Journal of Photochemistry and Photobzology, Be BloJogy, 2 (1988) 1 - 19

NEW TRENDS IN PHOTOBIOLOGY (Invited Review)

TIME-RESOLVED FLUORESCENCE IN PHOTOBIOLOGY

H SCHNECKENBURGER*, H K. SEIDLITZ and J. EBERZ

Gesellschaft fiir Strahlen- und Umweltforschung Miinchen, Institut fiir Angewandte Optik, Ingolstiidter Land&r. 1, D-8042 Neuherberg (F.R.G)

(Received December 3,1987, accepted February 12,1988)

Keywords. Fluorescence lifetimes, fluorescent biomolecules, fluorescence stainmg, photosensitization, microoptic devices, picosecond light sources, recording methods.

Summary

The article focuses on techniques and applications of time-resolved fluorescence spectroscopy in biology and medicine. Both novel methods and well-established ones are discussed and future trends are outlined.

Applications mcluding fluorescence detection of nucleic acids, proteins, coenzymes and plant pigments and fluorescence labelling of nuclei, mem- branes and antibodies are outlined. In addition the fluorescence properties of photosensitizers used in photodynamic therapy are discussed.

1. Introduction

Many organic molecules can be identified by their characteristic lumi- nescence after excitation by visible or near-UV radiation. A radiative transi- tion between electronic states of the same resulting spin (usually the first excited singlet state and the ground state) is termed fluorescence. Non- radiative (nr) transitions, intersystem crossing (ISC) between states of dif- ferent spin and energy transfer (ET) with neighbouring molecules compete with the radiative (r) transition. The rates k, of all these deactivation pro- cesses can be added up, yielding a total rate 12 of deactivation

k=k,+k,,+k,,+k,=L

r being the effective observed lifetime of the excited state.

*Present address for correspondence. Institut fiir Lasertechnologlen in der Medizin an der Universitiit Ulm, Postfach 4066, D-7900 Ulm, F.R.G.

loll-1344/88/$3.50 @ Elsewer Sequola/Prmted m The Netherlands

Fig 1 Fluorescence mlcrograph of methanogemc bacteria, excitation at 405 nm, detec- tion range 460 - 570 nm, image size 220 m X 340 pm Reproduced from ref. 31.

The present paper concentrates on complex fluorescence signals. An example is given in Fig. 1, which shows a fluorescence micrograph of bac- teria occurring in fermentation processes with different fluorescent co- enzymes. The fluorescence spectra of these coenzymes overlap; however, their lifetimes are different and can be used to distinguish and quantify individual fluorophores.

Moreover, different components of the same fluorophore can be dis- tinguished according to their*sites of binding in cells or tissues or to their states of aggregation. Changes in the micro-environment, solvent or aggrega- tion may have a drastic impact on the fluorescence lifetimes, even when they have only a minor influence on the optical spectra. In addition, different fluorescence lifetimes are obtained for different functional groups of certain molecules. For example, the lifetime of chlorophyll molecules depends on whether these are part of the intact photosystems I or II, or whether they are stopped from taking part in photosynthetic reactions (e.g. by blocking intermolecular energy transfer).

Numerous applications have been reported for the analysis of complex fluorescence signals and the measurement of fast kinetics (such as the forma- tion of aggregates or energy transfer) on the basis of fluorescence lifetimes. Only a few of them can be mentioned in the present paper, which empha- sizes some recent applications and novel techniques.

We will not discuss femtosecond methods which have been applied mainly to absorption spectroscopy and not to fluorescence measurements.

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2. Methods of nanosecond and picosecond fluorescence spectroscopy

2.1. Picosecond fluorescence recording methods Three methods are commonly applied in tune-resolved fluorescence

measurements: (a) single-photon counting; (b) the phase shift method; (c) streak camera detection.

2.1.1. Time-correlated single-photon counting Tune-correlated single-photon counting is a well-established technique

[ 11. The method is based on the concept that the probability for emission of a single photon after an excitation pulse mirrors the actual intensity-time profile of a fluorescence decay. The basic set-up of a single-photon counting instrument is shown in Fig. 2. The sample is repeatedly excited with short flashes of light and the emitted fluorescence is adjusted in intensity such that only a single photon arrives per excitation pulse, as otherwise the measure- ment is falsified and yields decays which are too short. The single photon events are detected by a fast high gam photomultiplier. The resulting signal is used, after some appropriate pulse shaping by a constant fraction dis- criminator (CFD), to stop the ramp generator of a time-to-amplitude converter (TAC) which has been triggered by the excitation pulse. Thus the time interval between excitation and fluorescence emission is converted into a correspondmg voltage. The repetitive measurements of this time interval are collected by a multichannel analyser (MCA) and displayed as a histo- gram, which represents the fluorescence decay. However, this “experimental” decay curve is distorted owing to the finite width of the excitation pulse and the limited time resolution of the photomultiplier and other electronic components.

start

Fig. 2. Schematic diagram of a single-photowzountmg v, amplifier; PMT, photomultlpher tube; CFD, constant time-to-amplitude converter; MCA, multlchannel analyser.

apparatus. BS, beamsplitter; fraction dwriminator; TAC,

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The “true” sample decay can be reconstructed by numerical deconvolu- tion of the experimental curve using the instrumental response function. This function is obtained by replacing the sample by a light scatterer or by the measurement of the decay of a material with a well-known lifetime. The algorithm most widely used is the non-linear least-squares deconvolution analysis of data. The deconvolution algorithm combines a theoretical model curve with the instrumental response function and calculates an optimal fit. The quality of the fit can be judged from a statistical quantity x2, which measures j the ratio between actual deviation and expected deviation caused by noise. A more detailed representation of the goodness of the result is obtained from a plot of the so-called weighted residuals which shows directly where misfits occur (see below, Figs. 8 and 9). The model function is chosen according to the physical processes expected. Pure decay reactions are usually fitted by monoexponentials or multiexponentials, and the physical significance of each component has to be discussed critically [ 21.

Tune-correlated single-photon counting is presently the most sensitive technique for measuring fluorescence decay, providing a very good signal-to- noise ratio, a wide range of intensities and a subnanosecond resolution time. The time resolution is limited mainly by the transit time spread of the photomultiplier, timing errors introduced by the pulse-shaping discrimina- tors and fluctuations of the excitation pulses. The advent of microchannel photomultipliers (MCP) has greatly improved the transit time spread error. An mstrumental response function with 50 ps FWHM can be obtained [3]. After deconvolution the time resolution is 10 ps or less.

The main problem of the single-photon counting technique is that not more than one fluorescence photon per excitation pulse can be detected. This means that, by using pulsed lasers or flashlamps with low repetition rates, rather long measuring times are necessary, which could cause degrada- tion of biological samples. In order to overcome this disadvantage, time- resolved detection of multiphoton events following each laser pulse was introduced. For this purpose all photon signals were fed directly mto the fast memory of a computer and added up over a certain number of cycles to obtain the fluorescence decay curves (Fig. 3) [ 41. The time resolution is

Photo - Multlpller

* Ampllfler +

Dwrlmmator

Expenment !

I zY-r

Address Fast -

Counter Memory

t

AmplIfter + Sample+Hold - PDP-11 + -

Pulse Former A/D Converter 4 Display

Fig. 3 Experimental scheme for time-resolved multiphoton counting. Reproduced from ref 4.

5

Ref

Fig. 4. Schematx diagram of a phase fluorometer MOD, light modulator; BS, beam- splitter; PMT, photomultlplier tube, Ref, reference channel; Slg, fluorescence signal.

limited so far by the cycle time of the memory of 100 ns, but in future it seems realistic to say that time resolutions of 10 ns or less will be obtained. The method of “multiphoton counting” therefore appears attractive for a time range from several nanoseconds to microseconds.

2.1.2. Phase shift method In the phase shift approach (Fig. 4) the sample is excited with light

modulated sinusoidally by a Pockels cell or an acusto-optic modulator. The resulting fluorescence emission detected by a photomultiplier varies sinu- soidally at the same frequency but is phase shifted. For a monoexponential decay the lifetime t can be calculated from the phase shift $J by the relation

t tan@) =-

where f is the modulation frequency. The frequency domain and the time domain are related by a Fourier transform. Phase fluorometry and pulse fluorometry are therefore equivalent in principle.

The details of phase fluorometry have been reviewed in ref. 5. Phase measurements are rapid and require only a few seconds of measuring time. The method provides very good time resolution down to 1 ps [ 61, but multi- component analysis was very difficult until recently. Phase fluorometry has regained attraction by the introduction of variable frequency phase modula- tion combined with cross-correlation. Up to three discrete exponentials can be resolved [ 71. A novel approach is based on the assumption that a popula- tion of molecules does not show a discrete spectrum but a continuous distri- bution of lifetimes. The lifetime distribution function can be recovered from variable frequency phase fluorometer data [ 81. This distribution may reflect the influence of the microenvironment of each molecule.

The phase shift method may also be combined with single-photon detection as reported previously [ 91.

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Streak Tube TV Camera

DmPlay Processor

Fig. 5 Basw set-up of a streak camera system. ES, entrance slit; PC, photocathode; AM, accelerator mesh, Defl, electron beam deflectlon plate; MCP, mlcrochannel plate; PS, phosphor screen.

2.1.3. Streak camera The streak camera registers an ultrafast light signal directly in real time

as opposed to the techniques described above. Figure 5 shows a block diagram of a streak camera. The slit image of the incident light is focused onto the photocathode and converted into an electron beam. Electrons are accelerated by the field between the photocathode and a mesh and then swept at high speed across a microchannel plate. After amplification by the microchannel plate the electrons impinge on the phosphor screen and form an optical Image. The time-varying light signal is thus transformed into a spatial variation at the image plane. In modem instruments [lo] the image is recorded by a TV system and adequately processed. The result is then the graphical display of the temporal intensity profile of the incident light. In addition, digitized data are also available for further evaluation. The time resolution of streak camera systems has recently been extended into the femtosecond range [ll] but it is still difficult to achieve the high resolution of less than 10 ps and a long recording duration (> 5 ns) at the same time.

A particularly useful aspect of streak cameras is the possibility of simultaneous recording of a second parameter in addition to time. A spectro- graph placed between.the sample and the entrance slit of the camera can be oriented m such a way that the spectrum occurs along the slit axis. The two-dimensional streak image then displays wavelength dependency perpen- dicular to the time direction. The complete time-resolved spectrum is obtained either by one shot or by addition of streak images after multiple- shot excitation (“synchroscan streak camera” [IO]). An application of this technique is described in ref. 12. Other arrangements may be chosen for simultaneous recording of spatial and temporal variations.

The streak camera exhibits the highest temporal performance of the three methods described; it is versatile but it is also a very expensive instru- ment.

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2.2. Light sources Single-photon counting methods require pulsed excitation sources with

repetition rates from 10 kHz to 100 MHz with a high degree of reproducibili- ty of pulse shape and intensity. The pulse width contributes directly to the width of the instrumental response function and ought to be as short as pos- sible. Tunability over a broad spectral range clearly increases the versatility of the light source. High pulse intensity is desirable if the excitation beam has to be guided through various optical elements, e.g. lenses, filters, polarizers, pin-holes etc. There is no ideal source which covers all require- ments and the appropriate choice depends on the specific application.

Three light sources are generally used in single-photon counting: (a) flashlamp; (b) synchrotron radiation; (c) mode-locked laser.

2.2.1. Flash lamps The most compact sources are flash lamps. They provide a broad

spectral output and are relatively cheap, but for a long time they suffered from poor stability making deconvolution of subnanosecond lifetimes virtually impossible. Several attempts have therefore been made to improve the performance of flash lamps. In up-to-date commercial equipment, low pressure lamps gated by thyratrons are usually employed. These excitation sources are now very stable over periods of several hours at rates of 20 kHz or more, providing a pulse width of 1 ns or less [ 131. As a filler gas nitrogen or hydrogen is used. Low pressure lamps have been applied successfully to routine measurements of lifetimes down to 200 ps.

2.2.2. Synchrotron radiation Synchrotron radiation is generated by radial acceleration of electrons

at high energy. It was originally an unwelcomed byproduct of circular elec- tron accelerators, as it causes an energy leak in such machines. But spec- troscopists soon began to appreciate the unique properties of synchrotron radiation. It possesses a very broad spectra continuum from the X-ray region to 10 pm. Most of the energy is confined within a narrow cone of 0.2 mrad pointing from a location of the electron orbit along the tangent. Thus a small bunch of electrons can produce a flash of less than 1 ns duration on a target. The repetition rate can be several 100 MHz. Storage rings which are especial- ly designed as synchrotron radiation factories produce highly stable pulses over many hours of operation and provide a nearly ideal excitation source for time-resolved spectroscopy. Because synchrotron or storage ring facilities are accessible only to a few research workers we will not discuss further details. The reader is referred to the literature [ 141.

2.2.3. Mode-locked lasers Mode-locked lasers are the state-of-the-art with respect to the genera-

tion of ultrashort light pulses. Several types of mode-locked lasers have been developed. In connection with single-photon counting, synchronously mode- locked (SML) dye lasers are usually employed. SML dye lasers actually

8

consist of a tandem arrangement of two lasers, an actively mode-locked gas laser and a cw dye laser. The extremely short dye laser pulses (minimum pulse duration several femtoseconds) are generated by gain modulation from the pumping gas laser. The most critical parameter of this system is the cavity length of the dye laser resonator which must be tuned such that a dye laser pulse on its round trip within the cavity arrives precisely at that particu- lar time at the dye jet when the pump laser pulse has built up maximum inversion. The adjustment of SML dye lasers is therefore very delicate and it takes several hours of warming up time for thermal balance of the complete laser system to be reached.

The natural pulse repetition rate of SML dye lasers, 50 - 80 MHz, is often too high for the excitation of samples with long overall deactivation time. The insertion of cavity dumpers, based on acousto-optic light switches reduces the repetition rate to the value desired. SML dye lasers provide only a restricted tunability over a range of approximately 50 nm if a certain dye is used. A dye change requires a tedious realignment procedure or even com- plete replacement of the resonator elements. The employment of such costly instrumentation can only be justified if ultimate time resolution is needed.

Phase fluorometry can be performed either with repetitively pulsed light sources as described above which can be regarded as intrinsically modulated sources or with cw light sources and suitable optical modulators.

3. Recent applications

3.1. Fluorescent biomolecules Fluorescent molecules occurring in cells and tissues include amino

acids, nucleic acids and certain metabolites (e.g. porphyrins, coenzymes and plant pigments). In addition, fluorescent dyes are being used for selective staining of intracellular parts or organelles. For both intrinsic fluorescence and staining dyes, time-resolved fluorescence spectroscopy is a powerful method of differentiation. Thus DNA constituents were recently differen- tiated on the basis of their picosecond lifetimes [ 151. In addition, remark- able results were reported for acridine dyes bound to DNA, since their fluorescence lifetimes depend significantly on whether they are intercalated between two adenine-thymine (AT) base pairs, two guanine-cytosine (GC) pairs or in a GC-AT sequence [16, 171. This does not only allow for deter- mination of the DNA base pair composition but also indicates an energy transfer from the a&dine molecules intercalating the AT-AT sequences to molecules intercalated adjacent to a guanine residue [ 161.

Intrinsic fluorescence of amino acids was reported mainly for trypto- phan and tyrosine [ 181. However, their lifetimes depend significantly on the micro-environment and also vary between different ionic species and con- formations (see, for example, ref. 19). This situation is even more complex for multiple-tryptophan-contaming proteins, showing a superposition of life- times between less than 1 ns and about 8 ns. Different lifetimes of tryptophan were also attributed to different energy transfer efficiencies from tryptophan

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to adjacent molecular groups, e.g. NADH or other coenzymes as well as heme groups in different forms of hemoglobin [20]. Taking into account the fact that tryptophan fluorescence in proteins is often composed by a large number of components, the usual two- or three-exponential curve fitting was recently replaced by a measurement of continuous distributions of ex- ponentially decaying components [21] using the method of phase shift fluorometry.

Time-resolved anisotropy studies, an appropriate method for measuring rotational properties of molecules, were also reported for tryptophan- containing proteins [18]. In this case the time course of fluorescence intensi- ty was measured parallel and perpendicular to the polarization of the excitation beam. After the so-called rotational correlation time the orienta- tions of preferentially aligned excited molecules are randomized by Brow- man motion. Two correlation times (0.14 ns and 14 ns) were reported for tryptophan-containing enzymes and attributed to local tryptophan motion and rotation of the entire protein respectively [22]. An apparatus for simultaneous measurement of two polarized fluorescence decays, together with the excitation pulse, has been described in ref. 23.

Time-resolved measurements of fluorescence and its polarization were also reported for micellar lipid phases and natural membranes [24]. In this case fluorescent probes were often used to provide information on bilayer structures, molecular rotation or the presence of certain fluorescence quenchers, such as ubiquinone or other coenzymes. 1-Anilinonaphthalene-8- sulphonate (ANS) [24] is commonly used as a fluorescent probe in mem- brane studies. This is mainly due to its low quantum yield in water and the much higher yield in non-polar environments. Therefore only membrane- bound molecules contribute significantly to the observed fluorescence signal. In addition, small changes within the lipid structure accompanied by varia- tions in polarity affect the fluorescence lifetime of ANS or N-phenyl-l- naphthylamine, another fluorescent probe for membranes.

Intermolecular distances and the localization of different fluorescent components in membranes can be probed by energy transfer measurements. Studies have been reported on energy transfer from tryptophan groups of proteins to bound ANS molecules and, more recently, from donor to acceptor dye molecules. The combination of energy transfer microscopy [25] with time-resolving techniques appears rather promising for this pur- pose.

3.2. Immunofluorescence Fluorescence staining has become a well-known technique for detecting

antigens or antibodies in solution, cells and tissues. An even more sensitive technique used in the clinical laboratory is based on linking antigens or anti- bodies to specific enzymes which typically produce lo4 - 10’ fluorescent molecules per minute. A large factor of multiplication is therefore attained when antibody concentrations are to be determined from these so-called enzyme-linked immunosorbent assays (ELISA).

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However, time-resolving detection methods were only introduced to immunology after development of antibody-coupled fluorophores with sufficiently long time constants in the range 10 - 1000 ps. By using chelates of europium and other rare earth metals as long-lived fluorescent probes [ 261, scattered light and short-lived background luminescence could be sup- pressed. This technique of background discrimination by setting an ap- propriate “time window” became more complicated when the lifetimes of the fluorescent probes were in the nanosecond range. This is the case for most fluorophores used in ELISA tests. In one of those tests, enzymatically produced 4-methylumbelliferone with a lifetime of about 2 ns was differen- tiated from stray light and fluorescent background using the single-photon counting method [27]. The detection limit could then be lowered by two orders of magnitude (as compared with conventional tests) to about lo-i5 mol of fluorescent product, corresponding to 200 - 500 enzyme molecules, after 40 min of reaction time. This limit was caused by non-specificity in the immune reaction and could be lowered further by using monoclonal anti- bodies.

3.3. Biotechnology Immunofluorometric methods have also become important in biotech-

nology, since antibodies against a broad spectrum of substances including proteins and bacteria have become available. However, fluorescence monitoring in biotechnology has so far been concentrated on “natural” fluorescent products, such as NADH or other coenzymes in cultures of microorganisms or bioreactors [28, 291. In addition changes in the fluo- rescence yield of certain hydrocarbons caused by the presence of oxygen or other metabolites are utilized in optical sensors [ 301.

Little work has so far been done in applying the methods of time- resolved fluorescence to the field of biotechnology. In cultures of metha- nogenic bacteria certain coenzymes were differentiated on the basis of their fluorescence lifetimes [31]. Moreover, a selective photobleaching effect of

. . the specific coenzyme Fez,, was measured and found to depend on the metabolic activity of these methane-producing bacteria. A quantification of bacterial activity by fluorescence measurements has so far been impossible owing to a superposition of the bacterial fluorescence by fluorescence from cell-free coenzymes, which accumulate in the culture medium. By use of time-resolved detection, however, different fluorescence lifetimes were recently obtained for intracellular (1.0 ns) and extracellular (2.5 ns) com- ponents of coenzyme F 420 (Fig. 6 [32]). A combmation of these decay time measurements with the detection of photobleaching is therefore proposed, not only for monitoring bacterial growth in culture media but also for surveying the bacterial activities in bioreactors or fermenters used for example for waste water cleaning or biogas production.

3.4. Photosynthesis Light incident on a green plant is absorbed by photosynthetic pig-

ments, mainly chlorophyll molecules, which are embedded m the thylacoid

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Time (ns)

Fig. 6. Time-resolved fluorescence tram microscopic samples of methanogemc bacteria, detection range 420 - 570 nm. (a) Intracellular fluorescence (b) Extracellular fluo- rescence. Roth curves show some overlap by longer-lived background luminescence. Re- produced from ref. 32

membranes of specific cell organelles called chloroplasts. Healthy plants convert the greater part of the excitation energy, some 80%, into chemical energy. The remainder is lost as heat or it is radiated as fluorescence. The emission of light is connected to processes involving the primary steps of energy and charge transfer. Picosecond fluorescence spectroscopy is there- fore an invaluable tool for analysing these mechanisms. It may also serve as an indicator for malfunction and damages of the photosynthetic apparatus.

Numerous studies performed mainly on algae, intact chloroplasts and chloroplast fragments with mode-locked laser excitation and sensitive single- photon detection give a picture of the photosynthetic kinetics [33 - 361. The model of the photosynthetic membrane which is currently most widely accepted includes two units: (a) photosystem I (PS I) with the reaction centre PTO,, (absorbance peak at 700 nm) ; (b) photosystem II (PS II) equip- ped with a reaction centre PdsO (absorbance peak at 680 nm), a pigment complex called antenna and a more loosely associated light-harvesting chlorophyll complex (LHCP). A modification of this model has been recent- ly proposed in which PS II appears in two forms, PS II, and PS I$ [36, 371.

After excitation is routed from the LHCP through the antenna to the reaction centre of PS II, charge separation takes place. The first steps are

P 6a,*PhQ - P68,,+Ph-Q - P,,+PhQ-

where Ph is the primary electron acceptor (pheophytin) and Q is the second- ary electron acceptor.

Fluorescence occurs as a reaction accompanying the energy transfer in the LHCP or as a result of the back transfer of energy from blocked reaction centres.

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There is still no complete agreement on the contribution of each unit to the fast fluorescence kinetics. At least three groups of components have been resolved where each component varies with the initial oxidation state of the secondary acceptor Q of PS II.

(a) Fast components with decay times from 80 to 180 ps with con- tributions probably from both PS I and PS II [ 331.

(b) Middle components ranging from 0.5 to 1.2 ns, which have been assigned to PS II at various oxidation states [33].

(c) Slow components from 1 to 2.6 ns. Some workers attribute these components to the recombination of the primarily formed radical pair Pdso+Ph- [38] h 1 th w i e 0 ers argue in favour of fluorescence originating from free or “dead” chlorophyll.

Only very few picosecond experiments have been carried out with whole mtact leaves or plants. Senorer [39] has studied the fluorescence of leaves of maize and spruce using a two-beam method. One beam provides subnanosecond excitation pulses at 435 nm while a strong cw light at 633 nm is used to induce variations in the fluorescence yield. The strong light- induced fluorescence was found to be monoexponential (t = 2 ns) and was explained m terms of recombmation processes. Schneckenburger et al. [40] investigated needles from pines and spruces exposed to environmental pollutants and partly infected by fungi. It was found that fluorescence intensity of the middle component with a decay time of 0.55 ns decreased sigmficantly whereas the fast component with 0.11 ns remained nearly constant if the needles were both mfected and exposed to high doses of ozone (Fig. 7). This might indicate a selective inhibition of PS II by a com- bination of a fungal infection and high ozone doses.

Results from picosecond fluorescence measurements appear to be very promising, to the extent that further advances in understanding of primary processes of photosynthesis and damagmg mechanisms of the photosynthetic apparatus may be expected.

3.5. Photosensitization Photosensitization is a field of apphcation of time-resolved fluorescence

spectroscopy which is expanding rapidly. Numerous photosensitizing dyes such as acridines, coumarins and, in particular, porphyrin-related compounds have been studied. During the past few years most of the interest has been centred on hematoporphyrin derivative (Hpd) and the more pure versions Photofrin II [41] or Photosan, which show tumour-localizing properties and are potential candidates for a photodynamic therapy of cancer. However, these substances were revealed to be complex mixtures of porphyrin mono- mers, “dimers” (mamly dihematoporphyrin ether and ester, DHE) and aggregates, and the most tumour-localizing components were not identical with the best photosensitizers.

To differentiate between these components, time-resolved fluorescence spectroscopy proved to be very helpful. At first Andreoni et al. [42] mea- sured hfetimes of about 16 ns and 4 ns in different solutions and attributed

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Decay times

7, = 120 ps

72 1 550 ps T3 II700 ps

‘-0i3-

r K f06- Y zl

wtthout fungal InfectIon

ZOb-

4 i= 0 2-*,*<__fi. ngal lnfectlon

I. ,. 1 *.L<m

0 i- I I I

0 200 400 600 WA TIME IPS)

Frg 7. Fluorescence decay curves of chlorophyll wlthm needles of spruce after 2 months of exposure to 100 l.(g mV3 ozone during 9 hours per day Upper curve, needle without infection, lower curve, needle Infected by Rhizosphaera kalkhoffii. The infected needle shows a relatively low intensity contrlbutlon of the component decaying with r2 = 550 ps (about 20% as compared with 70% for non-infected needles) Excltatlon wavelength 420 nm, detectron range 580 - 800 nm. Reproduced from ref. 40

them to monomeric and dimeric porphyrin species respectively. A more detailed analysis gave values of 14 ns, 2.7 ns and 0.7 ns for porphyrin solu- tions containing varrous concentrations of micelles [43]. Some different values were obtained from single cells [ 44, 451, and different intracellular distributions and retentions were measured for components decaying with time constants of 11 ns and 1.7 ns respectively [45].

Using streak camera detection, time-resolved fluorescence measure- ments were extended to the picosecond range. Time constants of about 90 ps [40] and 120 ps [12] were detected, but they could not be measured simultaneously with lqng-lived components owing to the limited time axis of the streak camera. Only after optimization of the single-photon technique were three components 7. = 0.14 ns, 71 = 1.7 ns and r2 = 11.7 ns measured simultaneously for porphyrin solutions. Figure 8 demonstrates that tri- exponential curve fitting gives a good correlation with the experimental data, whereas the corresponding curves for intracellular porphyrins are bi- exponential (Fig. 9). According to a previous discussion [46] an attribution of TV, r1 and r2 to aggregated, “dimeric” (DHE) and monomeric porphyrin species respectively appears reasonable and indicates that aggregated species are monomerized or dimerized when taken up by cells.

Preliminary time-resolved fluorescence measurements in uiuo were reported recently [46]. Further experiments of this kind are expected to

MI s. = 1.00 ny = 0.160 ns 7lN,= 167 ns Ii+ = 11.7 “S

‘2. MI so. = 1.m Ml, = 2.w “S

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1.5 20 25 0 0 0.5 1.0 I 5 2 0 2 5 x l&l IllIf Ins) x IL&I

i.802

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Fig. 8 Fluorescence decay of a HpD solutron, Photosan m methanol-water, 100 mg 1-l ; excitation 422 nm, detection range 620 - 685 nm. Upper curves experrmental data and triexponential fit with components TO = 0 16 ns, r1 = 1.7 ns and 72 = 11 7 ns, vertical axrs, counts Lower curve* werghted residuals, vertical axis, umts of standard deviation.

Frg 9 Fluorescence decay of Photosan m a tumour cell (QS 24). Upper curves experl- mental data and blexponentlal fit with components 71 = 2.4 ns and 72 = 10 6 ns Other data, same as m Frg. 8.

give more information about the uptake, retention, deaggregation etc. of individual porphyrin components in tumours and tumour-free tissues and to clarify the mechanisms of tumour-selective accumulation.

In addition, it will be possible to characterize some “new” photo- sensitizers on the basis of their fluorescence lifetimes. Of particular interest are porphyrin-like substances such as phthalocyanines [47] and chlorins [48] with higher absorption coefficients in the red part of the spectrum than hematoporphyrin derivatives. These substances can be excited more effi- ciently in tissues at penetration depths of some millimetres. Time-resolved in vitro measurements of porphyrins and chlorins were reported by Ridder and Wabnitz [49], who differentiated between the emission spectra of a short-lived and a longer-lived component, using a gated single-photon counting technique.

In contrast to porphyrins and related compounds which are located at different intracellular sites, furocoumarins are linked selectively to DNA, where they form photoproducts after UV excitation. For a further charac- terization of some angular furocoumarins their lifetimes were studied by Andreoni et al. [ 501.

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Endogeneous photosensitizers occurring in metabolism include several coenzymes, porphyrins, bile pigments and ocular pigments. In ophthal- mology lipofuscin was recently discriminated from background luminescence on the basis of its fluorescence lifetime [51]. As hpofuscin is a “waste product” occurring during aging of the eye, the time-resolved detection method may be very helpful in identifying the high risk population in senile macular degeneration.

Besides photosensitizers, the acceptors of excitation energy are of par- ticular interest. It is generally believed that an energy transfer from the excited triplet state of porphyrins to oxygen molecules takes place, thus producmg cytotoxic singlet oxygen ( ‘02). ‘02 can be identified by its luminescence at 1270 nm. All time-resolved measurements reported so far are baaed on pulsed laser excitation and detection by a germanium diode [52 - 541. The range of decay times was between approximately 3 ps m water [55] and 220 JLS in highly deuterated solutions [ 561. Such different lifetimes reflect very different quantum yields of ‘02 luminescence. This also indicates the problem of its in uiuo detection, e.g. as a measure of photo- sensitization of tumours. Nevertheless, first measurements of singlet oxygen luminescence in murine tumours were recently reported [ 571, thus encouraging further investigations of this kind. As an alternative method “time-resolved thermal lensing” [56] was introduced to measure the non- radiative transitions of ‘02 by heat production and changes in the refractive index of test solutions. But it remams uncertain whether this method can be applied to an in uiuo model.

4. Future aspects

In the near future an increasing number of fluorophores, pigments and photosensitizers will be investigated by time-resolving methods. Of particular interest are those measuring devices which combine high temporal and spatial resolution to get an insight into cellular and subcellular systems. Time-resolved fluorescence microscopy with single-photon detection already functions well as a method [16, 27, 44, 581. A combination of microscopic techniques with streak camera detection was used for picosecond spectros- copy of chlorophyll and porphyrins in conifers and cultured cells [40]. This measurmg device, however, is very complex, sensitive against vibrations and expensive. A more compact and low cost alternative may be a newly developed optical sampling oscilloscope [59] used as a picosecond detector in fluorescence microscopy. This sampling oscilloscope is based on streak camera technology, but instead of measurmg a temporally and spatially resolved streak image, the time profile is “sampled” point per point and detected by a photomultiplier tube.

The most severe limitation of the single-photon-counting technique is caused by the lack of IR sensitivity of photomultipliers. Many interesting problems, like the detection of singlet oxygen discussed above, require the

16

registration of weak luminescence in the IR spectral range above 900 nm. Advanced semiconductor devices developed mainly for fibre communication in the 0.8 - 2 I.trn range such as silicon or germanium avalanche diodes cannot detect single photon events owing to their low internal amplification (20 - 100). Much larger amplification factors (>lOOOO) can be achieved if semi- conductor diodes are biased above breakdown voltage. A self-sustaining avalanche current burst can then be triggered by the absorption of a single photon. Such a device is called a single-photon avalanche diode (SPAD). Practical applications of such devices have been prevented mainly by problems in terminating the avalanche and large dark count rates.

An active quenching method and an improved pn-junction geometry devised by Cova and coworkers [60, 611 have opened the way to a broad field of applications. With silicon SPADs they obtained at 833 nm an instrumental response of 60 ps which is comparable with that of a fast MCP. Dark count rates between 10 and 1000 pulses per second have been re- ported. The spectral limit is 1 pm; however, this is at the expense of tem- poral resolution and quantum efficiency. Germanium SPADs could extend the spectral range to 1.5 pm [ 621. Recently a silicon device capable of single- photon detection from 0.4 to 28 pm has been described [63]. The amplifica- tion process is based on impurity-impact ionization. The operating condi- tions (6 - 10 K) and the risetime of 50 ns are not yet satisfactory for general applications.

One main problem with practical work using biological samples is the small detector area (diameter about 10 pm) of solid state devices. However, in connection with fibre-optic arrangements and semiconductor radiation sources munature fluorescence spectrometers can be envisaged. Pulsed semi- conductor lasers have already been incorporated mto time-resolved IR fluorometry [ 641. Such compact devices offer many potential applications in the near future.

Acknowledgment

A purified version of HpD (Photosan) was kindly put at our disposal by Professor H. Mtiller van der Haegen.

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