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Time-resolved photoacoustic spectroscopy: New developments of an old idea
3
I. Photochem. Photobiol. B: Biol., 24 (1994) 3-15
New Trends in Photobiology
(Invited Review)
Time-resolved old idea
spectroscopy:
P.R.
Crippa,
photoacoustic
A. Vecli
of an
and C. Viappiani
Department of Physics, University of Puma, (Received
new developments
August 23, 1993; accepted
43100 Panna (Italy)
October
19, 1993)
Abstract Acoustic waves generated by heat emission in radiationless transitions from photoexcited molecules can be detected by suitable transducers. Their study allows the investigation of thermal relaxations, thus providing thermodynamic and kinetic data on short-lived species produced by the absorption of pulses of light. In this field of research the best technique has proved to be the so-called pulsed-laser, time-resolved photoacoustic spectroscopy, which is based on piezoelectric detection of pressure waves in the time domain. Deconvolution processing of the transient signals gives both the lifetimes of excited states and the energy content of the transitions, provided that decay times are in the range 5 ns-5 ps. Moreover, when compared with proper theoretical models emphasizing the energy balance, the photoacoustic results can help to build a complete picture of the deactivation pathways, including photochemical events. The biophysical applications, although numerous and widespread both in basic and applied research, offer the real possibility of giving information on photobiological processes in conditions very close to the living state. Among the more significant contributions obtained in this area, the results on photosynthesis and photosensitivity of plants and photosynthetic micro-organisms, structural and functional dynamics of respiratory proteins, photocycles of rhodopsin and bacteriorhodopsin and photophysical properties of several natural pigments are particularly relevant, together with some medical and biotechnological applications. Another promising field of application of photoacoustics concerns photoactive drugs and the photophysics of fluorescent probes for conformational studies of proteins, nucleic acids and membranes. In general terms, timeresolved photoacoustic spectroscopy promises to become one of the most powerful techniques in photobiophysics, provided that some limitations in data analysis and time resolution are removed by technical improvements.
Key words: Photoacoustics;
Optical spectroscopy;
Photophysics;
1. Introduction
The excitation of biomolecules by light - from UV to IR - triggers a complex sequence of phenomena whose investigation constitutes one of the main methods of studying their structure and interactions with the local environment. These events have been successfully explored by many experimental techniques, which as a whole define optical spectroscopy in its broadest sense, and we can actually affirm that the most relevant advances in the knowledge of biological molecular constit-
loll-1344/94/$07.00 0 1994 Elsevier SSDI loll-1344(93)06959-7
Molecular
biophysics
uents were achieved on the basis of data obtained by optical spectroscopy. At present, molecular photobiology can be considered an autonomous branch of biology, on account of the extent and the basic importance of light-induced processes in plants, micro-organisms and animals. From this point of view the study of photophysics and photochemistry of biomolecules involves many different approaches, each one potentially requiring an appropriate experimental technique. A comprehensive picture of such processes should in fact include determination of electronic energy levels (singlets and triplets), vi-
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4
P.R. Crippa et al. / Time-resolved photoacoustic spectroscopy
brational states, lifetimes, kinetic constants and efficiency of the different transitions. Moreover, also owing to the interactions with other chemical species (i.e. solvents, oxygen, etc.), the formation of photochemical transients and/or stable intermediates with a different energy content must be considered with particular attention. Two main areas of photobiology as studied by spectroscopic techniques can be identified. In the first one, attention is devoted to the effects induced by the primary event of photon absorption by biomolecules, supramolecular aggregates or isolated organelles that in the living organisms play the role of interfaces between the light world and the cell machinery. Chloroplasts and phytochrome in plants, and rhodopsin and melanins in animals are clear examples of such transductional intermediates. It is evident that biophysical description in quantitative terms of the sequence of molecular events following the absorption of light gives the reference points for the interpretation of the physiology of photosensitive organisms. The second area can be defined as optical spectroscopy of the biomolecules per se, and we can say that this approach has conclusively contributed to clarifying biomolecular structures (mainly by UV-visible absorption spectroscopy, dichroic spectroscopy, etc.) and dynamics (by fluorescence spectroscopy). The use of extrinsic chromophores has greatly enhanced the potential of this second aspect, thus enlarging the field of application of optical techniques beyond their natural limits. However, we must bear in mind that a relevant percentage of energy absorbed as light by matter is dissipated as heat by various molecular mechanisms and is consequently lost to optical studies. The recently developed methods of investigating this “dark side” of the problem, namely the photothermal methods, have greatly widened the application area of spectroscopy, allowing the direct quantitative detection of non-radiative transitions. In this sense we can refer to photothermal or photoacoustic methods as complementary to fluorescence and phosphorescence spectroscopy. As a matter of fact, the photoacoustic phenomenon was known over a century ago, and its fundamental principles are already well explained in the original papers by A.G. Bell, J. Tyndall and W.C. Roentgen giving notice of the discovery of the effect. Though for a long time the photoacoustic effect remained really little more than a scientific curiosity, from the turn of the century many significant contributions to the interpretation of the effect in gaseous and condensed matter have been published (for a historical review and
a summary of the oldest theories, see ref. 1) and we can see in this thorough analysis the basis of the more recent developments of photoacoustics as an analytical, structural and calorimetric technique for the investigation of matter. Essentially, photoacoustics reveals pressure waves generated by heat emission in radiationless transitions from excited states, and the theoretical treatment of the production of these acoustic waves by light pulses and their travelling in matter can be connected to the theory of Landau and Lifschitz [2] on pressure-wave propagation. The theory of photoacoustics was further developed in the 1970s [1,3-61, while from the early 1960s a variety of experimental apparatus and methods for experimental measurements has been proposed and some of them have emerged. The most widespread techniques at present are, in general, developments of the original one of Pate1 and Tam [7]. In particular, we can mention photorefractive techniques, such as thermal lensing and probe-beam deflection, and techniques based on both indirect (gas-microphone photoacoustics) and direct (piezoelectric photoacoustics) detection of the acoustic waves. In the application of these methods to the common case of solutions of biomolecules of low concentration, it should be emphasized that the solvent determines the thermal properties of the sample, while the solute affects its optical properties. The aim of the present review is to discuss briefly the principles and the methods of the socalled pulse-laser, time-resolved photoacoustic spectroscopy, with examples selected from the most significant of the biophysical literature and from researches in progress in our laboratory. We will present a short account for non-specialists of the photoacoustic phenomenon, the description of a typical experimental set-up and the problem of data management and analysis. For deeper details of both theoretical aspects and chemical applications we refer to some recent general reviews that present also other forms of photothermal spectroscopy and their applications, particularly in the field of organic photochemistry [f&10]. 2. Photoacoustic
signal generation
and analysis
2.1. Heat generation in absorbing media Light absorbed by molecules is converted to heat, luminescence or chemical energy, obeying the general condition of energy conservation. In Fig. 1 a scheme of the various processes that can act as heat sources is shown.
P.R. Crippa et al. / Time-resolved
~
Incident light +
Transmitted
light
Optical absorption Reflected light Prompt
Luminescence
keat
I-
V
Photochemical Energy
(1
\ , c-___--I
’ I I 1 I ’ I 1 1
Possible sources of delayed heat
Fig. 1. Block diagram showing the main processes bearing on the generation of heat following the absorption of light by a photoactive sample. The scheme demonstrates the various possible energetic pathways that can act as sources of delayed heat distinct from the mechanisms producing prompt heat, i.e. internal conversions and intersystem crossing.
In pulsed photoacoustics, the signal is analysed in the time domain and the signal-to-noise ratio is improved by averaging. With laser pulses of the order of nanoseconds, the length of propagation of the pressure wave is much less than the size of the sample cell: as a consequence, its shape is in general independent of reflections by the inner walls of the cell, and the sample dimensions can be assumed as infinite. As photothermal techniques are essentially calorimetric, they allow the monitoring of the input-output balance of the energy and, from conservation principles, the absorbed light energy is equivalent to the sum of the different output energies: N,hv,
= @,N,hv,+
aNAhve + c&, AE,
(1)
where NA and h are the Avogadro and Planck constants, v, and vf the frequencies of the incident and emitted light and af the quantum yield of fluorescence. The second term of the right-hand side represents the energy re-emitted as heat, and (Y the fraction of absorbed energy converted to heat within the instrumental integration time. The last term refers to the amount of energy stored in metastable species, AE, representing their molar energetic content, which can be evaluated by difference. The time resolution of the method depends on the possibility of separating the heat emitted in fast processes (Le. internal conversion, for example) from the heat produced by processes whose du-
photoacoustic
5
spectroscopy
ration is comparable with the integration time of the instrument, which in turn depends on the circuitry, the light pulse width, the experimental geometry and the time constants of the photophysical events. The geometry of the experiment, as in fluorescence, is generally 90”, i.e. the piezoelectric transducer detects pressure waves generated perpendicularly to the excitation-beam direction. This disposition is strictly valid only for solutions with low optical density; for high absorbances, the pressure waves are spherical, as generated by a quasipoint source situated near the inner surface of the cuvette, and a different geometry can be more suitable. In the case of scattering solutions, the experimental disposition is very critical and must be adapted to any particular situation. With low absorbance (A = ECZ-=x1) - the most common case - the pressure waves are cylindrical (Fig. 2). Several authors have treated formally the problem of the generation of acoustic waves in different approximations: see, for example, refs. 11-13. An elementary treatment of the time evolution of the photoacoustic signal can be developed introducing the transit time of the pressure front as Ta=
20
-
VS
where r. is the radius of the laser beam and vu, the sound velocity in the solution. The absorbed energy is E,=E(l-10-A)
(3)
and for a light pulse of duration T] and an excited state with non-radiative lifetime rnr greater than r,, an effective integration time can be defined, which determines amplitude, shape and time of arrival of the transient pressure to the transducer: ref= (r: + 7; + &)l’2 Experimentally, the instrumental lastic properties
(4)
the dependence of the signal on characteristics and the thermoeof the solvent involves a protransducer
laserp”,se
~ ~
~~~~~~~~~
Fig. 2. Scheme of the generation of the pressure pulse following the absorption of a pulse of light for the case of a solution with low absorbance (cylindrical geometry). For definitions of the parameters, see text.
6
P.R. Cnppa et al. / Time-resolved photoacoustic spectroscon
portionality constant that can be eliminated by the use of a reference substance that decays with 7 much less than T, and (Yequal to 1. In this case (Ycan be obtained easily from the ratio between the sample and the reference amplitudes, normalized for the absorbed energy E,. In fact, from the above discussion, a simplified form of the signal intensity S can be written as
I+ I
*i’con temprraturc
independent
AV=K(AV,+AVCon)
solution
S=kE(l-
lo-A)a
(5)
and the fraction Q for the sample can be evaluated by studying the dependence of the signal intensity upon A and/or E for both sample and reference. The knowledge of the value of (Yallows the evaluation of the energy stored in the metastable state(s), provided that the fluorescence quantum yield @r is known. In fact, the energy balance equation (1) can be written:
where Ef represents the average molar energy of the fluorescence emission and E, the molar energy of the laser pulses. Knowing one of the two terms aP or AZ?,, it is possible to determine the other. Moreover, from the dependence of CYon the ratio C&r, it is in principle easy to calculate T,,, varying the diameter r,, of the laser beam with proper pinholes. As the decays of the excited states are generally first-order, we can obtain: hE,=AE~exp
( 7,1 -
7 nr
(7)
where AE: represents the energy content of the transient species. Extrapolating the semi-logarithmic plot of 1 -(Y vus. T, at the limit of T, equal to 0, both rnr and aj,AE, can be evaluated.
T=20”C
T=4’C (@O)
Fig. with The ume
where AV, represents the volume change due to thermal relaxations, Q the amount of heat released in fast processes ( = &(l - lo-“)) and p the density of the solvent. p and cP, respectively the expansion volume coefficient and the specific heat at constant pressure, are markedly different in water than in most organic solvents and, as a consequence, the signal in water is smaller than in organics. However, the thermoelastic parameters of water, such as p, are strongly temperature dependent. So, in the case of photoinduced conformational change AV,, the total signal becomes S;k(bV.+AV.)=@
SaAV=pVAT= t t
@ CPP
(9)
+AVc)
If the reference has V, equal to 0 and Q/E equal to 1 (corresponding to (Y=l), its signal amplitude is: R=k
2.2. Molecular volume changes Another source of acoustic signal, sometimes superimposed on the thermal expansion discussed above, is the volume or conformational change of the photoexcited molecules, whose contribution to the total signal can be separated by a simple experiment involving variation of the temperature [14]. Figure 3 shows a sketch of the principles of the method. We can take as general expression for the photoacoustic signal amplitude S, due to fast thermal processes
3. Generation of the pressure signal in aqueous solution, its two components: the volume term and the thermal term. latter is temperature-dependent through the expansion volcoefficient /3 that becomes 0 at about 4 “C.
AV,=kE
p
(10)
CPP
and
S -=RE
Q
I
AK
cpp
(11)
E/3
Assuming cz (and Q) as independent of T, a linear plot can be obtained for ES/R vs. c,p/p, and the dependence of p on T can be used to calculate Q and AV, from the intercept and slope, respectively. 2.3. Time dependence deconvolution
of photoacoustic
signals:
A detailed analysis of the time dependence of photoacoustic signals can be based on the general
P.R. Crippa et al. I Time-resolved photoacoustic spectroscopy
achievements of the linear response theory: when a signal, in our case the heat release, stimulates a system, its time response is the convolution of the signal with the transfer function of the instrument. In photoacoustics the true acoustic pulse is convolved with the response function of the system 115,161, comprising both the ultrasonic microphone characteristics and the laser pulse shape. Experiments usually involve the measurement of the photoacoustic waveform R(t) for a reference compound, and of the waveform of the sample s(t) under identical conditions of absorbance, solvent and temperature. Generally speaking, R(t) will be a convolution of the laser pulse shape L(t) with the transducer response function: I R(t) =
L(t’)T(t-t’)
dt’
(12)
s 0
Since the reference compound undergoes fast nonradiative de-excitation, it has been assumed in the previous expression that the heat release follows the laser pulse exactly, and in this sense acts as a Dirac delta function (see Fig. 4). For a species having an excited state with finite lifetime, we have a time-dependent heat release, indicated by H(t); the corresponding expression of the photoacoustic waveform is therefore given by I’
f S(t)=
qt-t’) f
dt’x
L(t”)H(t’-t”) s
dt”
(13)
0
0
By interchanging we can write:
the order
of the convolutions,
f
S(t) =
s
R(t’)H(t-t’)
dr’
(14)
0
Fig. 4. Scheme of the generation of the photoacoustic signal S(t) as the convolution of the exponential release of heat H(t) with the signal produced by a reference compound R(t) giving only prompt heat with a=1 and T&T=.
7
It is worthwhile to note that the expression derived for s(t) is valid whichever pulse width L(t) is used. In the simple case of different channels of deexcitation starting from the same excited state, it is very easy to derive the expression for H(t); assuming first-order kinetics, the proper function that describes the heat decays is a sum of exponentials [17]:
H(r)=i$l : exp I
( 1 -
f
ri
(15)
The case of sequential decays too is described by eqn. (15). By analysing the time profile of the signal by means of the deconvolution technique it is thus possible to retrieve the parameters of the exponential decay. The real-time deconvolution is obtained by means of an iterative non-linear least-squares technique; the reference and the sample waveforms are input into the fitting program, and a portion of the waveform, which usually includes the first oscillations, is selected for analysis. The sample waveform is compared to the numerical convolution of the reference waveform and the assumed heat transient, which may be composed of one or two decays; the goodness of fit is judged by reduced 2, which is calculated for a determined set of fitting parameters and minimized in order to obtain the most likely values. The fitting procedure yields the pre-exponential factors C_X~ and the lifetimes 7i; a check on the goodness of the fit is provided, as stated, by the value of the reduced 2 and by visual inspection of the residuals, as well as by their autocorrelation. An example of superimposed reference and sample signals is shown in Fig. 5. As there will generally be a certain amount of energy released as heat in internal conversion processes, a fast decay will be present in the signal, whose lifetime is in the range of femtoseconds to picoseconds, well below the experimental integration time of the instrument. For these prompt processes, deconvolution allows the determination of the pre-exponential (Y, but the lifetime cannot be defined precisely; it can only be said that this decay is characterized by a lifetime below the experimental integration time. For longer lifetimes, up to about 5 ps, it is possible to determine both the rate and magnitude of the process. We will not enter into further details of the data analysis; the reader may conveniently find more elaborate descriptions of the deconvolution process in the literature [18].
P.R. Crippa et al. I Time-resolved photoacoustic spectroscopy
channel number
Fig. 5. Example of waveforms as obtained in a typical experiment. Curve 1, reference signal (ferrocene in CH,CN, OD = 0.2 (A,,= 308 nm), averaged over 1000 pulses); curve 2, simulated signal with a=0.6 and r= 100 ns (the dotted curve nearly coincident with curve 2 represents the convolved waveform); curve 3, residuals.
pulsed dye laser beam splitter
transducer
f
+,
digital oscilloscope
Eizl R
c
t
_
S
computer
t Fig. 6. Block diagram of the experimental set-up. As the examples reported in Section 4 refer to results obtained in our laboratory, our apparatus may conveniently describe a typical experimental arrangement. Laser, Lambda Physik EMGSO (excimer XeCl laser that delivers pulses of about 5 ns length) followed by a dye laser, Lambda Physik FL3100; energy meter, Laser Precision RjP-7620 with probe head RjP-735; 1 MHz piezoelectric transducer (PZT), with preamplifier Panametrics V103; digital oscilloscope, LeCroy 9450450))))))))))))))) (350 MHz) with sampling rate of 400 Msamples s-‘, corresponding to 2.5 ns per channel, fully programmable through GPIB (IEEE-488) or RS232C.
The pulsed-laser light source should give pulses of a few nanoseconds (or in special cases, a few picoseconds) duration. The choice of a pulsed laser is in general a compromise between practical and economical considerations and the specific characteristics of the process under investigation. Owing to the specific absorption properties of a sample containing biological molecules, tunability of the light source is very important. The pulse energy must be maintained below the threshold of non-linear effects; the repetition rate should be a few Hertz in order to maintain the thermal equilibrium in the sample. Transduction from mechanical to electrical signal is carried out by piezoelectric devices directly clamped on the measurement cell, with a thin layer of silicon grease interposed in order to ensure good acoustic matching. Lead zirconate-titanate (PZT) or polyvinylidene difluoride (PVF,) are the most common piezoelectric elements, the first being the more sensitive while the second has a higher resonance frequency, allowing better time resolution. The structure of the sample cell is obviously related to the instrumental transfer function, but no general rule can be stated about cell optimization: experience and serendipity are the two pillars of cell design. In many cases a simple fluorescence quartz cuvette gives good results and allows easy temperature control. On the other hand, the range of temperatures accessible is limited by the temperature sensitivity of the transducers. In order to normalize each photoacoustic signal to a fixed reference value of incident light intensity, a fraction of the laser pulse must be deviated by a beam splitter to the probe head of an energy meter whose output is sent to a personal computer managing the experiment and performing the acquisition and the first processing of the output data of the transducers, suitably amplified and digitized by a digital oscilloscope. In this way the averaged waveform (100-1000 acquisitions) is displayed and stored. Successively, the processing of the experimental signal gives the parameters cx and r.
3. Basic instrumentation
4. Biophysical
A typical instrumental set-up for time-resolved photoacoustic spectroscopy is schematized in Fig. 6: in relation to different needs corresponding to minor variations can be different experiments, introduced.
4.1. General overview From their beginnings, photoacoustic techniques proved to be a very useful and sensitive tool for experimental studies in biophysics and related fields: a number of interesting results were in fact
applications
P.R.
Ctippa
et al. / Time-resolved
obtained from the early 1970s on isolated biomolecules, cell suspensions and intact or physically and chemically treated tissues such as fresh leaves, dried algae and phytoplankton. Moreover, identification of metabolic states of bacteria and characterization of normal, pathological and drugtreated animal tissues, both soft and hard, demonstrated the possibility of applying these techniques as diagnostic subsidiary instruments in several branches of medicine (for reviews, see refs. 19-22). All these results were obtained by so-called C.W. photoacoustic spectroscopy, and the main advantage - unanimously pointed out - in comparison with classical optical techniques, during this pioneering stage of the biomedical application of photoacoustics, was the possibility of obtaining photoacoustic absorption spectra as accurate and informative as optical ones, and in addition on samples too opaque or transparent. In the same period an intense effort was made by several groups to adapt photoacoustic detection to special spectroscopic techniques that were successfully applied to biological studies, such as double-beam, IR Fourier transform and dichroic forms of spectroscopy. Detection of trace amounts of metal ions, the estimation of thermal diffusion lengths, the determination of absolute quantum yields of fluorescence and the first significant evaluation of energy storage in photosynthesis are some of the prominent results obtained in biological systems in this way, on both in vitro and in vivo samples. Incidentally, we wish to emphasize the opportunity to consider again this line of instrumental improvement with the more advanced detectors and light sources now available. A substantial improvement in photothermal methods is related to the analysis of the time profile of the heat emission by photoexcited samples. The lifetimes and the energy contents of metastable molecular species can be easily determined, and quantitative information on enthalpic changes and reaction volumes modifications induced by light absorption in the samples can be obtained. The relevance of these achievements for biophysical studies is rather obvious, and extensive reviews of the results obtained on this basis during the 1980s and the early 1990s have been published [10,17,23-261 and can usefully be consulted. It is interesting to underline that photoacoustic spectroscopy in its different instrumental configurations has been applied to biophysical studies in order to obtain structural, analytical, morphological and functional information in any case in which the presence or the interaction or the
photoacoustic
spectroscopy
9
time evolution of intrinsic or extrinsic chromophores accessible to photoexcitation can contribute to the study of biological molecules, organelles, cells and tissues. Some illustrative examples certainly not exhaustive, because of the abundance of the scientific literature in the field - may clarify the significance and the potentialities of the technique. The study of photosynthetic molecular mechanisms and structures is probably the most popular area of the application of photoacoustic spectroscopy in biophysics. Measurements in gas and liquid phases with modulated and pulsed actinic light on intact or treated plant tissues and bacteria made it possible to obtain a very complete description of basic phenomena such as the time course of energy flow and storage, oxygen evolution, electron flow, and interactions between photosysterns I and II, as well as a screening of processes relevant for biotechnological applications, such as inhibiting effects on photosynthetic yield by pollutants, herbicides and fertilizers, heat and chilling stress, water stress and photoinhibition. Also the photochemical and photophysical properties of phytochrome have been accurately determined, giving a relevant impulse to the definition of the plant photosensitivity problem. According to Fork and Herbert [26], photoacoustic methods “may become some of the most useful methods in photosynthesis research”. The respiratory proteins haemoglobin and myoglobin have been extensively studied by photoacaustic spectroscopy as regards their structures in solution and in the crystalline state and the mechanism of their interaction with binding substrates. In general we can say that photoacoustic calorimetry must be considered as a very important tool for the general problem of binding to biomolecules, i.e. for the determination of changes in extent of ligand, enthalpy and volume characterizing specific interactions of chromoproteins, including hydration and more general solvent-interaction phenomena. The use of extrinsic labels can give a tremendous impulse to the study of these processes that trigger many metabolic pathways. Other specific applications deserving a special mention have been the detailed analysis of the photocycles of rhodopsin and bacteriorhodopsin, including for the latter the dynamics of the protonpumping mechanism. Photoacoustic data obtained in all these applications and many others of biological interest, such as organic and organometallic reactions, possibly combined with fluorimetric and flash photolysis data, guided the formulation of models providing
10
P.R. Ctippa et al. / Time-resolved photoacoustic spectroscopy
new global insights into biological problems and stimulating a more realistic view of the underlying molecular mechanisms. Taking advantage of the above-mentioned technical developments, several medical applications of photoacoustic methods have been developed, mainly in dermatological and haematological studies [27,28] and in the assessment of the photodynamic therapy of tumours.
4.2. Photoacoustics of tryptophan [29] The interest in a photoacoustic study of tryptophan arises from its well-recognized relevance as an intrinsic optical probe in proteins. In spite of the large amount of work on the fluorescence and phosphorescence of this amino acid in peptides and proteins, many aspects of its photophysics are still not clarified and, in particular, the anomalous trend of fluorescence emission that strongly depends on the solvent. The most satisfactory model includes the formation of exciplexes between excited indole and one or more solvent molecules [30,31]. The only reported indole derivative that should not form exciplexes is 5-methoxyindole (5 MOI), because of the presence of the methoxy group, which significantly lowers the value of the dipole moment. time-resolved photoacoustic A comparative study of TRP and 5 MO1 was performed in order to assess: 1. the possible formation of intermediate excited states that can be precursors of the triplet state, and 2. the role of molecular oxygen in the thermal relaxation of these excited states and the consequent formation of singlet Oz. The experiments were performed with 5 ns laser pulses at a wavelength of 308 nm in order to avoid photoionization working on the extreme tail of the absorption band, and allowed the conclusion that: 1. the kinetic behaviour of photoexcited tryptophan can be described by introducing an intermediate state with energy below the first excited singlet state of the solvent (3.73 eV in cyclohexane, 3.43 eV in water) and a lifetime much shorter in water (17 ns) than in organic solvents (107 ns in cyclohexane, 68 ns in MeOH). The efficiency of formation is higher in organic solvent. Also 5 MO1 in Hz0 generates such a precursor with high yield (@= 0.74) These data strongly support the exciplex hypothesis even if at present their nature (strong or weak exciplexes) is not yet well defined;
2. the role of oxygen is elucidated by comparison of the results obtained in deoxygenated and oxygenated samples. The shorter lifetime and the enhanced signal amplitude in aerated solutions confirm the presence of a dynamic quenching process with formation of singlet oxygen both in TRP and 5 MO1 solutions. From energy-balance equations the quantum yield for singlet O2 formation can be evaluated and appears to be rather high, particularly in H,O: 0.74 for 5 MO1 and 0.80 for TRP. In conclusion, time-resolved photoacoustics allowed the clarification of the photophysics of this indole-like molecule also in quantitative terms. The application of these results to the study of tryptophan residues in peptides and proteins is at present under investigation. 4.3. Fluorescence quenching of free dyes and protein probes A straightforward application of photoacoustics is the determination of the absolute fluorescence quantum yield [32-351. Recently the possibility of investigating the effect of chemical-physical parameters on the photophysics of a molecule has also been investigated by performing fairly accurate analysis of collisional fluorescence quenching by KI on several organic dyes [36-381. In the simple case of free dyes in ethanol solutions, the good agreement between fluorescence and photoacaustic data shows that the two processes can be regarded as complementary to each other; in fact the quenching constants for the two effects are identical, and the fluorescence quantum yield, as determined by the fitting of the photoacoustics data, is in accordance with the literature data. These results clearly show that a combined fluorescence and photoacoustic approach to the quenching problem gives a good knowledge of the fate of the absorbed energy within the fluorophore. Protein fluorescence quenching of both aminoacid side chains and bound probes has become a widely used technique in protein biochemistry, and has allowed the determination of both structural and dynamic properties of local environments inside the macromolecule [39-41], while determination of quenching parameters allows the estimation of the degree of exposure of the molecule to the solvent [42]. Recently one of us has demonstrated [43] that a combined fluorescence and photoacoustic technique can be usefully employed to study protein fluorescent probes. Three different proteins containing a single free thiol have been selectively labelled with fluorescein-5-isothiocyanate (FITC),
P.R.Crtppa et al. / Time-resolved photoacoustic spectroscopy
and its fluorescence quenched with KI. Unmodified fluorescein is known to undergo intersystem crossing with very low efficiency (0.05) when free in solution. Fluorescein derivatives show a large increase in intersystem crossing efficiency, which assumes values of 0.49 for eosin, 0.71 for dibromofluorescein and even very close to unity for erythrosin. FITC itself is known to undergo intersystem crossing to a triplet state with low efficiency and long lifetime, outside the range detectable, at least at present, by photoacoustics. On the other hand, fluorescence quenching alone is not sensitive to the particular deactivation pathway of the molecule, the Stern-Volmer plot for dynamic quenching being generally linear. Non-linearities usually arise because of emission heterogeneity and/or static quenching. As a consequence, fluorescence spectroscopy is unable to distinguish an enhanced intersystem crossing from any other process depleting the excited singlet state. In contrast, photoacoustics is extremely sensitive to the different pathways that the energy follows in the deactivation process. In Fig. 7 the effect of KI on the photoacoustic signal of FITC, free and protein-bound, is shown, while, in the concentration range used for each sample, the Stern-Volmer plots of fluorescence data were linear. Obviously the quenching constants for the process determined from fluorescence and photoacoustic data, assuming intersystem crossing to the triplet state as negligible, are substantially different, even in the case of free FITC,
0.9
1
0.8
-
0.7
-
0.6
-
0.2
’
I
I
I
,
I
1
@
I
1
I
I
I
0.0
0.2
0.4
0.6
0.8
I
WI1 (Ml Fig. 7. Amplitude of signals after deconvolution for monoexponential decays obtained on fluorescein-S-isothiocyanate, free and bound to proteins quenched by KI: 0, free FITC, 0, BSAFITC, +), carbonic anhydrase-FITC; 0, papain-FITC.
11
TABLE 1. Fluorescence quenching by KI on free FITC and protein-FITC complexes as determined from fluorescence and photoacoustic data. All the measurements were carried out with A,,=490 nm, corresponding to a molar energy content of 58.37 kcal Sample
Free FITC FITC-Carbonic FITC-Papain FITC-BSA
anhydrase
K from fluorescence W-‘)
K from photoacoustics
10.40 4.90 4.24 5.81
10.2 5.8 4.2 5.7
W’)
whose triplet state is known to be very poorly populated. Invoking the presence of significant intersystem crossing gave good results for all the cases considered, indicating a sharp change in the photophysics of the molecule upon binding to the proteins, with a strong increase in the intersystemcrossing quantum yield. The quenching constants reported in Table 1 give information concerning the shielding of the binding site from the solvent in agreement with independent measurements [44]. An indication on local pH at the binding site was obtained from the value of the fluorescence quantum yield. 4.4. Photophysical parameters of drugs for phototherapy A large amount of research is presently being devoted to the study of photosensitized reactions in medicine, particularly in the photodynamic therapy of tumours. In these processes the photosensitizer is excited by absorption of light and undergoes intersystem crossing to a triplet state from which it either reacts with the nearby molecules or produces cytotoxic oxygen derivatives, such as singlet oxygen or superoxide anion. Photothermal sensitization by the heat release of non-fluorescent endogenous or externally-added dyes has been investigated recently [45] with interesting results directly related to the presumed therapeutic mechanism. In order to have good sensitizing efficiency, all these drugs must show very low fluorescent quantum yield and complementary non-radiative decays with very high efficiency; in this sense, photoacoustics is a suitable tool for studying the photophysical characteristics of these drugs and their interaction with biological molecules. 4.5. Interactions between melanins and porphyrin Porphyrins are among the most promising photoactive drugs in the phototherapy of malignant
12
P.R. Crippa et al. / Time-resolved photoacowtic
melanoma, and therefore a detailed knowledge of their transport and uptake in melanoma tissue as well as of their binding to biomolecules and, in particular, to the melanoma melanins is a necessary prerequisite in order to understand their mechanism of action at a subcellular level. The photochemical behaviour of porphyrins after binding to melanins deserves particular interest because, even if melanins are not the target of the therapeutic action, the strength of their interaction with the pigments suggests a potential source of photophysical side-effects overlapping the main photoactivity. Binding of zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) to synthetic melanins results in the formation of a non-fluorescent complex [46] whose non-radiative decay has been investigated by means of photoacoustics [47]. The titration of a fixed quantity of ZnTBzPyP with increasing concentrations of eumelanin resulted in the expected red shift and hypochromism in the absorbance spectrum of the dye, on account of the greater delocalization of the rr electrons in the central ring of the porphyrin. Addition of eumelanin to the dye quenches its fluorescence centred at about 630 nm; the Stern-Volmer plot for the quenching of porphyrin fluorescence is non-linear and exhibits an upward curve (Fig. 8): this behaviour is in good agreement with a model that takes into account static quenching by melanin and is best described by F
T/ being proportional to the volume of the action sphere and M being the melanin concentration. The best fit gave the results reported in Table 2. The photoacoustic data showed a feature of the quenching process that was not evident from the fluorescence data: in order to get a good fit, we had to assume that the quenching process affects, to a certain degree, also the intersystem crossing probability. The fraction of the prompt heat (Yis reported in Fig. 9 along with the best fit (solid line) to the following expression
rem
3.5 A 3 \2!
@T+(l+KcMI) E-l-
@*
OL=v,,1+qMj
-
U+K[MI)
-E
2.5
0 -_ 1.5
0.5
1.0
1
1
I
0
1
2
I
,
I
1
A_ 0
7. 2
-
ti 0
-2
0.7
0.4
L 0
(16)
- =exPwwl) FCI
spectroscopy
Fig. 8. Direct (A) and reciprocal (B) Stern-Volmer plots for the quenching of ZnTBzPyP flucrescence by eumelanin. A,,=475 nm; porphyrin concentration, 3.8 PM. TABLE 2. Quenching of ZnTBzPyP fluorescence with eumelanin: results of fitting experimental data with the expression for a
345 475
V (ml mg-‘)
[ZnTBzPyP] (cLM)
[Melanin] (pg ml-‘)
0.27 0.25
3.8 3.8
O-l.9 O-4.2
(17)
where K is the quenching constant of the process and ET is the energy of the triplet state. This equation contains a term describing the action of the enhanced intersystem crossing: exponential quenching of the fluorescence term was tried but gave indistinguishable results, the reason for this being the small weight of this term in the expression
of CC Fitting yields the following parameters: K= 0.30 ml pg- ‘; ET/E = 0.48; aIsc = 0.42; we assumed for cD~a value of 0.031 [46]. The photoacaustic data show that binding of porphyrin to eumelanin results in an enhanced degree of intersystem crossing of the porphyrin to a long-lived state, undetectable by the instrument, corresponding to a quenching of (Y. We are not however in
13
P.R. Crippa et al. / Time-resolved photoacoustic spectroscopy 0.8
r
a
\
0.7
0.6
< 0
0.5 0
I
I
I
I
2
4
6
8
10
[Melanin](pg/ml) Fig. 9. Fraction of prompt heat emitted function of concentration of melanin.
by ZnTEkzPyP as a
a position to decide if this long-lived state is the result of the formation of a triplet state of the porphyrin or of the melanin. Further investigations are in progress to assess the identity of the longlived species, in order to elucidate the fate of the energy stored in it. Oxygen plays a major role in this system, certainly related to its intrinsic ability to quench triplet states of photodynamic sensitizers by energy transfer processes; in fact deoxygenation dramatically lowered the prompt non-radiative emission of the mixed solutions, whereas for the single chromophores we did not observe any significant change. 4.6. Photoreceptor pigment from Blepharisma japonicum [48] The ciliate protozoan Blepharisma japonicum exhibits a complex and typical photobehaviour triggered by the photoactivation of blepharismin, a hypericin-like pigment present in strings of granules arranged just below the cell surface [49]. Action spectra of the photophobic response of the micro-organism and spectroscopic studies of photodynamic sensitization induced by intense illumination in vivo suggest that all these photoresponses are under the control of blepharismin. In particular, comparative studies of the optical properties of blepharismin and hypericin indicate the possibility of different deactivation pathways in pigment and reference molecule, but do not allow the formulation of a complete model of the molecular mechanisms of the energy transduction towards the photomotile and photochemical responses [50-531.
A photoacoustic analysis of the released prompt heat in isolated pigment in different conditions (oxygenated and deoxygenated solutions, ethanolic and mixed water-ethanol solutions) was carried out according to a model taking into account the intersystem crossing to the triplet state of the pigment and a possible subsequent energy transfer to OZ. The results (Fig. 10) strongly support the assumption of different deactivation mechanisms in pigment and model molecule and demonstrates the presence in blepharismin of a second fraction of released heat, poorly affected by the presence of water and suggesting the possibility of an energy transfer process from the triplet state in competition with the O2 singlet-state activation. Such a deactivation mechanism does not seem to be active in blue blepharismin, a modified form of the pigment induced in the micro-organisms by prolonged illumination in the presence of oxygen, and is probably due to intermolecular interactions in molecular aggregates of pigment whose presence in solution was confirmed by light-scattering measurements.
1.5
1
/
I
I
I
1.0
0.5
0.0
-0.5
-1.0
1
0
300
600
900
1200
1500
0
300
600
900
1200
1500
channel
number
Fig. 10. Normalized photoacoustic signal of red blepharismin in ethanol upon excitation at 308 nm. The upper plot shows the reference (higher curve), the sample and the convolved waveforms along with the residuals; the lower plot shows the autocorrelation function of the residuals.
14
P.R. Crtppa et al. / Time-resolved photoacoustic spectroscopy
Further measurements on pigment granules and artificial models, now in progress in our laboratory, will give us the possibility of formulating a more detailed model of the primary events in the energy transduction chains active in vivo. Such a model should be, in principle, adaptable also to other phototransduction events of biological relevance.
5. Conclusions From this short account of the experimental principles presented above and the examples we have selected from our laboratory experience, some general considerations emerge, allowing the judgement that time-resolved photoacoustic spectroscopy is a technique that has now reached a mature stage, and should be considered at the same level as other optical methods having wider application. In fact it is possible, with the presently available technology, to obtain sensitivity and time-resolution levels that are fully comparable with the performance of other common techniques such as flash-photolysis and fluorescence. Particularly when used in parallel with such forms of spectroscopy, time-resolved photoacoustics shows its special characteristics in the study of the complex phenomenology involved in the fate of the energy after photoexcitation processes. Among the ancillary techniques that can significantly improve such knowledge, time-resolved thermal lensing (not discussed in this short review) deserves particular attention, allowing the extension of the temporal scale to hundreds of seconds, a range where important events may occur such as decay of singlet oxygen. Though instrumental improvements can be predicted, in particular as a consequence of developments of new piezoelectric materials, in our opinion the most relevant progress will concern data management, where the introduction of new computer programs, such as global analysis methods, may in principle be decisive in the obtaining of better-refined data. Prospects of more extensive applications to biological problems are obvious, but probably require coordination between different laboratories in order to realize instrumental set-ups dedicated to specific themes, because of the necessary superposition of various kinds of expertise in various fields such as experimental physics and biology. The organization of a few well-equipped laboratories could be at present a good prospect, also in consonance with the more recent developments of modern biophysics whose efforts are mainly in
the direction of study of systems in conditions near to the real state, and not only of isolated molecules. In this sense, photoacoustics, as a borderline technique between physical chemistry and the physics of condensed matter, could play a role more important than we think at present.
References A. Rosencwaig, Photoacoustic spectroscopy, Adv. Electron. Electron Phys., 46 (1978) 207-263. D.L. Landau and E.M. Lifschitz, Fluid Mechanics, Pergamon Press, Oxford, 1959, Chap. VIII. A. Rosencwaig and A. Gersho, Theory of photoacoustic effect with solids, J. Appt. Phys., 4 (1976) 64-69. F.A. McDonald and G.C.J. Wetsel, Generalized theory of the photoacoustic effect, J. Appl Phys., 49 (1978) 2313-2322. S. Malkin and D. Cahen, Photoacoustic spectroscopy and radiant energy conversion: theory of the effect with special emphasis on photosynthesis, Photochem. Photobiol., 29 (1979) 803-813. 6 A. Mandelis, Y.C. Teng and B.S.H. Royce, Phase measurements in the frequency domain photoacoustic spectroscopy of solids, J. Appl. Phys., 50 (1979) 7138-7146. C.K.N. Pate1 and A.C. Tam, Pulsed optoacoustic spectroscopy of condensed matter, Rev. Mod. Phys., 53 (1981) 517-550. V.P. Zharov and VS. Letokhov, Laser Optoacoustic Spectroscopy, Springer, Berlin, 1986. S.E. Braslavsky and K. Heihoff, Photothermal methods, in J.C. Scaiano (ed.), CRC Handbook of Organic Photochemistry, Vol. I, CRC Press, Boca Raton, FL, 1989, pp. 327-355. 10 S.E. Braslavsky and G.E. Heibel, Time-resolved photothermal and photoacoustic methods applied to photoinduced processes in solutions, Chem. Rev., 92 (1992) 1381-1410. 11 A.C. Tam, Applications of photoacoustic sensing techniques, Rev. Mod. Phys., 58 (1986) 381-431, and references cited therein. gen12 D.A. Hutchins, Mechanisms of pulsed photoacoustic eration, Can. J. Phys., 64 (1986) 1247-1264. 13 D.A. Hutchins, Ultrasonic generation by pulsed lasers, in Physical Acoustics, Academic Press, New York, NY, 1988, and references cited therein. 14 J.B. Callis, W.W. Parson and M. Gouterman, Fast changes of enthalpy and volume of flash excitation of Chromatium chromatophores, Biochim. Biophys. Acta, 267 (1972) 348-362. 1.5 J.E. Rudzki, J.L. Goodman and K.S. Peters, Simultaneous determination of photoreaction dynamics and energetics using pulsed, time-resolved photoacoustic calorimetry, J. Am. Chem. Sot., 107 (1985) 7849-7854. 16 L.A. Melton, T. Ni and Q. Lu, Photoacoustic calorimetry: a new cell design and improved analysis algorithms, Rev. Sci. Instr., 60 (1989) 3217-3223. 17 KS. Peters, T. Watson and K. Marr, Time resolved photoacoustic calorimetry: a study of Myoglobin and Rhodopsin, Ann. Rev. Biophys. Biophys. Chem., 20 (1991) 343-362. 18 J.R. Small, L.J. Libertini and E.W. Small, Analysis of photoacousticwaveforms using the nonlinear least squares method, Biophys. Chem., 42 (1992) 24-48. 19 A. Rosencwaig, Photoacoustics andphotoacoustic spectroscopy, Wiley, New York, NY, 1980. 20 D. Cahen, G. Bults, H. Garty and S. Malkin, Photoacoustics in life sciences, J. Biochem. Biophys. Meth., 3 (1980) 293-310.
P.R. Crippa et al. / Time-resolved photoacoustic spectroscopy 21 D. Cahen, G. Bults, S.R. Caplan, H. Garty and S. Malkin, Photoacoustic methods applied to biological systems, in C. Helene, M. Charlier, T. Mortenay-Garestier and G. Laustriat (eds.), Trends in Photobiology, Plenum Press, New York, 1982, pp. 21-32. 22 T.A. Moore, Photoacoustic spectroscopy and related techniques applied to biological materials, Photochem. Photobiol. Rev., 7 (1983) 187-221. 23 D. Balasubramanian and Ch. Mohan Rao, Photoacoustic spectroscopy of biological systems, Photochem. PhotobioZ., 34 (1981) 749-752. 24 D. Balasubramanian and Ch. Mohan Rao, Applications of photoacoustics to biology: some specific systems and methods, Can. J. Phys., 64 (1986) 1132-1135. 25 S.E. Braslavsky, Photoacoustic and photothermal methods applied to the study of the radiationless deactivation processes in biological systems and in substances of biological interest, Photochem. Photobiol., 43 (1986) 667-675. 26 D.C. Fork and S.K. Herbert, The application of photoacoustic techniques to studies of photosynthesis, Photo&em. Photobiol., 57 (1993) 207-220. 27 P. Poulet and J.E.J. Chambron, In vivo cutaneous spectroscopy by photoacoustic detection, Med. Biol. Eng. Cornput., 23 (1985) 585-588. 28 P. Poulet, Spectroscopic photoacoustique et science biomedicale: contribution aux etudes du sang, de la peau et de la photosynthtse, Dissertation, Universite Louis Pasteur, Strasbourg, France, 1985. 29 L. Brancaleon, P.R. Crippa and C. Minari, Time resolved photoacoustic spectroscopy of tryptophan and 5-methoxyndole: solvent dependence, submitted for publication in Photochem. Photobiol. 30 Ming Sun and Pill Soon Song, Solvent effects on the fluorescent states of indole derivatives-dipole moments, Photochem. Photobiol, 25 (1977) 3-9. 31 K.J. Willis, A.G. Szabo and D.T. Kracjarski, Excited-state reaction and the origin of the biexponential fluorescence decay of tryptophan zwitterion, Chem. Phys. Len., 182 (1991) 614-616. 32 J.E. Sabol and M.G. Rockley, Absolute fluorescence quantum yields by relative fluorescence and photoacoustic measurements of low level luminescence quenching, _r. Photochem. Photobiol. A: Chem., 40 (1987) 245-257. 33 W. Lahmann and H.J. Ludewig, Opto-acoustic determination of absolute quantum yields in fluorescent solutions, Chem. Phys. Lett., 45 (1977) 177-179. 34 J.R. Small, J.J. Hutchings and E.W. Small, Determination of fluorescent quantum yields using pulsed-laser photoacoustic calorimetry, in E.R. Menzel (ed.), Fluorescence detection III, SPIE, Bellingham, WA, 1989, pp. 26-35. 35 J.R. Small and S.L. Larson, Photoacoustic determination of fluorescent quantum yields of protein probes, in J.R. Lakowicz (ed.), Time-resolved laser spectroscopy in biochemistry II, SPIE, Bellingham, WA, 1990, pp. 126-136. 36 K. Heihoff and S.E. Braslavsky, Triplet lifetime determination by laser induced optoacoustic spectroscopy. Benzophenone/ iodide revisited, Chem. Phys. Lett., 131 (1986) 183-188.
15
37 T.Q. Ni and L.A. Melton, Non-radiative decay lifetime standards, J. Photochem. Photobiol. A: Chem., 67 (1992) 167-172. and 38 C. Viappiani and J.R. Small, Combined photoacoustic fluorescent quenching studies on organic dyes, in J.R. Lakowicz (ed.), Time-resolved laser spectroscopy in biochemistry III, SPIE, Bellingham, WA, 1992, pp. 285-294. 39 M.R. Eftink, Transient effects in the solute quenching of tryptophan residues in proteins, in J.R. Lakowicz (ed.), Timeresolved laser spectroscopy in biochemistty II, SPIE, Bellingham, WA, 1990, pp. 40-14. 40 M.R. Eftink and K.A. Hagaman, Viscosity dependence of the solute quenching of the ttyptophanyl fluorescence of proteins, Biophys. Chem., 25 (1986) 277-282. 41 M.R. Eftink and C.A. Ghiron, Fluorescence quenching studies with proteins, Anal. Biochem., 114 (1981) 199-227. 42 D.A. Johnson and J. Yguerabide, Solute accessibility to Nfluorescein isothiocyanate-lysine-23 cobra-toxin bound to the acetylcoline receptor. A consideration of the effect of rotational diffusion and orientational constraints on fluorescence quenching, Biophys. J., 48 (1985) 949-955. 43 C. Viappiani, Use of non-radiative decays of extrinsic fluorophores as structural and dynamical probes in protein environments: fluorescence quenching, (1993), submitted for publication in Biophys. Chem. 44 F.G. Prendergast, M. Meyer, G.L. Carlson, S. Iida and J.D. Potter, Synthesis, spectral properties and use of 6-acryloyl2-dimethylaminonaphtalene (Acrylodan), J. Biol. Chem., 258 (1983) 7541-7544. 45 G. Jori and J.D. Spikes, Photothermal sensitizers: possible use in tumor therapy, J. Photochem. Photobiol. B: Biol., 6 (1990) 93-101. 46 A.S. Ito, E.C. Azzellini, S.C. Silva, 0. Serra and A.G. Szabo, Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrins complexes, Biophys. Chem., 45 (1992) 79-89. 47 A. Losi, R. Bedotti, L. Brancaleon and C. Viappiani, Porphyrin-melanin interaction: effect on fluorescence and nonradiative relaxations, J. Photochem. Photobiol. B: Biol., 21 (1993) 69-76. 48 A. Losi, A. Vecli, C. Viappiani, N. Angelini, F .Ghetti and F. Lenci, Photoacoustic spectroscopy of non-radiative transitions in photoreceptor pigments fromBZephartkma japonicum, (1993), submitted for publication in Med. Biol. Environ. 49 A.C. Giese, The photobiology of Blephatisma, Photochem. Photobiol. Rev., 5 (1981) 139-188. 50 R. Cubeddu, F. Ghetti, F. Lenci, R. Ramponi and P. Taroni, Time-gated fluorescence of Blepharismin, the photoreceptor pigment for photomovement of Blepharisma, Photochem. Photobioi., 52 (1990) 567-573. 51 F. Ghetti, G. Checcucci, F. Lenci and P.F. Heelis, A laser flash photolysis study of the triplet states of the red and blue forms of Blepharisma japonicum pigment, J. Photochem. Photobiol. B: Bioi., 13 (1992) 315-321. 52 T. Matsuoka, Y. Murakami, T. Furukori, M. Ishida and K. Taneda, Photoreceptor pigment in Blepharisma: H+ release from red pigment, Photochem. Photobiol., 56 (1992) 399-402. 53 G. Checcucci, G. Damato, F. Ghetti and F. Lenci, Action spectra of the photophobic response of blue and red forms of Blepharisma japonicum, Photochem. Photobiol., 57 (1993) 686-689.
I. Photochem. Photobiol. B: Biol., 24 (1994) 3-15
New Trends in Photobiology
(Invited Review)
Time-resolved old idea
spectroscopy:
P.R.
Crippa,
photoacoustic
A. Vecli
of an
and C. Viappiani
Department of Physics, University of Puma, (Received
new developments
August 23, 1993; accepted
43100 Panna (Italy)
October
19, 1993)
Abstract Acoustic waves generated by heat emission in radiationless transitions from photoexcited molecules can be detected by suitable transducers. Their study allows the investigation of thermal relaxations, thus providing thermodynamic and kinetic data on short-lived species produced by the absorption of pulses of light. In this field of research the best technique has proved to be the so-called pulsed-laser, time-resolved photoacoustic spectroscopy, which is based on piezoelectric detection of pressure waves in the time domain. Deconvolution processing of the transient signals gives both the lifetimes of excited states and the energy content of the transitions, provided that decay times are in the range 5 ns-5 ps. Moreover, when compared with proper theoretical models emphasizing the energy balance, the photoacoustic results can help to build a complete picture of the deactivation pathways, including photochemical events. The biophysical applications, although numerous and widespread both in basic and applied research, offer the real possibility of giving information on photobiological processes in conditions very close to the living state. Among the more significant contributions obtained in this area, the results on photosynthesis and photosensitivity of plants and photosynthetic micro-organisms, structural and functional dynamics of respiratory proteins, photocycles of rhodopsin and bacteriorhodopsin and photophysical properties of several natural pigments are particularly relevant, together with some medical and biotechnological applications. Another promising field of application of photoacoustics concerns photoactive drugs and the photophysics of fluorescent probes for conformational studies of proteins, nucleic acids and membranes. In general terms, timeresolved photoacoustic spectroscopy promises to become one of the most powerful techniques in photobiophysics, provided that some limitations in data analysis and time resolution are removed by technical improvements.
Key words: Photoacoustics;
Optical spectroscopy;
Photophysics;
1. Introduction
The excitation of biomolecules by light - from UV to IR - triggers a complex sequence of phenomena whose investigation constitutes one of the main methods of studying their structure and interactions with the local environment. These events have been successfully explored by many experimental techniques, which as a whole define optical spectroscopy in its broadest sense, and we can actually affirm that the most relevant advances in the knowledge of biological molecular constit-
loll-1344/94/$07.00 0 1994 Elsevier SSDI loll-1344(93)06959-7
Molecular
biophysics
uents were achieved on the basis of data obtained by optical spectroscopy. At present, molecular photobiology can be considered an autonomous branch of biology, on account of the extent and the basic importance of light-induced processes in plants, micro-organisms and animals. From this point of view the study of photophysics and photochemistry of biomolecules involves many different approaches, each one potentially requiring an appropriate experimental technique. A comprehensive picture of such processes should in fact include determination of electronic energy levels (singlets and triplets), vi-
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4
P.R. Crippa et al. / Time-resolved photoacoustic spectroscopy
brational states, lifetimes, kinetic constants and efficiency of the different transitions. Moreover, also owing to the interactions with other chemical species (i.e. solvents, oxygen, etc.), the formation of photochemical transients and/or stable intermediates with a different energy content must be considered with particular attention. Two main areas of photobiology as studied by spectroscopic techniques can be identified. In the first one, attention is devoted to the effects induced by the primary event of photon absorption by biomolecules, supramolecular aggregates or isolated organelles that in the living organisms play the role of interfaces between the light world and the cell machinery. Chloroplasts and phytochrome in plants, and rhodopsin and melanins in animals are clear examples of such transductional intermediates. It is evident that biophysical description in quantitative terms of the sequence of molecular events following the absorption of light gives the reference points for the interpretation of the physiology of photosensitive organisms. The second area can be defined as optical spectroscopy of the biomolecules per se, and we can say that this approach has conclusively contributed to clarifying biomolecular structures (mainly by UV-visible absorption spectroscopy, dichroic spectroscopy, etc.) and dynamics (by fluorescence spectroscopy). The use of extrinsic chromophores has greatly enhanced the potential of this second aspect, thus enlarging the field of application of optical techniques beyond their natural limits. However, we must bear in mind that a relevant percentage of energy absorbed as light by matter is dissipated as heat by various molecular mechanisms and is consequently lost to optical studies. The recently developed methods of investigating this “dark side” of the problem, namely the photothermal methods, have greatly widened the application area of spectroscopy, allowing the direct quantitative detection of non-radiative transitions. In this sense we can refer to photothermal or photoacoustic methods as complementary to fluorescence and phosphorescence spectroscopy. As a matter of fact, the photoacoustic phenomenon was known over a century ago, and its fundamental principles are already well explained in the original papers by A.G. Bell, J. Tyndall and W.C. Roentgen giving notice of the discovery of the effect. Though for a long time the photoacoustic effect remained really little more than a scientific curiosity, from the turn of the century many significant contributions to the interpretation of the effect in gaseous and condensed matter have been published (for a historical review and
a summary of the oldest theories, see ref. 1) and we can see in this thorough analysis the basis of the more recent developments of photoacoustics as an analytical, structural and calorimetric technique for the investigation of matter. Essentially, photoacoustics reveals pressure waves generated by heat emission in radiationless transitions from excited states, and the theoretical treatment of the production of these acoustic waves by light pulses and their travelling in matter can be connected to the theory of Landau and Lifschitz [2] on pressure-wave propagation. The theory of photoacoustics was further developed in the 1970s [1,3-61, while from the early 1960s a variety of experimental apparatus and methods for experimental measurements has been proposed and some of them have emerged. The most widespread techniques at present are, in general, developments of the original one of Pate1 and Tam [7]. In particular, we can mention photorefractive techniques, such as thermal lensing and probe-beam deflection, and techniques based on both indirect (gas-microphone photoacoustics) and direct (piezoelectric photoacoustics) detection of the acoustic waves. In the application of these methods to the common case of solutions of biomolecules of low concentration, it should be emphasized that the solvent determines the thermal properties of the sample, while the solute affects its optical properties. The aim of the present review is to discuss briefly the principles and the methods of the socalled pulse-laser, time-resolved photoacoustic spectroscopy, with examples selected from the most significant of the biophysical literature and from researches in progress in our laboratory. We will present a short account for non-specialists of the photoacoustic phenomenon, the description of a typical experimental set-up and the problem of data management and analysis. For deeper details of both theoretical aspects and chemical applications we refer to some recent general reviews that present also other forms of photothermal spectroscopy and their applications, particularly in the field of organic photochemistry [f&10]. 2. Photoacoustic
signal generation
and analysis
2.1. Heat generation in absorbing media Light absorbed by molecules is converted to heat, luminescence or chemical energy, obeying the general condition of energy conservation. In Fig. 1 a scheme of the various processes that can act as heat sources is shown.
P.R. Crippa et al. / Time-resolved
~
Incident light +
Transmitted
light
Optical absorption Reflected light Prompt
Luminescence
keat
I-
V
Photochemical Energy
(1
\ , c-___--I
’ I I 1 I ’ I 1 1
Possible sources of delayed heat
Fig. 1. Block diagram showing the main processes bearing on the generation of heat following the absorption of light by a photoactive sample. The scheme demonstrates the various possible energetic pathways that can act as sources of delayed heat distinct from the mechanisms producing prompt heat, i.e. internal conversions and intersystem crossing.
In pulsed photoacoustics, the signal is analysed in the time domain and the signal-to-noise ratio is improved by averaging. With laser pulses of the order of nanoseconds, the length of propagation of the pressure wave is much less than the size of the sample cell: as a consequence, its shape is in general independent of reflections by the inner walls of the cell, and the sample dimensions can be assumed as infinite. As photothermal techniques are essentially calorimetric, they allow the monitoring of the input-output balance of the energy and, from conservation principles, the absorbed light energy is equivalent to the sum of the different output energies: N,hv,
= @,N,hv,+
aNAhve + c&, AE,
(1)
where NA and h are the Avogadro and Planck constants, v, and vf the frequencies of the incident and emitted light and af the quantum yield of fluorescence. The second term of the right-hand side represents the energy re-emitted as heat, and (Y the fraction of absorbed energy converted to heat within the instrumental integration time. The last term refers to the amount of energy stored in metastable species, AE, representing their molar energetic content, which can be evaluated by difference. The time resolution of the method depends on the possibility of separating the heat emitted in fast processes (Le. internal conversion, for example) from the heat produced by processes whose du-
photoacoustic
5
spectroscopy
ration is comparable with the integration time of the instrument, which in turn depends on the circuitry, the light pulse width, the experimental geometry and the time constants of the photophysical events. The geometry of the experiment, as in fluorescence, is generally 90”, i.e. the piezoelectric transducer detects pressure waves generated perpendicularly to the excitation-beam direction. This disposition is strictly valid only for solutions with low optical density; for high absorbances, the pressure waves are spherical, as generated by a quasipoint source situated near the inner surface of the cuvette, and a different geometry can be more suitable. In the case of scattering solutions, the experimental disposition is very critical and must be adapted to any particular situation. With low absorbance (A = ECZ-=x1) - the most common case - the pressure waves are cylindrical (Fig. 2). Several authors have treated formally the problem of the generation of acoustic waves in different approximations: see, for example, refs. 11-13. An elementary treatment of the time evolution of the photoacoustic signal can be developed introducing the transit time of the pressure front as Ta=
20
-
VS
where r. is the radius of the laser beam and vu, the sound velocity in the solution. The absorbed energy is E,=E(l-10-A)
(3)
and for a light pulse of duration T] and an excited state with non-radiative lifetime rnr greater than r,, an effective integration time can be defined, which determines amplitude, shape and time of arrival of the transient pressure to the transducer: ref= (r: + 7; + &)l’2 Experimentally, the instrumental lastic properties
(4)
the dependence of the signal on characteristics and the thermoeof the solvent involves a protransducer
laserp”,se
~ ~
~~~~~~~~~
Fig. 2. Scheme of the generation of the pressure pulse following the absorption of a pulse of light for the case of a solution with low absorbance (cylindrical geometry). For definitions of the parameters, see text.
6
P.R. Cnppa et al. / Time-resolved photoacoustic spectroscon
portionality constant that can be eliminated by the use of a reference substance that decays with 7 much less than T, and (Yequal to 1. In this case (Ycan be obtained easily from the ratio between the sample and the reference amplitudes, normalized for the absorbed energy E,. In fact, from the above discussion, a simplified form of the signal intensity S can be written as
I+ I
*i’con temprraturc
independent
AV=K(AV,+AVCon)
solution
S=kE(l-
lo-A)a
(5)
and the fraction Q for the sample can be evaluated by studying the dependence of the signal intensity upon A and/or E for both sample and reference. The knowledge of the value of (Yallows the evaluation of the energy stored in the metastable state(s), provided that the fluorescence quantum yield @r is known. In fact, the energy balance equation (1) can be written:
where Ef represents the average molar energy of the fluorescence emission and E, the molar energy of the laser pulses. Knowing one of the two terms aP or AZ?,, it is possible to determine the other. Moreover, from the dependence of CYon the ratio C&r, it is in principle easy to calculate T,,, varying the diameter r,, of the laser beam with proper pinholes. As the decays of the excited states are generally first-order, we can obtain: hE,=AE~exp
( 7,1 -
7 nr
(7)
where AE: represents the energy content of the transient species. Extrapolating the semi-logarithmic plot of 1 -(Y vus. T, at the limit of T, equal to 0, both rnr and aj,AE, can be evaluated.
T=20”C
T=4’C (@O)
Fig. with The ume
where AV, represents the volume change due to thermal relaxations, Q the amount of heat released in fast processes ( = &(l - lo-“)) and p the density of the solvent. p and cP, respectively the expansion volume coefficient and the specific heat at constant pressure, are markedly different in water than in most organic solvents and, as a consequence, the signal in water is smaller than in organics. However, the thermoelastic parameters of water, such as p, are strongly temperature dependent. So, in the case of photoinduced conformational change AV,, the total signal becomes S;k(bV.+AV.)=@
SaAV=pVAT= t t
@ CPP
(9)
+AVc)
If the reference has V, equal to 0 and Q/E equal to 1 (corresponding to (Y=l), its signal amplitude is: R=k
2.2. Molecular volume changes Another source of acoustic signal, sometimes superimposed on the thermal expansion discussed above, is the volume or conformational change of the photoexcited molecules, whose contribution to the total signal can be separated by a simple experiment involving variation of the temperature [14]. Figure 3 shows a sketch of the principles of the method. We can take as general expression for the photoacoustic signal amplitude S, due to fast thermal processes
3. Generation of the pressure signal in aqueous solution, its two components: the volume term and the thermal term. latter is temperature-dependent through the expansion volcoefficient /3 that becomes 0 at about 4 “C.
AV,=kE
p
(10)
CPP
and
S -=RE
Q
I
AK
cpp
(11)
E/3
Assuming cz (and Q) as independent of T, a linear plot can be obtained for ES/R vs. c,p/p, and the dependence of p on T can be used to calculate Q and AV, from the intercept and slope, respectively. 2.3. Time dependence deconvolution
of photoacoustic
signals:
A detailed analysis of the time dependence of photoacoustic signals can be based on the general
P.R. Crippa et al. I Time-resolved photoacoustic spectroscopy
achievements of the linear response theory: when a signal, in our case the heat release, stimulates a system, its time response is the convolution of the signal with the transfer function of the instrument. In photoacoustics the true acoustic pulse is convolved with the response function of the system 115,161, comprising both the ultrasonic microphone characteristics and the laser pulse shape. Experiments usually involve the measurement of the photoacoustic waveform R(t) for a reference compound, and of the waveform of the sample s(t) under identical conditions of absorbance, solvent and temperature. Generally speaking, R(t) will be a convolution of the laser pulse shape L(t) with the transducer response function: I R(t) =
L(t’)T(t-t’)
dt’
(12)
s 0
Since the reference compound undergoes fast nonradiative de-excitation, it has been assumed in the previous expression that the heat release follows the laser pulse exactly, and in this sense acts as a Dirac delta function (see Fig. 4). For a species having an excited state with finite lifetime, we have a time-dependent heat release, indicated by H(t); the corresponding expression of the photoacoustic waveform is therefore given by I’
f S(t)=
qt-t’) f
dt’x
L(t”)H(t’-t”) s
dt”
(13)
0
0
By interchanging we can write:
the order
of the convolutions,
f
S(t) =
s
R(t’)H(t-t’)
dr’
(14)
0
Fig. 4. Scheme of the generation of the photoacoustic signal S(t) as the convolution of the exponential release of heat H(t) with the signal produced by a reference compound R(t) giving only prompt heat with a=1 and T&T=.
7
It is worthwhile to note that the expression derived for s(t) is valid whichever pulse width L(t) is used. In the simple case of different channels of deexcitation starting from the same excited state, it is very easy to derive the expression for H(t); assuming first-order kinetics, the proper function that describes the heat decays is a sum of exponentials [17]:
H(r)=i$l : exp I
( 1 -
f
ri
(15)
The case of sequential decays too is described by eqn. (15). By analysing the time profile of the signal by means of the deconvolution technique it is thus possible to retrieve the parameters of the exponential decay. The real-time deconvolution is obtained by means of an iterative non-linear least-squares technique; the reference and the sample waveforms are input into the fitting program, and a portion of the waveform, which usually includes the first oscillations, is selected for analysis. The sample waveform is compared to the numerical convolution of the reference waveform and the assumed heat transient, which may be composed of one or two decays; the goodness of fit is judged by reduced 2, which is calculated for a determined set of fitting parameters and minimized in order to obtain the most likely values. The fitting procedure yields the pre-exponential factors C_X~ and the lifetimes 7i; a check on the goodness of the fit is provided, as stated, by the value of the reduced 2 and by visual inspection of the residuals, as well as by their autocorrelation. An example of superimposed reference and sample signals is shown in Fig. 5. As there will generally be a certain amount of energy released as heat in internal conversion processes, a fast decay will be present in the signal, whose lifetime is in the range of femtoseconds to picoseconds, well below the experimental integration time of the instrument. For these prompt processes, deconvolution allows the determination of the pre-exponential (Y, but the lifetime cannot be defined precisely; it can only be said that this decay is characterized by a lifetime below the experimental integration time. For longer lifetimes, up to about 5 ps, it is possible to determine both the rate and magnitude of the process. We will not enter into further details of the data analysis; the reader may conveniently find more elaborate descriptions of the deconvolution process in the literature [18].
P.R. Crippa et al. I Time-resolved photoacoustic spectroscopy
channel number
Fig. 5. Example of waveforms as obtained in a typical experiment. Curve 1, reference signal (ferrocene in CH,CN, OD = 0.2 (A,,= 308 nm), averaged over 1000 pulses); curve 2, simulated signal with a=0.6 and r= 100 ns (the dotted curve nearly coincident with curve 2 represents the convolved waveform); curve 3, residuals.
pulsed dye laser beam splitter
transducer
f
+,
digital oscilloscope
Eizl R
c
t
_
S
computer
t Fig. 6. Block diagram of the experimental set-up. As the examples reported in Section 4 refer to results obtained in our laboratory, our apparatus may conveniently describe a typical experimental arrangement. Laser, Lambda Physik EMGSO (excimer XeCl laser that delivers pulses of about 5 ns length) followed by a dye laser, Lambda Physik FL3100; energy meter, Laser Precision RjP-7620 with probe head RjP-735; 1 MHz piezoelectric transducer (PZT), with preamplifier Panametrics V103; digital oscilloscope, LeCroy 9450450))))))))))))))) (350 MHz) with sampling rate of 400 Msamples s-‘, corresponding to 2.5 ns per channel, fully programmable through GPIB (IEEE-488) or RS232C.
The pulsed-laser light source should give pulses of a few nanoseconds (or in special cases, a few picoseconds) duration. The choice of a pulsed laser is in general a compromise between practical and economical considerations and the specific characteristics of the process under investigation. Owing to the specific absorption properties of a sample containing biological molecules, tunability of the light source is very important. The pulse energy must be maintained below the threshold of non-linear effects; the repetition rate should be a few Hertz in order to maintain the thermal equilibrium in the sample. Transduction from mechanical to electrical signal is carried out by piezoelectric devices directly clamped on the measurement cell, with a thin layer of silicon grease interposed in order to ensure good acoustic matching. Lead zirconate-titanate (PZT) or polyvinylidene difluoride (PVF,) are the most common piezoelectric elements, the first being the more sensitive while the second has a higher resonance frequency, allowing better time resolution. The structure of the sample cell is obviously related to the instrumental transfer function, but no general rule can be stated about cell optimization: experience and serendipity are the two pillars of cell design. In many cases a simple fluorescence quartz cuvette gives good results and allows easy temperature control. On the other hand, the range of temperatures accessible is limited by the temperature sensitivity of the transducers. In order to normalize each photoacoustic signal to a fixed reference value of incident light intensity, a fraction of the laser pulse must be deviated by a beam splitter to the probe head of an energy meter whose output is sent to a personal computer managing the experiment and performing the acquisition and the first processing of the output data of the transducers, suitably amplified and digitized by a digital oscilloscope. In this way the averaged waveform (100-1000 acquisitions) is displayed and stored. Successively, the processing of the experimental signal gives the parameters cx and r.
3. Basic instrumentation
4. Biophysical
A typical instrumental set-up for time-resolved photoacoustic spectroscopy is schematized in Fig. 6: in relation to different needs corresponding to minor variations can be different experiments, introduced.
4.1. General overview From their beginnings, photoacoustic techniques proved to be a very useful and sensitive tool for experimental studies in biophysics and related fields: a number of interesting results were in fact
applications
P.R.
Ctippa
et al. / Time-resolved
obtained from the early 1970s on isolated biomolecules, cell suspensions and intact or physically and chemically treated tissues such as fresh leaves, dried algae and phytoplankton. Moreover, identification of metabolic states of bacteria and characterization of normal, pathological and drugtreated animal tissues, both soft and hard, demonstrated the possibility of applying these techniques as diagnostic subsidiary instruments in several branches of medicine (for reviews, see refs. 19-22). All these results were obtained by so-called C.W. photoacoustic spectroscopy, and the main advantage - unanimously pointed out - in comparison with classical optical techniques, during this pioneering stage of the biomedical application of photoacoustics, was the possibility of obtaining photoacoustic absorption spectra as accurate and informative as optical ones, and in addition on samples too opaque or transparent. In the same period an intense effort was made by several groups to adapt photoacoustic detection to special spectroscopic techniques that were successfully applied to biological studies, such as double-beam, IR Fourier transform and dichroic forms of spectroscopy. Detection of trace amounts of metal ions, the estimation of thermal diffusion lengths, the determination of absolute quantum yields of fluorescence and the first significant evaluation of energy storage in photosynthesis are some of the prominent results obtained in biological systems in this way, on both in vitro and in vivo samples. Incidentally, we wish to emphasize the opportunity to consider again this line of instrumental improvement with the more advanced detectors and light sources now available. A substantial improvement in photothermal methods is related to the analysis of the time profile of the heat emission by photoexcited samples. The lifetimes and the energy contents of metastable molecular species can be easily determined, and quantitative information on enthalpic changes and reaction volumes modifications induced by light absorption in the samples can be obtained. The relevance of these achievements for biophysical studies is rather obvious, and extensive reviews of the results obtained on this basis during the 1980s and the early 1990s have been published [10,17,23-261 and can usefully be consulted. It is interesting to underline that photoacoustic spectroscopy in its different instrumental configurations has been applied to biophysical studies in order to obtain structural, analytical, morphological and functional information in any case in which the presence or the interaction or the
photoacoustic
spectroscopy
9
time evolution of intrinsic or extrinsic chromophores accessible to photoexcitation can contribute to the study of biological molecules, organelles, cells and tissues. Some illustrative examples certainly not exhaustive, because of the abundance of the scientific literature in the field - may clarify the significance and the potentialities of the technique. The study of photosynthetic molecular mechanisms and structures is probably the most popular area of the application of photoacoustic spectroscopy in biophysics. Measurements in gas and liquid phases with modulated and pulsed actinic light on intact or treated plant tissues and bacteria made it possible to obtain a very complete description of basic phenomena such as the time course of energy flow and storage, oxygen evolution, electron flow, and interactions between photosysterns I and II, as well as a screening of processes relevant for biotechnological applications, such as inhibiting effects on photosynthetic yield by pollutants, herbicides and fertilizers, heat and chilling stress, water stress and photoinhibition. Also the photochemical and photophysical properties of phytochrome have been accurately determined, giving a relevant impulse to the definition of the plant photosensitivity problem. According to Fork and Herbert [26], photoacoustic methods “may become some of the most useful methods in photosynthesis research”. The respiratory proteins haemoglobin and myoglobin have been extensively studied by photoacaustic spectroscopy as regards their structures in solution and in the crystalline state and the mechanism of their interaction with binding substrates. In general we can say that photoacoustic calorimetry must be considered as a very important tool for the general problem of binding to biomolecules, i.e. for the determination of changes in extent of ligand, enthalpy and volume characterizing specific interactions of chromoproteins, including hydration and more general solvent-interaction phenomena. The use of extrinsic labels can give a tremendous impulse to the study of these processes that trigger many metabolic pathways. Other specific applications deserving a special mention have been the detailed analysis of the photocycles of rhodopsin and bacteriorhodopsin, including for the latter the dynamics of the protonpumping mechanism. Photoacoustic data obtained in all these applications and many others of biological interest, such as organic and organometallic reactions, possibly combined with fluorimetric and flash photolysis data, guided the formulation of models providing
10
P.R. Ctippa et al. / Time-resolved photoacoustic spectroscopy
new global insights into biological problems and stimulating a more realistic view of the underlying molecular mechanisms. Taking advantage of the above-mentioned technical developments, several medical applications of photoacoustic methods have been developed, mainly in dermatological and haematological studies [27,28] and in the assessment of the photodynamic therapy of tumours.
4.2. Photoacoustics of tryptophan [29] The interest in a photoacoustic study of tryptophan arises from its well-recognized relevance as an intrinsic optical probe in proteins. In spite of the large amount of work on the fluorescence and phosphorescence of this amino acid in peptides and proteins, many aspects of its photophysics are still not clarified and, in particular, the anomalous trend of fluorescence emission that strongly depends on the solvent. The most satisfactory model includes the formation of exciplexes between excited indole and one or more solvent molecules [30,31]. The only reported indole derivative that should not form exciplexes is 5-methoxyindole (5 MOI), because of the presence of the methoxy group, which significantly lowers the value of the dipole moment. time-resolved photoacoustic A comparative study of TRP and 5 MO1 was performed in order to assess: 1. the possible formation of intermediate excited states that can be precursors of the triplet state, and 2. the role of molecular oxygen in the thermal relaxation of these excited states and the consequent formation of singlet Oz. The experiments were performed with 5 ns laser pulses at a wavelength of 308 nm in order to avoid photoionization working on the extreme tail of the absorption band, and allowed the conclusion that: 1. the kinetic behaviour of photoexcited tryptophan can be described by introducing an intermediate state with energy below the first excited singlet state of the solvent (3.73 eV in cyclohexane, 3.43 eV in water) and a lifetime much shorter in water (17 ns) than in organic solvents (107 ns in cyclohexane, 68 ns in MeOH). The efficiency of formation is higher in organic solvent. Also 5 MO1 in Hz0 generates such a precursor with high yield (@= 0.74) These data strongly support the exciplex hypothesis even if at present their nature (strong or weak exciplexes) is not yet well defined;
2. the role of oxygen is elucidated by comparison of the results obtained in deoxygenated and oxygenated samples. The shorter lifetime and the enhanced signal amplitude in aerated solutions confirm the presence of a dynamic quenching process with formation of singlet oxygen both in TRP and 5 MO1 solutions. From energy-balance equations the quantum yield for singlet O2 formation can be evaluated and appears to be rather high, particularly in H,O: 0.74 for 5 MO1 and 0.80 for TRP. In conclusion, time-resolved photoacoustics allowed the clarification of the photophysics of this indole-like molecule also in quantitative terms. The application of these results to the study of tryptophan residues in peptides and proteins is at present under investigation. 4.3. Fluorescence quenching of free dyes and protein probes A straightforward application of photoacoustics is the determination of the absolute fluorescence quantum yield [32-351. Recently the possibility of investigating the effect of chemical-physical parameters on the photophysics of a molecule has also been investigated by performing fairly accurate analysis of collisional fluorescence quenching by KI on several organic dyes [36-381. In the simple case of free dyes in ethanol solutions, the good agreement between fluorescence and photoacaustic data shows that the two processes can be regarded as complementary to each other; in fact the quenching constants for the two effects are identical, and the fluorescence quantum yield, as determined by the fitting of the photoacoustics data, is in accordance with the literature data. These results clearly show that a combined fluorescence and photoacoustic approach to the quenching problem gives a good knowledge of the fate of the absorbed energy within the fluorophore. Protein fluorescence quenching of both aminoacid side chains and bound probes has become a widely used technique in protein biochemistry, and has allowed the determination of both structural and dynamic properties of local environments inside the macromolecule [39-41], while determination of quenching parameters allows the estimation of the degree of exposure of the molecule to the solvent [42]. Recently one of us has demonstrated [43] that a combined fluorescence and photoacoustic technique can be usefully employed to study protein fluorescent probes. Three different proteins containing a single free thiol have been selectively labelled with fluorescein-5-isothiocyanate (FITC),
P.R.Crtppa et al. / Time-resolved photoacoustic spectroscopy
and its fluorescence quenched with KI. Unmodified fluorescein is known to undergo intersystem crossing with very low efficiency (0.05) when free in solution. Fluorescein derivatives show a large increase in intersystem crossing efficiency, which assumes values of 0.49 for eosin, 0.71 for dibromofluorescein and even very close to unity for erythrosin. FITC itself is known to undergo intersystem crossing to a triplet state with low efficiency and long lifetime, outside the range detectable, at least at present, by photoacoustics. On the other hand, fluorescence quenching alone is not sensitive to the particular deactivation pathway of the molecule, the Stern-Volmer plot for dynamic quenching being generally linear. Non-linearities usually arise because of emission heterogeneity and/or static quenching. As a consequence, fluorescence spectroscopy is unable to distinguish an enhanced intersystem crossing from any other process depleting the excited singlet state. In contrast, photoacoustics is extremely sensitive to the different pathways that the energy follows in the deactivation process. In Fig. 7 the effect of KI on the photoacoustic signal of FITC, free and protein-bound, is shown, while, in the concentration range used for each sample, the Stern-Volmer plots of fluorescence data were linear. Obviously the quenching constants for the process determined from fluorescence and photoacoustic data, assuming intersystem crossing to the triplet state as negligible, are substantially different, even in the case of free FITC,
0.9
1
0.8
-
0.7
-
0.6
-
0.2
’
I
I
I
,
I
1
@
I
1
I
I
I
0.0
0.2
0.4
0.6
0.8
I
WI1 (Ml Fig. 7. Amplitude of signals after deconvolution for monoexponential decays obtained on fluorescein-S-isothiocyanate, free and bound to proteins quenched by KI: 0, free FITC, 0, BSAFITC, +), carbonic anhydrase-FITC; 0, papain-FITC.
11
TABLE 1. Fluorescence quenching by KI on free FITC and protein-FITC complexes as determined from fluorescence and photoacoustic data. All the measurements were carried out with A,,=490 nm, corresponding to a molar energy content of 58.37 kcal Sample
Free FITC FITC-Carbonic FITC-Papain FITC-BSA
anhydrase
K from fluorescence W-‘)
K from photoacoustics
10.40 4.90 4.24 5.81
10.2 5.8 4.2 5.7
W’)
whose triplet state is known to be very poorly populated. Invoking the presence of significant intersystem crossing gave good results for all the cases considered, indicating a sharp change in the photophysics of the molecule upon binding to the proteins, with a strong increase in the intersystemcrossing quantum yield. The quenching constants reported in Table 1 give information concerning the shielding of the binding site from the solvent in agreement with independent measurements [44]. An indication on local pH at the binding site was obtained from the value of the fluorescence quantum yield. 4.4. Photophysical parameters of drugs for phototherapy A large amount of research is presently being devoted to the study of photosensitized reactions in medicine, particularly in the photodynamic therapy of tumours. In these processes the photosensitizer is excited by absorption of light and undergoes intersystem crossing to a triplet state from which it either reacts with the nearby molecules or produces cytotoxic oxygen derivatives, such as singlet oxygen or superoxide anion. Photothermal sensitization by the heat release of non-fluorescent endogenous or externally-added dyes has been investigated recently [45] with interesting results directly related to the presumed therapeutic mechanism. In order to have good sensitizing efficiency, all these drugs must show very low fluorescent quantum yield and complementary non-radiative decays with very high efficiency; in this sense, photoacoustics is a suitable tool for studying the photophysical characteristics of these drugs and their interaction with biological molecules. 4.5. Interactions between melanins and porphyrin Porphyrins are among the most promising photoactive drugs in the phototherapy of malignant
12
P.R. Crippa et al. / Time-resolved photoacowtic
melanoma, and therefore a detailed knowledge of their transport and uptake in melanoma tissue as well as of their binding to biomolecules and, in particular, to the melanoma melanins is a necessary prerequisite in order to understand their mechanism of action at a subcellular level. The photochemical behaviour of porphyrins after binding to melanins deserves particular interest because, even if melanins are not the target of the therapeutic action, the strength of their interaction with the pigments suggests a potential source of photophysical side-effects overlapping the main photoactivity. Binding of zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) to synthetic melanins results in the formation of a non-fluorescent complex [46] whose non-radiative decay has been investigated by means of photoacoustics [47]. The titration of a fixed quantity of ZnTBzPyP with increasing concentrations of eumelanin resulted in the expected red shift and hypochromism in the absorbance spectrum of the dye, on account of the greater delocalization of the rr electrons in the central ring of the porphyrin. Addition of eumelanin to the dye quenches its fluorescence centred at about 630 nm; the Stern-Volmer plot for the quenching of porphyrin fluorescence is non-linear and exhibits an upward curve (Fig. 8): this behaviour is in good agreement with a model that takes into account static quenching by melanin and is best described by F
T/ being proportional to the volume of the action sphere and M being the melanin concentration. The best fit gave the results reported in Table 2. The photoacoustic data showed a feature of the quenching process that was not evident from the fluorescence data: in order to get a good fit, we had to assume that the quenching process affects, to a certain degree, also the intersystem crossing probability. The fraction of the prompt heat (Yis reported in Fig. 9 along with the best fit (solid line) to the following expression
rem
3.5 A 3 \2!
@T+(l+KcMI) E-l-
@*
OL=v,,1+qMj
-
U+K[MI)
-E
2.5
0 -_ 1.5
0.5
1.0
1
1
I
0
1
2
I
,
I
1
A_ 0
7. 2
-
ti 0
-2
0.7
0.4
L 0
(16)
- =exPwwl) FCI
spectroscopy
Fig. 8. Direct (A) and reciprocal (B) Stern-Volmer plots for the quenching of ZnTBzPyP flucrescence by eumelanin. A,,=475 nm; porphyrin concentration, 3.8 PM. TABLE 2. Quenching of ZnTBzPyP fluorescence with eumelanin: results of fitting experimental data with the expression for a
345 475
V (ml mg-‘)
[ZnTBzPyP] (cLM)
[Melanin] (pg ml-‘)
0.27 0.25
3.8 3.8
O-l.9 O-4.2
(17)
where K is the quenching constant of the process and ET is the energy of the triplet state. This equation contains a term describing the action of the enhanced intersystem crossing: exponential quenching of the fluorescence term was tried but gave indistinguishable results, the reason for this being the small weight of this term in the expression
of CC Fitting yields the following parameters: K= 0.30 ml pg- ‘; ET/E = 0.48; aIsc = 0.42; we assumed for cD~a value of 0.031 [46]. The photoacaustic data show that binding of porphyrin to eumelanin results in an enhanced degree of intersystem crossing of the porphyrin to a long-lived state, undetectable by the instrument, corresponding to a quenching of (Y. We are not however in
13
P.R. Crippa et al. / Time-resolved photoacoustic spectroscopy 0.8
r
a
\
0.7
0.6
< 0
0.5 0
I
I
I
I
2
4
6
8
10
[Melanin](pg/ml) Fig. 9. Fraction of prompt heat emitted function of concentration of melanin.
by ZnTEkzPyP as a
a position to decide if this long-lived state is the result of the formation of a triplet state of the porphyrin or of the melanin. Further investigations are in progress to assess the identity of the longlived species, in order to elucidate the fate of the energy stored in it. Oxygen plays a major role in this system, certainly related to its intrinsic ability to quench triplet states of photodynamic sensitizers by energy transfer processes; in fact deoxygenation dramatically lowered the prompt non-radiative emission of the mixed solutions, whereas for the single chromophores we did not observe any significant change. 4.6. Photoreceptor pigment from Blepharisma japonicum [48] The ciliate protozoan Blepharisma japonicum exhibits a complex and typical photobehaviour triggered by the photoactivation of blepharismin, a hypericin-like pigment present in strings of granules arranged just below the cell surface [49]. Action spectra of the photophobic response of the micro-organism and spectroscopic studies of photodynamic sensitization induced by intense illumination in vivo suggest that all these photoresponses are under the control of blepharismin. In particular, comparative studies of the optical properties of blepharismin and hypericin indicate the possibility of different deactivation pathways in pigment and reference molecule, but do not allow the formulation of a complete model of the molecular mechanisms of the energy transduction towards the photomotile and photochemical responses [50-531.
A photoacoustic analysis of the released prompt heat in isolated pigment in different conditions (oxygenated and deoxygenated solutions, ethanolic and mixed water-ethanol solutions) was carried out according to a model taking into account the intersystem crossing to the triplet state of the pigment and a possible subsequent energy transfer to OZ. The results (Fig. 10) strongly support the assumption of different deactivation mechanisms in pigment and model molecule and demonstrates the presence in blepharismin of a second fraction of released heat, poorly affected by the presence of water and suggesting the possibility of an energy transfer process from the triplet state in competition with the O2 singlet-state activation. Such a deactivation mechanism does not seem to be active in blue blepharismin, a modified form of the pigment induced in the micro-organisms by prolonged illumination in the presence of oxygen, and is probably due to intermolecular interactions in molecular aggregates of pigment whose presence in solution was confirmed by light-scattering measurements.
1.5
1
/
I
I
I
1.0
0.5
0.0
-0.5
-1.0
1
0
300
600
900
1200
1500
0
300
600
900
1200
1500
channel
number
Fig. 10. Normalized photoacoustic signal of red blepharismin in ethanol upon excitation at 308 nm. The upper plot shows the reference (higher curve), the sample and the convolved waveforms along with the residuals; the lower plot shows the autocorrelation function of the residuals.
14
P.R. Crtppa et al. / Time-resolved photoacoustic spectroscopy
Further measurements on pigment granules and artificial models, now in progress in our laboratory, will give us the possibility of formulating a more detailed model of the primary events in the energy transduction chains active in vivo. Such a model should be, in principle, adaptable also to other phototransduction events of biological relevance.
5. Conclusions From this short account of the experimental principles presented above and the examples we have selected from our laboratory experience, some general considerations emerge, allowing the judgement that time-resolved photoacoustic spectroscopy is a technique that has now reached a mature stage, and should be considered at the same level as other optical methods having wider application. In fact it is possible, with the presently available technology, to obtain sensitivity and time-resolution levels that are fully comparable with the performance of other common techniques such as flash-photolysis and fluorescence. Particularly when used in parallel with such forms of spectroscopy, time-resolved photoacoustics shows its special characteristics in the study of the complex phenomenology involved in the fate of the energy after photoexcitation processes. Among the ancillary techniques that can significantly improve such knowledge, time-resolved thermal lensing (not discussed in this short review) deserves particular attention, allowing the extension of the temporal scale to hundreds of seconds, a range where important events may occur such as decay of singlet oxygen. Though instrumental improvements can be predicted, in particular as a consequence of developments of new piezoelectric materials, in our opinion the most relevant progress will concern data management, where the introduction of new computer programs, such as global analysis methods, may in principle be decisive in the obtaining of better-refined data. Prospects of more extensive applications to biological problems are obvious, but probably require coordination between different laboratories in order to realize instrumental set-ups dedicated to specific themes, because of the necessary superposition of various kinds of expertise in various fields such as experimental physics and biology. The organization of a few well-equipped laboratories could be at present a good prospect, also in consonance with the more recent developments of modern biophysics whose efforts are mainly in
the direction of study of systems in conditions near to the real state, and not only of isolated molecules. In this sense, photoacoustics, as a borderline technique between physical chemistry and the physics of condensed matter, could play a role more important than we think at present.
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