is detected with a microphone.In the case of samples that produce both photothermaland photobaric signals, leaf tissue for example, thetwo may be separated kinetically, since the diffu-sion of heat through biological samples is many timesfaster than the diffusion of gas (Poulet et al. 1983;Mauzerall 1990). In liquids, the photothermal signalmay also be combined with a PA effect that resultsfrom light-induced conformational changes of photo-chemically active proteins (Callis et al. 1972; Arataand Parson 1981; Peters and Snyder 1988). For re-action centers and photosystems, this conformationalPA signal results from a protein contraction occur-ring with charge separation. The contraction generatesa negative pressure, opposing the positive pressureof the photothermal signal. In aqueous media, thephotothermal signal is strongly influenced by temper-ature while the conformational signal is insensitiveto temperature in the biological range (Malkin et al.1994). This difference allows the photothermal andconformational signals to be separated by comparingmeasurements made at different temperatures (Calliset al. 1972; Arata and Parson 1981; Delosme et al.1994; Edens et al. 2000).

Photoacoustic measurement systems

A wide variety of photoacoustic systems have beenused for photosynthesis research. Studies that presentdesigns for PA systems include Cahen et al. (1980)Bults et al. (1982a), Jabben and Schaffner (1985),Canaani et al. (1988), Peters and Snyder (1988),Fork and Herbert (1991), Kolbowski et al. (1990),Delosme et al. (1994), Pereira et al. (1994),Puchenkov et al. (1995), Tabrizi et al. (1998), and Hanand Vogelmann (1999). Schematic diagrams that il-lustrate the basic elements of different types of pho-toacoustic systems are available in other reviews(Buschmann and Prehn 1990; Malkin and Canaani1994). In general, photoacoustic systems are of twotypes: Continuously modulated and pulsed. Continu-ously modulated PA systems use a measuring light thatis continuously modulated at frequencies ranging from1 Hz to 1 kHz by means of a mechanical chopper orflashing LEDs. The upper frequency limit is set by thedecrease of the PA signal with increasing frequencyas the energy of the progressively shorter measuringflashes and the interval during which heat emissionis detected both decline. The lower frequency limitis established by the microphone detectors used incontinuously modulated PA systems, which do notwork well below 1 Hz. Continuously modulated PAsystems enclose the sample in a small volume of gaswith the microphone detector. They have a practicaltime resolution of a few seconds, and are best suitedto measurements of slow induction phenomena andphotosynthesis in the steady state (e.g. Katoh andYamagishi 1984; Buschmann 1987; Snel et al. 1990).

Pulsed PA systems use short pulses of light fromlasers or LEDs to produce the PA effect. For meas-urements in gaseous media, pulsed PA systems use amicrophone detector and the sample is enclosed in aphotoacoustic cell similar to that used for continuouslymodulated measurements. For measurements in liquidmedia, a piezoelectric detector is appressed to thephotoacoustic cell, which is sometimes no more thana spectrophotometer cuvette filled with a suspensionof the sample. Pulsed PA measurements can achievemicrosecond to nansecond time resolution and allowmeasurement of fast induction phenomena in isolatedreaction centers, photosystems, thylakoid membranes,intact cells and leaf tissue. Fast phenomena that can bemeasured by pulsed photoacoustics include S states ofthe oxygen evolving complex in leaf tissue (Canaaniet al. 1988; Mauzerall 1990), and the earliest stepsof photosynthetic electron transport in photosystems

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(Callis et al. 1972; Arata and Parson 1981; Delosme etal. 1994; Delosme 1998; Edens et al. 2000). Pulsed PAsystems can also measure steady state photosynthesisin intact cells and leaf tissue if the measuring pulsesare given in combination with continuous backgroundlight (e.g. Kolbowski et al. 1990).

Terminology

A somewhat confusing variety of symbols and termshave been used to describe the features of photoacous-tic signals. A similar situation existed not long ago forfluorescence yield methodology, prompting a formaldiscussion and acceptance of standard terms for fluor-escence yield parameters (Van Kooten and Snel 1990).As photoacoustic methods become more widely usedby non-specialists, a similar consensus on photoacous-tic terms and symbols will need to be established.Table 1 provides a list of terms and abbreviations usedin this review, as well as synonyms that may be foundin the photoacoustics literature. References in whichthe terms have been used are included in Table 1.In some cases, the term or its description has beenmodified from its original usage.

Interpretation of photoacoustic signals

The application of photoacoustics to photosynthesisresearch has grown steadily but slowly. Several hypo-theses may be advanced to explain why photoacousticmethods have not been more widely adopted. Oneof these is that photoacoustic signals are complexand their interpretation can appear uncertain. Thisapparent uncertainty may discourage the adoptionof photoacoustic methodologies when more famil-iar techniques will suffice (Kramer and Crofts 1996;Havaux and Malkin 1998). A similar situation exis-ted for fluorescence yield kinetics 20 years ago. Itwas not until a large body of empirical work definedsimple rules for the interpretation of fluorescence yieldsignals that fluorescence methods were embraced bynon-specialists (Schreiber et al. 1986; Krause andWeiss 1991; Schreiber et al. 1995). Similar painstak-ing work on photoacoustic methods is still in progress(Havaux and Malkin 1998; Malkin 1998; Reising etal. 1998; Delosme 1998; Edens et al. 2000). The fol-lowing sections of this review discuss interpretation ofthe photothermal, photobaric and conformational PAsignals in the light of recent refinements.

Figure 1. Representative drawing of a photothermal PA signal froma continuously modulated photoacoustic system showing photo-chemical loss. Similar signals can be obtained from leaves at 300 to400 Hz or from algal samples at 30 to 50 Hz modulation frequencies(Herbert et al. 1990). At these frequencies, the photobaric signal isabsent from these samples. Upward and downward dashed arrowsindicate the onset and end, respectively, of a weak modulated meas-uring light. Upward and downward open arrows indicate the onsetand end, respectively, of a strong non-modulated background light.Terms are defined in Table 1.

Interpretation of photothermal signals

The photothermal PA signal is used to measure ab-sorption of light by a sample and storage of energyby its photochemical reactions. Light absorption canbe measured accurately in optically dense or highlyscattering samples when it is not possible to calculateabsorption from light transmission (Rosencwaig 1973;Cahen et al. 1980). Storage of energy can be meas-ured in a variety of photosynthetic samples includ-ing isolated reaction centers, photosystems, thylakoidmembranes, intact cells and leaf tissue. Storage ofenergy is determined by comparing the magnitude ofthe photothermal signal from a photochemically act-ive sample to an inactive reference (Figure 1). Thedifference between the sample and reference values isdivided by the reference value, yielding the approx-imate fraction of absorbed light energy stored by thephotochemistry of the sample. For most photosyn-

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Table 1. Terms and symbols used in this review and in the photoacoustics literature

Term Description Reference

AOX The photoacoustic oxygen signal. Malkin and Canaani (1994)

APT The photoacoustic thermal signal. Malkin and Canaani (1994)

ES Energy storage. Equal to (PAM − PAS) / PAM after Malkin et al. (1990)

correction for fluorescence and prompt heat loss. Malkin and Canaani (1994)

Equal to the fraction of absorbed energy that is

stored on a quantum basis.

ESM The maximum value of ES exhibited by a sample at Malkin et al. (1981)

very low light intensity. Proportional to the Carpentier et al. (1988)

maximum efficiency of the energy storing Collier et al. (1994)

processes.

ESSAT The maximum rate of the energy storage exhibited Collier et al. (1994)

by a sample in saturating light. Also described as

ES capacity.

ESV The rate of energy storage. Equal to PAM − PAS after Collier et al. (1994)

correction for fluorescence and prompt heat loss.

LIOAS Laser Induced Optoacoustic Spectroscopy. Refers Braslavsky and Heibel (1992)

to pulsed PA measurements in a liquid medium

using laser pulses.

Optoacoustic A synomym for photoacoustic measurement of Braslavsky and Heibel (1992)

calorimetry energy storage. Nitsch et al. (1989)

Optoacoustic A synonym for photoacoustic spectroscopy. Braslavsky and Heibel (1992)

spectroscopy

PAM The photothermal PA signal in the presence of Collier et al. (1994)

saturating background light. Used as an internal,

photochemically-inactive reference for the

calculation of ES. Proportional to absorption of

measuring light by the sample after correction for

fluorescence.

PAS The photothermal PA signal in the absence of Collier et al. (1994)

saturating background light.

PL Photochemical loss. Equal to (PAM − PAS) / PAM Malkin and Canaani (1994)

Sometimes used as a synonym for ES. Malkin et al. (1981)

Ø′r A synonym for PL. Carpentier et al. (1985,

1989)

QC A synonym for PAS. Carpentier et al. (1985,

1989)

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Table 1. Continued

Term Description Reference

QM A synonym for PAM. Carpentier et al. (1985,

1989)

Thermally In continuously modulated PA measurements, the Cahen et al. (1980)

active layer depth in a sample from which the thermal signal can Tam (1986)

be detected. Decreases as modulation frequency Buschmann and Prehn (1990)

increases.

TE Thermal efficiency. A synonym for ES. Cha and Mauzerall (1992)

Berges et al. (1996)

Charlebois and Mauzerall (1999)

thetic samples, an accurate internal reference can becreated by simply using a strong background light tosaturate photochemistry and provide the photochem-ically ‘inactive’ reference value from the same samplein which energy storage is being measured (Callis etal. 1972; Lasser-Ross et al. 1980; Malkin et al. 1981,Figure 1).

Corrections for fluorescence and photon energy of themeasuring light

The difference of the photothermal signals from a pho-tochemically active sample and its inactive referencedivided by the reference value has been termed the‘photochemical loss’ (PL, Figure 1, Table 1). In manycases, it is useful to convert PL to energy storage(ES, Table 1), which represents the storage of lightenergy on a quantum basis. For calculation of ES,PL must be corrected for losses of excitation fromthe pigment antennae as prompt heat and chlorophyllfluorescence. Correction for prompt heat loss is partic-ularly important when the measuring light is of shortwavelength. When photons are absorbed that havemore energy than is required for charge separation, theexcess energy is lost as prompt heat from the antennapigments and contributes to the photothermal signal.Thus, PL values for blue measuring light are signific-antly lower than for red measuring light even whenan equal amount of electron transport is occurring. Tocorrect for this effect, PL values can be multiplied bythe difference in photon energy of the measuring lightrelative to the energy required for charge separation.Neglecting fluorescence, in the case of energy storageby PS II and PS I combined, ES = PL × (690 nm/λ)where λ is the average measuring wavelength in nm

and 690 nm is the wavelength intermediate betweenthe absorption peaks of P680 and P700 (Herbert et al.1990). This calculation yields the fraction of absorbedquantum energy retained in photochemical products.PL may also be multiplied by the factor 1240eV/λ toyield the energy stored per absorbed quantum in unitsof electron volts (Delosme et al. 1994; Delosme 1998).

Highly accurate calculation of ES requires correc-tion for chlorophyll fluorescence in addition to correc-tion for prompt heat loss. The yield of chlorophyllfluorescence increases when saturating backgroundlight is added to the measuring light. This effect candecrease the apparent ES by as much as 13% below itstrue value, depending upon the sample and measuringconditions (Malkin and Canaani 1994). Fluorescencequenching phenomena also alter fluorescence yieldand, therefore, influence its effect on energy storage.For these reasons, accurate correction flor fluores-cence requires that fluorescence yield be monitoredsimultaneously with the photothermal signal. Detaileddiscussions of fluorescence correction are presentedby Cha and Mauzerall (1992) and also by Malkin andCanaani (1994). In many cases, the errors caused byfluorescence are relatively small and have often beenignored.

The nature of energy storage

Photoacoustic measurements of energy storage areuniquely valuable because they provide an empiricaldetermination of the conservation of light energy byphotosynthetic electron transport. The challenge of en-ergy storage data is that the quantitative relationshipbetween the photothermal signal and energy storingevents is difficult to establish. For this reason, energy

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Figure 2. Representative drawing of energy storage and energy stor-age rate by intact photosynthetic cells (see Herbert et al. 1990;Collier et al. 1994; Herbert et al. 1995). Energy storage is pro-portional to the quantum yield of photochemistry and declinesasymptotically as light intensity increases. Energy storage rate isproportional to the rate at which stable products of photochemistryaccumulate and therefore increases asymptotically with light intens-ity. Light intensity refers to the intensity of the modulated measuringlight for continuously modulated PA systems or the intensity of acontinuous background light for pulsed PA systems.

storage is best used as a relative indicator of elec-tron transport and care must be taken to define theelectron transport steps observed. The following para-graphs discuss approaches for defining the electrontransport steps observed by energy storage measure-ments. A survey of measurements in different types ofphotosynthetic samples follows this discussion.

Maximum rate and maximum efficiency of energystorage

In many cases, it is useful to determine both the max-imum efficiency of energy storage and the maximumrate of energy storage in a sample. The maximum ef-ficiency of energy storage is the ES value at very lowlight intensity (ESM, Table 1). ES values decrease withincreasing light intensity as the energy storing reac-tions become light saturated and progessively moreabsorbed light is dissipated as heat (Figure 2). ESMcan be determined from a series of ES measurementsat different light intensities using one of two analyses.Malkin et al. (1981) and Carpentier et al. (1985, 1988,1989, 1990) plotted 1/ES versus light intensity anddetermined ESM from the y-intercept of the resultingline, which is the extrapolated ES at infinitely low lightintensity (Figure 3A). Alternatively, Cha and Mauzer-all (1992) and Charlebois and Mauzerall (1999) fittedES versus light intensity values to a Poisson distri-bution and extrapolated to the ES at infinitely low

Figure 3. Representative drawings of linearized plots of en-ergy storage and energy storage rate versus light intensity.Semi-reciprocal plots of 1/ES versus light intensity (A) yield a linethat can be extrapolated to the y-axis to obtain ESM (Carpentier et al.1985, 1989, 1990). Double reciprocal plots of 1/ESV versus 1/lightintensity (B) yield a line that can be extrapolated to the y-axis toobtain ESSAT (Collier et al. 1994; Herbert et al. 1995). Terms aredefined in Table 1.

light intensity. In several of these studies, the energystoring steps of electron transport observed by thephotoacoustic measurements were inferred from ESMvalues.

The maximum rate of energy storage by a samplecan also be determined from a series of ES measure-ments at different light intensities. The difference inphotothermal signals between an active sample andits inactive reference is equal to the energy storagerate, after correction for prompt heat loss and fluores-cence (ESV, Figure 1, Table 1). Graphing ESV versuslight intensity yields the familiar asymptotic responseof photosynthesis to light intensity, with light-limitedand light-saturated regions (Collier et al. 1994; Fig-ure 2). The maximum, light-saturated rate of energystorage (ESSAT, see Table 1) may be determined fromthe y-intercept of a double reciprocal plot: 1/ ESV

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versus 1/light intensity (Figure 3B). The equivalent ofESSAT can also be derived from the slope of a semi-reciprocal plot: 1/ES versus light intensity (Carpentieret al. 1985, 1988, 1989, 1990; Charlebois and Mauzer-all 1999, Figure 3A). The value of the ESSAT is evidentin samples treated with photosynthetic inhibitors. Forexample, the ESM in algae treated with DCMU was80–100% of the ESM in untreated controls, reflectingsimilar photochemical efficiencies of PS I-driven cyc-lic electron transport and the DCMU-sensitive linearpathway (Herbert et al. 1990). In contrast, the ESSATof the DCMU-treated samples was only 3–15% of theuntreated controls, reflecting the much smaller capa-city of the cyclic electron transport pathway relative tothe linear pathway.

Time scale of measurements

Photoacoustics observes energy storage within a cer-tain time scale. This is an important parameter indefining the electron transport steps under observation.For continuously modulated PA systems, time resolu-tion has two aspects. The first is the time resolutionwith which changes in the photothermal signal may befollowed, e.g. the change induced by addition of satur-ating background light (Figure 1). This time resolutionis determined by the time constant used in the lock-in amplifier and is typically 1–3 seconds because of alow signal–noise ratio. A second aspect of time resolu-tion in continuously modulated PA systems is the timescale within which energy from each cycle of measur-ing light is retained in photochemical products. This istheoretically limited to 1/2π f, where f is the frequencyof light modulation in Hz (Cahen et al. 1978; Malkinand Cahen 1979; Cahen et al. 1980; Carpentier et al.1985; Buschmann and Prehn 1990). As an example,measuring light modulated at 35 Hz observes energystored in products of electron transport that persist for4.5 ms after excitation, such as reduced plastoquinone(Carpentier et al. 1985). The continuously modulatedPA signal declines with frequency, making it diffi-cult to use frequencies above 1 kHz. For this reason,continuously modulated PA measurements cannot ob-serve energy storage in products of photochemistrythat persist for less than 200 µs after excitation.

Pulsed PA systems have much better time resol-ution than continuously modulated systems. In gasphase with a microphone detector, the response of themicrophone sets a maximum time resolution of about30 µs (Kolbowski et al. 1990; Mauzerall 1990). Forpulsed PA measurements in liquid phase, use of piezo-

electric detectors and very short laser pulses allowtime resolution into the nanosecond range (Peters andSnyder 1988; Nitsch et al. 1988; Bruce and Salehian1992; Delsome et al. 1994; McClean et al. 1998).

Depth of measurement

For the case of continuously modulated photoacous-tics applied to non-uniform samples, such as leaftissue, it is important to define what regions of thesample gives rise to the photothermal signal. Thevolume of a sample that contributes to the photo-thermal signal is determined by the depth to whichthe measuring light penetrates and the depth of thethermally active layer, which is the depth in the samplefrom which modulated heat emission can be detec-ted (Cahen et al. 1980; Carpentier et al. 1983; Tam1986; Buschmann and Prehn 1990; Table 1). Lowmodulation frequency allows heat from relatively deepin the sample to diffuse to its surface and contributeto the PA effect. High frequencies measure shallowerparts of the sample because heat from deep parts ofthe sample does not diffuse to the surface within thecycle of the measuring light. For example, a modula-tion frequency of 32 Hz observes a thermally activelayer of 237 µm while a frequency of 1024 Hz ob-serves a thermally active layer of 42 µm, assumingthat the sample has the same thermal properties aswater (Buschmann and Prehn 1990). Utilizing thiseffect, depth profiles of light absorption and energystorage have been acquired from leaf tissue by PAmeasurements at different frequencies. A clear dis-tinction was observed between the absorption spectraof the mesophyll cells and epidermal cells of leaf tis-sue but greater resolution was not possible (Malkinet al. 1981; Buschmann and Prehn 1983; Buschmannand Prehn 1990; Han and Rongfu 1991). Given thatleaves are comprised of 20–50% air space, they donot meet the ideal criteria of a homogeneous mediumfor either light or heat transmission. Further work isrequired to clarify the relationship between choppingfrequency and depth profiles in leaves. Recently, thedevelopment of a microphotoacoustic spectrometerhas allowed much better resolution of the light absorp-tion and photosynthetic activity of different cell layersin leaf cross-sections (Han and Vogelmann 1999).

Energy storage measurement in different types ofphotosynthetic samples

The use of simplified samples, narrow-wavelengthmeasuring light and electron transport inhibitors help

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to define the electron transport steps that contribute toenergy storage. The simplest samples are purified re-action centers and photosystems. A number of studieshave examined reaction centers from purple bacteriausing pulsed photoacoustics. In these samples, the en-ergy storing reactions are known to be the early stepsof electron transport occurring within the reaction cen-ter. Depite this knowledge, accurate quantification ofthe energy stored by these electron transport stepshas proven challenging and different laboratories haveobtained different results. (Note that the ESM val-ues below were recalculated for comparison from thecited references assuming a quantum yield of 1 forelectron transport.) In chromatophores of Chromatiumvinosum, Callis et al. (1972) calculated an ESM closeto zero for QA reduction and concluded that the freeenergy change of this reaction resulted almost entirelyfrom a decrease in entropy. Similarly, Arata and Par-son (1981) calculated an ESM of less than 10% forreduction of QA in reaction centers of Rhodopseudo-monas sphaeroides. In contrast, Malkin et al. (1994)and Puchenkov et al. (1995) calculated ESM valuesof 58% and 39%, respectively, for QA reduction byreaction centers of Rhodobacter sphaereoides. Nitschet al. (1989) calculated an ESM of 43% for QA reduc-tion by intact cells of Rhodospirillum rubrum. Thislast study differed from the others in that no correc-tion was made for possible conformational PA signals.Most recently, Edens et al. (2000) arrived at an ESMof 68% for QA reduction by Rhodobacter sphaeroidesand discussed sources of error in their measurementsand those of others. Possible sources of error includedthe use of improper external reference solutions, fail-ure to account for changes in the compressibility ofwater with temperature, use of short wavelength pulsesfor which much of the photothermal signal is promptheat loss, and multiple excitation of reaction centersby excessively strong measuring pulses.

Energy storage by preparations of purified PS IIand PS I has been measured with both pulsed andcontinuously modulated photoacoustics. Nitsch et al.(1988) observed an ESM of 83% for PS I and 65% forPS II in preparations from a thermophilic cyanobac-terium using pulsed photoacoustics. As noted by De-losme et al. (1994), these values were not correctedfor the conformational PA signals known to occur inisolated photosystems and may have been erroneouslyhigh. Taking care to correct for the conformationalPA signal, Delosme et al. (1994) calculated ESM val-ues of 50–55% for cyanobacterial PS I and 60% forPS II from spinach. These values were close to those

expected for photoreduction of FAFB in the case ofPS I and photoreduction of QA in the case of PSII. Energy storage by photosystems can be observedwith continuously modulated photoacoustics if careis taken to avoid degradation of the sample by themodulated light. Carpentier et al. (1985, 1989) meas-ured PS II from spinach at 35 Hz and found ESMvalues of 22–42%. The higher values were ascribedto better preparations that had been measured verybriefly to avoid degradation of the sample by thecontinuously modulated measuring light. Both the ad-dition of PS II inhibitors and PS II electron acceptorsdecreased the ESSAT of the PS II preparations butneither inhibitors nor acceptors altered the ESM, aresult that will be discussed below. Energy storageby spinach PS I preparations was also observed bycontinuously modulated photoacoustics (Owens et al.1990). The ESM of this preparation was 58% and,like the spinach PS II preparations, was insensitive toelectron transport inhibitors and acceptors. Completechemical oxidation of P700 and complete chemical re-duction of FAFB abolished energy storage in the PSI preparations, however. The continuously modulatedmeasurements described above had a time resolutionof 4.5 ms, while the pulsed PA measurements wereresolved to about 1.5 µs. Also, the PS II prepara-tions of Carpentier et al. (1985, 1989) included nativeplastoquinone while those, of Delosme et al. (1994)did not. For these reasons, the PS II energy storageobserved with continuous modulation likely resultedfrom plastoquinone photoreduction while the pulsedmethod observed the earlier and larger energy storageof QA photoreduction.

A few studies have used photoacoustics to measureenergy storage by isolated thylakoids and chloroplasts.Delosme et al. (1994) applied pulsed photoacous-tics to spinach chloroplasts and observed an ESM of47%, less than was observed by the same methodsin purified PS I or PS II (see above). Using con-tinuously modulated photoacoustics, Carpentier et al.(1990) made measurements of spinach thylakoids at35 Hz. The ESM observed in this case was 16%,also much less than in spinach PS II or PS I pre-parations measured similarly (Carpentier et al. 1985,1989; Owens et al. 1990; see above). The low ESMexhibited by thylakoids and chloroplasts relative totheir isolated photosystems may have resulted fromgreater heterogeneity of reaction centers and anten-nae in the more complex thylakoid or chloroplastsamples. The ESSAT of thylakoids was tenfold greaterthan that of isolated PS I or PS II, however, pre-

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sumably because pools of native electron acceptorswere present (Carpentier et al. 1990). As had beenobserved in PS II and PS I preparations, the ESM ofthe spinach thylakoids was insensitive to addition ofelectron transport inhibitors and electron transport ac-ceptors. In contrast, the ESSAT of both photosystemsand thylakoids was reduced by such additions, in mostcases (Carpentier et al. 1989, 1990). These results canbe explained as follows. The rates of energy-storingelectron transport observed as ESM are very low. Theycan proceed unaffected by electron transport inhibitorsif those inhibitors act subsequent to the energy-storingsteps (Charlebois and Mauzerall 1999) and these low,light-limited rates are not improved by added electronacceptors. The much higher rates of energy-storingelectron transport observed as ESSAT are significantlydecreased by electron transport inhibitors and also byacceptors because the acceptors decrease the lifetimeof the reduced intermediates in which energy is stored(Carpentier et al. 1990).

Most studies of energy storage have been donewith intact cells and leaf tissues. The energy stor-ing processes in such complex samples can be partlydefined by using inhibitors of electron transport or byusing monochromatic measuring light. Energy storagehas been used extensively to observe PS I-driven cyc-lic electron transport in intact cells. The PS I cyclecan be isolated from PS II-driven processes by us-ing far-red measuring light and confirming the resultswith DCMU and DBMIB (Lasser-Ross et al. 1980;Yamagishi and Katoh 1984; Carpentier et al. 1984;Canaani et al. 1989; Canaani 1990; Herbert et al.1990; Cha and Mauzerall 1992; Havaux 1992; Collieret al. 1994; Herbert et al. 1995). In general, DCMUhas little effect on energy storage in far-red light whileDMBIB exerts a strong inhibition. These results areconsistent with energy storage of far-red light oc-curring by the PS I-driven cyclic electron transportpaths. Ravenel et al. (1994), Charlebois and Mauzerall(1999) and Van Thor et al. (2000) used a variety ofinhibitors to further define PS I-driven cyclic electrontransport into multiple pathways observed by energystorage. Based upon the ESM and the time scale of theobservation, Charlebois and Mauzerall (1999) furtherproposed that much of the energy storage by the PSI cycle resulted from ferredoxin reduction. In addi-tion to PS I cycles, cyclic electron transport in PS IIhas been observed by photoacoustics as energy storageby PS II without oxygen evolution (Cha and Mauzer-all 1992; Allakhverdiev et al. 1997; Havaux 1998;Boucher and Carpentier 1999). State transitions have

also been studied in intact cells by means of energystorage (Malkin et al. 1990; Fork et al. 1991; Mullin-eaux et al. 1991; Bruce and Salehian 1992; Delosme etal. 1996) The latter two studies are of particular note inthat nanosecond-resolved, pulsed photoacoustics wasused to prove that phycobilisomes do not become ener-getically disconnected from reaction centers in State IIas it occurs in cyanobacteria. The photobaric PA signalhas also been applied to the study of state transitions(Canaani 1986; Malkin and Canaani 1994).

Use of the photothermal signal to observephotoprotection

The photothermal PA signal has often been used tostudy stress effects in leaves or intact algae (Havauxet al. 1986; Havaux et al. 1987; Jansen et al. 1989;Havaux and Eyeletters 1991; Herbert et al. 1992; Col-lier et al. 1994; Berges et al. 1996; Ouzounidou et al.1997). Most notably, several studies have used pho-toacoustic thermal signals to examine the increase ofprompt heat loss that is associated with zeaxanthinaccumulation and other photoprotective mechanismsinduced by strong light (Havaux 1989; Havaux 1990;Dau and Hansen 1990; Havaux et al. 1991b; Havauxand Tardy 1997; Veeranjaneyulu and LeBlanc 1998;Yahyaoui et al. 1998; Buschmann 1999). These stud-ies focused on increases in the photothermal referencesignal (PAM) during prolonged exposure to stronglight and interpreted them as increased rates of pho-toprotective heat dissipation. In some cases, as notedpreviously by Malkin and Canaani (1994), this inter-pretation has been flawed. In the presence of strongbackground light, the photothermal reference signalalready represents a conversion of excitation to heatthat approaches 100%, which is the basis for usingit as a reference signal for calculating energy stor-age and also for using it to suppress the photobariccomponent of the PA signal (Malkin and Cahen 1979;Lasser-Ross et al. 1980; Bults et al. 1982; Malkinand Canaani 1994). If the background light is trulysaturating for photochemistry, a decrease of the max-imum fluorescence yield from 0.10 to 0.02 caused byphotoprotective mechanisms would increase PAM byno more than 8% (Dau and Hansen 1990; Malkinand Canaani 1994). Increases of PAM greater than 8%likely result from heating of the sample or other ef-fects of strong light exposure that alter its absorptionor thermal diffusion properties (Malkin and Canaani1994). Notably, Havaux (1989) found that exposureto photoinhibitory light caused a 50–100% increase in

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PAM but this increase did not occur if samples werecooled below 12 ◦C during treatment.

In several studies, photoprotection has been moreclearly observed as an increase of PAS and a decreasein energy storage following exposure to strong light(Buschmann 1987; Buschmann and Kocsanyi 1989;Snel et al. 1990). In a recent example, Delosme (1998)noted low energy storage in the 450–550 nm regionof photoacoustic action spectra in a variety of photo-synthetic samples. These results suggested that betacarotene routinely contributed less than 20% of its ex-citation to charge separation, reflecting a constitutivephotoprotective function.

Interpretation of photobaric signals

Photoacoustic measurements of oxygen evolution canbe time-resolved to less than 100 microseconds and,under most circumstances, they are not significantlyaffected by respiratory or photorespiratory consump-tion of oxygen. Absorption of light by the sample isalso accurately measured at the same time, allowingsimple calculation of the relative quantum yield foroxygen evolution. For measurements of leaves, thesefeatures are advantages over the most common po-larographic methods for measuring oxygen evolution.The disadvantage of photoacoustic oxygen measure-ments is that they are restricted to leaf tissue, whichoffers a very short diffusion path for oxygen from PSII complexes to the gas spaces of the tissue. Algaeand thylakoid preparations collected on filter paperyield only very weak photobaric signals (Carpentieret al. 1984; Canaani 1986; Herbert et al. 1990). Insuch samples, the path of oxygen diffusion to the sur-face of the sample is long enough to damp out thephotobaric signal, though excellent photothermal sig-nals can be obtained. Infiltration of leaves with wateralso abolishes the photobaric signal by lengthening thediffusion path of oxygen to the gas phase and thusdamping out the signal (Bults et al. 1982; Malkin etal. 1992). For algal cells or chloroplasts in liquid me-dium, it should be noted that the Joliot polarographicsystem can also measure oxygen evolution with a timeresolution of a few milliseconds and can discriminatebetween oxygen evolution and most oxygen uptakeprocesses (Joliot and Joliot 1968).

Separation of photothermal and photobaric signals

When present, photobaric PA signals are always com-bined with photothermal signals, requiring a method

Figure 4. Representative drawings of a low frequency, continuouslymodulated PA measurement of leaf tissue (see Bults et al. 1981;Kanstad et al. 1983; Poulet et al. 1983; Malkin et al. 1992). Meas-urements that are in-phase for the photothermal signal consist ofa PA signal that includes both photothermal and photobaric com-ponents (A). Measurements that are at quadrature phase for thephotothermal signal (B) consist only of the photobaric signal thatpersists after the photothermal signal has declined to zero in itscycle of modulation. The drawing represents a measurement at a lowintensity of modulated light. The photothermal signal would declinerelative to the photobaric signal if the intensity of modulated lightwere increased.

for their separation. In the case of continuously mod-ulated PA measurements, the strongest photobaricsignals are obtained at modulation frequencies from3 to 100 Hz (Bults et al. 1982; Frandas et al. 1997). In

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Figure 5. A pulsed photoacoustic measurement from leaf tissue us-ing an LED flash and microphone detector. The solid line representthe photothermal reference signal taken with saturating backgoundlight applied. The dashed line represents the signal in the absenceof saturating background light, consisting of both photothermal andphotobaric components. Deconvolution allows the combined signalsto be separated. A gas uptake component is absent from this sig-nal, as would occur if the measurement were made at normal CO2concentrations. Redrawn from Kolbowski et al. (1990).

this frequency range, the photothermal signal can besubtracted from the total PA signal by the expedientof measuring at the quadrature phase angle, which isthe phase angle 90◦ out of phase from the in-phaseangle of the pure photothermal signal (Poulet et al.1983; Figure 4). Addition of strong background lightsaturates oxygen evolution and abolishes the photo-baric signal but the pure photothermal signal remains,allowing its in-phase angle to be established. Adjust-ing the phase angle of measurement to 90◦ relativeto the in-phase angle of the pure photothermal signalabolishes the photothermal signal and only a part ofthe slower photobaric signal remains, allowing it tobe measured in isolation (Figure 4). An alternative tomeasurement at quadrature phase is to measure energystorage by the sample at a frequency high enough todamp out the photobaric signal (300–400 Hz). The PLvalue obtained at high frequency can then be used tocorrect the photobaric signal at low frequency. A thirdway to isolate the photobaric signal is to use pulsedphotoacoustics. In pulsed PA signals, the slower pho-tobaric component of the acoustic wave is obvious andcan be isolated from the photothermal components bydeconvolution (Figure 5). Deconvolution yields separ-ate curves for thermal expansion, gas evolution, andalso gas uptake (Kolbowski et al. 1990; Tabrizi et al.1998).

Figure 6. A pulsed photoacoustic measurement from bacterial reac-tion centers in aqueous medium using a laser flash and piezoelectricdetector. The upward arrow shows the laser flash. The solid lineis the photothermal PA signal from a photochemically inactivereference. The dashed line is the combined photothermal and con-formational PA signal from a reaction center sample. The negativeconformational signal from the reaction centers is larger than thepositive photothermal signal, making the combined signal negative,as would occur at 25 ◦C. At 4 ◦C, the photothermal would be absentfrom both reference and reaction center samples. Thus, the referencewould show very little signal at 4 ◦C, while the reaction centerswould show a more negative conformational signal. The oscillationof the signal represents the characteristic resonance of piezoelectricdetectors. The PA signal is quantified as either the height of the firstpeak or the amplitude of the first oscillation. Redrawn after Edenset al. (2000) and Malkin et al. (1994).

Gas uptake

Photosynthetic oxygen evolution by PS II is ordin-arily the major contributor to the photobaric signalfrom leaves. Under some conditions, however, gasuptake phenomena clearly affect the photobaric sig-nal. Gas uptake effects are a serious concern, sincethey compromise the accuracy of oxygen evolutionmeasurements. Uncertainty about gas uptake effectshas slowed wide acceptance of photoacoustic methodsfor observing oxygen evolution in leaves (Kramer andCrofts 1996). The gas uptake issue has been recentlyresolved, however, and is now limited to special cases.

Gas uptake was first noted by Malkin (1987) ina study of photosynthetic induction phenomena usingcontinuously modulated photoacoustics. Upon expos-ure to light, dark-adapted leaves showed an initially

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strong photobaric signal followed by a depression ofthe signal before it recovered to normal levels. Thisdepression of the photobaric signal during inductionwas ascribed to a period of oxygen uptake by photore-duction at PS I, which was consistent with earlierstudies of photosynthetic induction (Radmer and Kok1976). If modulated far-red light was used in theseexperiments, the photobaric signal actually becamenegative, seeming to confirm the hypothesis that oxy-gen uptake at PS I could produce a photobaric signal(Havaux 1988). With the application of pulsed pho-toacoustics to leaves, a negative excursion of the PAsignal was obvious at low flash energies (Canaani et al.1988; Mauzerall 1990). This negative excursion wasascribed to oxygen uptake at PS I for three reasons: Itwas of the appropriate magnitude, it could be excitedwith 695 nm light, and gas uptake by the oxygena-tion or carboxylation reactions of RUBISCO was tooslow to contribute to the PA signal in the time scale ofobservation (Mauzerall 1990).

Kolbowski et al. (1990) first proposed that rapidCO2 uptake might produce a gas uptake signal, notby RUBISCO activity but by rapid solubilization ofCO2 in the chloroplast stroma as it was alkalized byphotosynthetic proton movement. Since proton move-ment results from linear and cyclic electron transportalike, CO2 solubilization could account for observa-tions of gas uptake in far-red light, as well as in visiblewavelengths. Using pulse-modulated photoacoustics,Reising and Schreiber (1992) developed the CO2 sol-ubilization hypothesis further, showing that gas uptakewas greatly enhanced if the CO2 concentration wasraised above 1%. The stimulation of gas uptake byhigh CO2 was consistent with observations that heattreatment and dark adaptation enhanced gas uptake(Havaux 1987; Malkin 1987), since both heat anddarkness would cause CO2 from leaf respiration to ac-cumulate in a sealed PA cell. Furthermore, gas uptakewas observed to persist under low oxygen but to beinhibited by ethoxyzolamide, an inhibitor of carbonicanhydrase (Reising and Schreiber 1992, 1994). Allthese observations supported the hypothesis that CO2solubilization contributed to the gas uptake componentof the photobaric PA signal.

Recently, gas uptake was studied in tobacco anti-sense mutants having a greatly reduced carbonic an-hydrase activity (Reising et al. 1998; Havaux andMalkin 1998). These mutants exhibited little or nogas uptake, strongly supporting the CO2 solubiliza-tion hypothesis. These studies also confirmed that,in wild type tobacco, CO2 solubilization was negli-

gible in air, occurring only when CO2 levels wereraised above 1%. Thus, photoacoustic measurementsof oxygen evolution are not affected by CO2 solubil-ization if measurements are made under normal levelsof CO2. This conclusion removes a significant con-cern regarding the use of photoacoustics to observephotosynthetic oxygen evolution by leaves under mostexperimental conditions.

Two additional gas uptake issues have been raised.The first is whether oxygen uptake by photoreduc-tion at PS I detracts from the photobaric signal inleaves. The second is whether non-modulated oxygenuptake by respiratory electron transport or RUBISCO-catalyzed oxygenation can detract from pulses of oxy-gen evolution as they diffuse through leaf cells to theexternal gas phase (Charland et al. 1992). Both theseprocesses potentially detract from the photobaric sig-nal but, if they occur at all, they are very small relativeto oxygen evolution and can be ignored (Havaux andMalkin 1998; Malkin 1998).

Interpretation of conformational signals

Changes in molecular volume caused by photochem-ical reactions in proteins can be detected by pulsedphotoacoustics in liquid media (Figure 6). Callis et al.(1972) were the first to observe these conformationalPA signals. They noted a contraction of Chromatiumvinosum chromatophores upon excitation. The neg-ative pressure of this contraction could be separatedfrom the positive pressure of the photothermal signalby measuring in aqueous solution at its temperature ofmaximum density, which is roughly 4 ◦C dependingon the presence of dissolved salts. At this temper-ature, thermal expansion of the solution is zero andthe photothermal signal is abolished, leaving a pureconformational PA signal. The conformational PA sig-nal from reaction centers is temperature insensitiveover the range of 0–23◦C so measurements at 4 ◦Creflect events at more normal biological temperatures(Malkin et al. 1994). Reaction centers of purple bac-teria exhibit a contraction of 28–35 cubic angstromsper molecule upon charge separation. (Arata and Par-son 1981; Puchenkov et al. 1995; Edens et al. 2000).Several manipulations of reaction centers have beenobserved to affect this contraction. Quinone-depletedreaction centers exhibited no contraction while mutantreaction centers in which tyrosine 210 is replaced withtryptophan exhibited twice the contraction of wild typereaction centers (Malkin et al. 1994). Substitution of

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Figure 7. Spatial resolution of a microphotoacoustic spectrometer.The 40 µm wide measuring beam was scanned across a test gridof black lines on a photographic negative. The black lines were43.7 µm wide separated by transparent spaces of the same width.Redrawn from Han and Vogelmann (1999).

the native ubiquinone with other quinones decreasedor increased the contraction, depending on the quinoneused (Mauzerall et al. 1995; Edens et al. 2000). Inaddition to bacterial reaction centers, conformationalPA signals have been observed in isolated photosys-tems and isolated chloroplasts (Delosme et al. 1994).In intact cyanobacteria, however, the conformationalPA signal was absent (Bruce and Salehian 1992).

The conformational PA signal may interfere some-what with detection of the photothermal signal but itis potentially very useful. The light-induced contrac-tion of reaction centers or photosystems can be usedas a quantitative indicator of charge separation (De-losme et al. 1994). Also, the conformational PA signalcan be used to understand the reaction dynamics of avariety of photochemically active proteins (McCleanet al. 1998). Of particular interest are the early studiesof Ort and Parson (1978, 1979a, b) on the conform-ational changes that are caused by the light-drivenproton pumping of bacteriorhodopsin.

Recent innovations in photoacoustics

Several studies of photosynthesis using novel or in-crementally improved photoacoustic approaches havebeen published recently. They are summarized in thefollowing paragraphs.

Figure 8. Diffusion of CO2 into a gas permeable photoacoustic cell.Photoacoustic signals were generated from absorption of a modu-lated infrared beam by CO2 in the photoacoustic cell. The spaceoutside the gas-permeable PA cell was flushed with nitrogen andthen with either 500 ppm CO2 (open symbols) or 10 000 ppm CO2(closed symbols). In both cases, equilibrium was achieved withinless than 10 min. Redrawn from Han and Vogelmann (1999).

Microphotoacoustics

Han and Vogelmann (1999) have developed a modu-lated photoacoustic system that uses a focused laserline as the measuring beam. The narrowness of thebeam allows photoacoustic measurements of bothphotothermal and photobaric signals from leaves andleaf cross-sections with a spatial resolution of 40 µm(Figure 7). By measuring different cell layers in leafcross sections, microphotoacoustics adds a third di-mension to photosynthesis measurements in leaves.Such observations are significant because when a leafis irradiated normally (i.e. from one direction) thespectrum and intensity of light changes sharply fromone surface of the leaf to another. Photosynthetic cellsin different vertical regions of a leaf would, therefore,be expected to exhibit modifications of their photo-synthetic systems that suit them to their particularlight environment. Microphotoacoustics contributessignificantly to observing such modifications and tounderstanding the contributions of different cell lay-ers to photosynthesis and productivity by the leaf as awhole.

The first use of the microphotoacoustic systemwas to determine profiles of chlorophyll concentra-tion and oxygen evolution capacity in cross sectionsof spinach leaves (Han and Vogelmann 1999; Hanet al. 1999). The data revealed that light-saturatedoxygen evolution, measured by the photobaric signal,was relatively uniform from the upper to the lower

26

surface of the leaf. This relatively flat profile of oxy-gen evolution capacity differed unexpectedly from theprofile for chlorophyll concentration, which showed abell-shaped curve peaking at 300 µm from the uppersurface of the 700 µm-wide cross sections. The profileof CO2 assimilation capacity, measured as light satur-ated 14C fixation in paradermal sections of the leaves,was also bell-shaped, roughly in parallel with the dis-tribution of chlorophyll (Nishio et al. 1993). The flatprofile for light-saturated oxygen evolution relative toCO2 assimilation suggested that the uppermost andlowermost regions of leaf cross sections direct morephotosynthetic electron transport to processes otherthan carbon fixation than do the middle regions. Mi-crophotoacoustics has also been applied to a studyof photoinhibition by combined stresses in leaves ofBrassica napus (Bornmann et al. 2000). Photoinhib-ition was shown to be surprisingly uniform throughthe leaf section, decreasing only at the deepest, mostshaded cell layers in the spongy mesophyll.

Gas permeable photoacoustic cells

Photoacoustic measurements in the gas phase are typ-ically made in gas-tight PA cells having a volumeof less than 1 ml. In such cells, an intact photosyn-thetic sample rapidly exhausts the available CO2 andall measurements must be assumed to be at the CO2compensation point. To avoid this limitation, Fork andHerbert (1991) constructed a gas-permeable PA cellin which one wall of the cell was made of sinteredglass covered by a thin Teflon membrane. Flushingthe outside of the gas-permeable wall with 3% CO2in air caused rates of both energy storage and oxy-gen evolution to improve significantly, especially inleaf samples from C4 plants. Two drawbacks of thisapproach were that the gas permeable wall of the PAcell decreased the signal–noise ratio by half and theactual concentration of CO2 inside the PA cell duringthe measurement was uncertain. Han and Vogelmann(1999) remedied the latter problem by measuring ratesof CO2 diffusion into a gas-permeable PA cell atdifferent concentrations of external CO2 (Figure 7).Concentrations of CO2 inside the cell were determ-ined with a modulated measuring light of 4.7 µmwavelength, which is absorbed strongly by CO2 andgenerates a clear photothermal signal. Use of a 4.7 µmmeasuring beam required the addition of sapphire win-dows to the PA cell and CO2 measurements couldnot be made with a photosynthetic sample in the cellbecause water vapor from the sample absorbed the

4.7 µm measuring beam and contributed to the pho-tothermal signal. Despite this limitation, if the rate ofCO2 consumption by the sample is known or can beestimated, it is possible to calculate the CO2 equilib-rium inside the PA cell from the known rate of CO2diffusion into it. The CO2 equilibrium in the cell canbe controlled by altering the external CO2 concentra-tion. Manipulation of oxygen and other gases in thePA cell can also be accomplished in this fashion.

Other methods have been used to control the gasenvironment inside a gas phase PA cell. Small gasvalves added to the cell allow it to be flushed with thedesired gas composition prior to measurements (Tab-rizi et al. 1998). Using such valves allows the PA cellto be sealed during measurements, increasing the sig-nal to noise ratio relative to gas permeable cells. Thedisadvantage of this approach is that frequent interrup-tion of the measurement for gas flushing is needed tomaintain a consistent internal gas environment. A thirdalternative for controlling gas composition is to use an‘open’ PA cell in which the sample itself forms onewall of the cell, leaving its other surfaces exposed tothe outer gas environment (Kanstad and Nordal 1978;Da Silva et al. 1987; Pereira 1994; de Paula 1997).Open PA cells exhibit very poor signal–noise ratios,though they do allow measurements of intact leavesattached to plants. Another alternative that applies toleaves is to have part of the leaf sample remain outsidethe PA cell so that gas diffusion can occur throughthe leaf mesophyll to the enclosed portion of the leaf(Havaux and Malkin 1998).

Phytoplankton sample analysis

Photoacoustic spectroscopy was applied to freeze-dried samples of phytoplankton nearly three decadesago by Ortner and Rosencwaig (1977). The rationalefor the study was that photoacoustic spectroscopycould readily measure absorption in highly scatter-ing or opaque materials, allowing freeze-dried phyto-plankton samples from the field to be rapidly char-acterized by their photosynthetic pigments. This ap-proach has recently been revived with improvements(Dubinsky et al. 1998; Mauzerall et al. 1998). Themore recent studies used pulsed PA measurementsof living phytoplankton suspended in water. Absorp-tion of light by chlorophyll was measured in algalsuspensions that varied by more than two orders ofmagnitude in chlorophyll a concentration. Tests werealso performed to show that the relative proportionsof chlorophytes, chromophytes and cyanobacteria in

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the sample could be determined from the ratio ofabsorbances at 540, 650 and 677 nm. In the samemeasurement, photosynthetic energy storage by thesample was determined, allowing its productivity andgeneral health to be assessed. The influence of nutrientstarvation on energy storage by PS I and PS II hasalso been examined in microalgae by photoacoustics(Collier et al. 1994; Berges et al. 1996). A significantconclusion of these studies was that PS II abundanceand activity declined dramatically with nutrient star-vation while PS I was maintained. The prospect forthis application of photoacoustics is that natural watersamples can be rapidly assessed for their phytoplank-ton biomass, primary productivity, nutrient status andthe relative proportions of major photosynthetic taxa.All of these paramters are useful indicators of thequality of natural waters.

Protein dynamics

Reaction centers and other photochemically activeproteins exhibit light-induced changes of volume thatgive rise to a conformational PA signal (Peters andSnyder 1988; McClean et al. 1998). The photo-thermal and conformational PA signals together canyield information about the dynamics and energet-ics of photochemically active proteins. This capacityof photoacoustics to rapidly assess multiple aspectsof protein function may have applications in ‘post-genomics’ biology. As the genomic sequences ofmodel organisms are completed, accurate predictionof the structure and function of their proteins fromgene sequence data will become a priority. This willrequire detailed understanding of the structure andfunction of a relatively small number of model pro-teins. For proteins that are photochemically active, orthat can be made so by addition of a chromophore,the relatively simple tests of protein dynamics thatare possible with photoacoustics can contribute to thiseffort.

The future of photoacoustics in photosynthesisresearch

With the resolution of gas uptake issues, photoacousticmethods are the best available for measuring photo-synthetic oxygen evolution in leaves without interfer-ence from oxygen-consuming processes. A remainingtask is to calibrate the photobaric signal such thatPA measurements of oxygen evolution can be made

quantitatively rather than on a relative basis. Addi-tion of a polargraphic electrode to PA cells makes thissimple (Havaux 1998). Combined with fluorescenceand absorption measurements, PA measurements ofoxygen evolution can contribute significantly to un-derstanding the many integrated processes occurringin intact photosynthetic cells and tissues. Develop-ment of a field portable PA system (Tao Han, workin progress) will render photoacoustic measurementof oxygen evolution suitable for ecological studiesof photosynthesis, much as pulse-modulated fluores-cence techniques are now widely used.

Photoacoustic measurement of energy storage byphotosynthetic samples remains somewhat problem-atic because of the many factors that can affect thephotothermal signal in complex biological samples.For studies of processes that are difficult to observeotherwise, such as PS I- or PS II-driven cyclic electrontransport, photoacoustic measurements of energy stor-age are a useful adjunct to fluorescence and absorptiondifference measurements.

Technical and methodological developments, suchthe microphotoacoustic spectrometer or the use ofphotoacoustics to study protein dynamics, are ongo-ing. Over time, these will further expand and so-lidify the contribution of photoacoustic methods tophotosynthesis research.

Acknowledgements

This work was supported by an NSF Major ResearchInstrumentation grant, DBI-9724499, to T.C Vogel-mann, J.N. Nishio, and S.K. Herbert. The authorsthank professors Shmuel Malkin and David Mauzer-all for helpful discussion. The thorough and help-ful comments of two anonymous reviewers are alsoacknowledged.

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