STREAK CAMERA MEASUREMENT OF TRYPTOPHAN AND RHODAMINE MOTIONS WITH PICOSECOND TIME RESOLUTION

We have used a signal averaging laser (30 ps pulse)-streak camera system to measure fluorescence anisotropy decays of dyes in solution and protein tryptophan chromophores with picosecond time resolution. Emission polarization as a function of time can be measured directly and simultaneously for both polarization directions and stored in an optical multichannel analyzer. The corrected anisotropy is computed after background subtraction. Rotations of free dyes in solution (Sulforhodamine 101, tryptophan) and the temperature-dependent internal motions of human serum albumin (tryptophan emission) are displayed.     

PICOSECOND TIME RESOLVED FLUORESCENCE OF CHLOROPHYLL IN VIVO

Abstract. The published data concerning the fluorescence kinetics of chlorophyll a in various photosynthetic species are reviewed. The effects of singlet-singlet and singlet-triplet annihilation induced by excessively high incident light intensities are discussed and related to the changes produced in the fluorescence lifetimes and quantum yields. We also review the fluorescence lifetimes of Chlorella pyrenoidosa and spinach chloroplast fragments under a variety of experimental conditions; these measurements were performed at single pulse excitation intensities of less than 5 × 10¹³ photons cm^–2 where distortion due to annihilation processes is negligible. Evidence for and against a time dependent rate equation for energy migration will be discussed with reference to the authors' work on in vitro systems.     

PICOSECOND FLUORESCENCE FROM PHYCOCYANIN 612

The biliprotein, phycocyanin 612, was purified from a cryptomonad, Hemiselmis virescens. The protein, which is an α₂β₂ dimer having four spectrally different tetrapyrrole chromophores, was studied using picosecond fluorescence by exciting the various chromophores at three wavelengths, 565, 585 and 615 nm. These wavelengths were chosen to excite selectively the three highest energy chromophores. Decay times were measured as the excitation energy migrated from each of the three excited chromophores to the lowest-energy chromophore. The ps decay times were found to be 9, 13, and 12 ps for excitations at 565, 585, and 615 nm, respectively. A comparison is made between phycocyanin 612 and phycocyanin 645 with regard to the causes of their differing absorption maxima.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

PICOSECOND FLUORESCENCE DECAY TIME MEASUREMENTS OF NUCLEIC ACIDS AT ROOM TEMPERATURE IN AQUEOUS SOLUTION

Abstract— We report the room-temperature fluorescence decay times of calf thymus DNA when native and when 16% of its guanine residues are methylated at theN–7 position. The samples were excited with single, 25 ps, 266 nm laser pulses from a frequency-quadrupled Nd: YAG laser. Fluorescence was detected with a streak camera-optical multichannel analyzer system that has a time jitter of about 2 ps. For DNA and methylated DNA we detected a major component that has a decay time of about 10 and 20 ps, respectively. A second component has a corresponding decay time of about 65 and 80 ps and makes a contribution of0–10% and20–40% depending on the transmission characteristics of the emission filter employed. In contrast, the decay time of 7-methyl GMP, which contains the same fluorophore as methylated DNA, is approximately single exponential and has a decay time of180–210 ps depending on the emission filter. The absence of a pronounced time delay between the fluorescence decay profiles of the nucleic acids and the exciting light pulse points against the formation of excited-state complexes (excimers).     

PICOSECOND FLUORESCENCE KINETICS OF N-ALL-TRANS-RETINYLIDENE-AT-BUTYLAMINE

Fluorescence lifetimes are reported for N-all- trans -retinylidene- n -butylamine in hexane. Picosecond resolution was obtained by excitation with a single 355-nm pulse, 25 ps FWHM, from a passive mode-locked YAG-laser and detection with a high speed streak camera. The lifetime dependence on temperature and the spectral characteristics support a vibrational-torsional deactivation model of the singlet excited state of two different conformers. Additional data in decalin are consistent with this model, while the quenching in alcoholic solutions may be supported by the solvent reorientation.     

PICOSECOND FLUORESCENCE AND ELECTRON TRANSFER IN PRIMARY PHOTOSYNTHETIC PROCESSES

Abstract. Under conditions that drive the reaction centers (RC's) into the "closed" state, the lifetime ( T ) of the fluorescence emitted by antenna molecules increases from 80 to 200 ps in PS I, from 300 to 600 ps in PS II, and from 200 to 500 ps in bacterial chromatophores. In Rhodopseudomonas sphaeroides strain 1760-1, the decay curve for fluorescence from the RC's has a component with T ₂= 15 ps due to the bacteriochlorophyll of the RC, and a second component with T ₂= 250 ps due to bacteriopheophytin.
Data on electron transfer at low temperatures and under different redox conditions are analyzed. along with the ps fluorescence kinetics. The hypothesis is discussed that electron transfer in RC's is coupled to conformation changes in the interacting molecules.     

PICOSECOND FLUORESCENCE KINETICS IN SPINACH CHLOROPLASTS AT LOW TEMPERATURE

Abstract— At 77 K the fluorescence from spinach chloroplasts excited using picosecond mode-locked laser pulses at 620 nm is made up of 5 separate kinetic components. Three of these are predominant at short wavelengths. between 650 and 690 nm, and they appear to correspond to the 3 decay phases seen at room temperature. The 2 new components. a 100 ps rise and a 3000 ps decay, characterize the longer (730–770 nm) wavelength fluorescence. The temperature dependence of the kinetic components of the long wavelength fluorescence shows that the 3000 ps decay accounts for essentially all of the large increase in fluorescence yield observed at low temperature. Furthermore, it appears that this increase does not result entirely from an increase in the fluorescence lifetime, as has been proposed. The dependences of these 2 new components (the 100 ps rise and 3000 ps decay) on emission wavelength and temperature are similar enough to suggest they have a common origin, presumably the chlorophyll pigment component C705. The amplitudes (yield/lifetime) of these 2 phases are approximately equal, and they are opposite in sign. Thus. we see evidence of time-resolved excitation transfer from those pigment molecules that absorb the 620 nm radiation to those that give rise to the long wavelength fluorescence at low temperature.     

ENERGY TRANSFER IN TRIMERIC C-PHYCOCYANIN STUDIED BY PICOSECOND FLUORESCENCE KINETICS

Abstract— The excited state kinetics of trimeric C-phycocyanin from Mastigocladus laminosus has been measured as a function of the emission and excitation wavelength by the single-photon timing technique with picosecond resolution and simultaneous data analysis. A fast decay component of 22 ps (C-phycocyanin with linker peptides) and 36 ps (C-phycocyanin lacking linker peptides) is attributed to efficient energy transfer from sensitizing to fluorescing chromophores. At long detection wavelengths the fast decay components are found to turn into a rise term. This finding further corroborates the concept of intramolecular energy transfer. Previous reports on the conformational heterogeneity of the chromophores and/or proteins in C-phycocyanin are confirmed. Our data also provide indications for the importance of the uncoloured linker peptides for this heterogeneity.     

PICOSECOND FLUORESCENCE STUDIES OF P700-ENRICHED PARTICLES OF SPINACH CHLOROPLASTS

Dynamic properties of the picosecond fluorescence of highly enriched reaction-center particles of photosystem I (8 - 10 chlorophylls/P700) prepared from spinach have been investigated. The number (N) of photons used to excite chlorophyll molecules per reaction center was controlled between 0.06 and 80. The 1/e lifetime was ca. 25 ps for N 1. which is much shorter than previously measured lifetimes of photosystem I particles. The initial fluorescence intensity saturated at higher excitation intensities (N ≲ 1). This was interpreted in terms of interaction and annihilation among excited chlorophyll molecules which occur almost entirely within the duration of a laser flash. The spectrum-resolved fluorescence decay was faster at 690 than at 680 nm. This implies that two kinds of antenna chlorophylls, apart from and in close proximity to P700, have different lifetimes. Upon heat treatment a component with a much longer fluorescence decay time was observed. The growth of this component upon heat treatment at increasing temperatures showed a correlation with a decrease in the amount of P700 that could be photooxidized.     

USE OF A PULSED LASER DIODE TO MEASURE PICOSECOND FLUORESCENCE LIFETIMES

Abstract— The use of an inexpensive pulsed laser diode (Hamamatsu picosecond light pulser PLP-01) as the excitation source for a single photon timing spectrolluorimeter with microchannel plate photomultiplier detection was dem-onstrated. The performance of the instrument was tested with two very short-lived fluorescent dyes and two pho-tosynthetic systems with wcll-defined decay characteristics. Individual fluorescence decays were analyzed by modeling with a convolution of the instrument response function to a sum of exponential decay components. Accurate fluorcscence lifetimcs of the dyes cryptocyanine (55 ps in acetone and 83 ps in ethanol) and 1,1'-diethyl-2,2'-dicarbocyanine iodide (13 ps in acetone and 26 ps in ethanol) were obtained by analysis of the decay kinetics with a single exponential component. Fits to the fluorescence decay kinetics of isolated photosystem I particles and intact cyanobacterial cells required three and four decay components. respectively. The decay kinetics of the isolated photosystem I preparation were dominated (99%) by a very fast 9 ps lifetime, reflecting the preparation's small antenna size of approximately 30 chlorophyll a . The cyanobackria showed decay components of 35 ps, 160 ps, 400 ps and 1.95 ns similar to those described previously by Mullincaux and Holzwarth ( Rinchim. Biophys. Acfa 1098 , 68–78, 1991). The performance of the pulsed laser diode as an excitation source for single photon timing is discussed in comparison with conventional sources of picosecond light pulses.     

FLUORESCENCE QUENCHING FOR FLAVINS INTERACTING WITH EGG WHITE RIBOFLAVIN-BINDING PROTEIN

Abstract— The effect of flavin structure variation upon the binding process of flavin to hen egg white riboflavin was studied using fluorescence methods for formylmethylflavin (FMF), riboflavin (RF) and flavomononucleotide (FMN).
Measurements of flavin fluorescence intensities (steady state and phase-sensitive) and lifetimes were performed in a variety of RBP concentrations and temperatures (4 to 40°C). No fluorescence of flavoproteins was detected, while the fluorescence of flavins was found to be quenched by RBP. The overall quenching process is dominated by the static quenching (about 88%) due to the flavoprotein complex formation in the ground state, presumably a charge transfer complex.     

PICOSECOND FLUORESCENCE OF R3230AC MAMMARY CARCINOMA MITOCHONDRIA AFTER TREATMENT WITH HEMATOPORPHYRIN DERIVATIVE and PHOTOFRIN® II in vivo

Hematoporphyrin derivative (HPD) and other porphyrin samples were excited by 20-ps 532-nm laser pulses. Fluorescence was detected using a low-jitter streak camera. Data were fitted to a sum of exponential decay times on the order of picoseconds. Fluorescence of porphyrins in aqueous solution show various behaviors depending on the hydrophobicity of the porphyrins. The most hydrophilic porphyrins show long decays only (greater than 500 ps). Porphyrins intermediate in hydrophobicity have intensity-dependent fast decays. The most hydrophobic have fast decays (less than 20 ps). Picosecond fluorescences of mitochondria prepared from rat tumors treated in vivo with HPD or Photofrin II show an increase in the ratio of fast to slow decays when compared to the injected porphyrins. These results are consistent with the concentration of the more hydrophobic porphyrins in mitochondria in photosensitization treatment. Thus picosecond fluorescence studies of porphyrins may provide a means to obtain photoproperties which differentiate between effective and ineffective in vivo photosensitizers.     

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild-type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild-type C. reinhardii fluorescence decay data when the absorption cross-sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild-type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.     

TEMPERATURE DEPENDENCE OF PICOSECOND FLUORESCENCE KINETICS OF A CYANOBACTERIAL PHOTOSYSTEM I PARTICLE*

Picosecond time-resolved fluorescence of photosystem I particles isolated from Synechococcus sp. was recorded in the wavelength range from 680 nm to 736 nm for temperatures of 6°C to 42°C and - 100°C using the single-photon-timing technique. By global analysis of the data we found four contributing lifetime components at the higher temperatures (T₁ ' 12 ps, T₁= 35 ps, T₃ ' 65 ps, T₄ ' 1000 ps). We attribute T₁ to an energy transfer between two pigment pools, T₂ to the charge separation process in the reaction center, component T₃ is assigned to aggregate and T₄ to uncoupled chlorophyll emission. The corresponding decay-associated spectra are presented. We also applied a target analysis procedure to fit parameters of a kinetic model directly to the data. The resulting rate constants and species-associated spectra are discussed. The data indicate substantial spectral heterogeneity in the antenna with at least three substantially different chlorophyll pools. The overall exciton decay kinetics (by charge separation) is trap-limited.     

PICOSECOND FLUORESCENCE KINETICS and ENERGY TRANSFER IN THE ANTENNA CHLOROPHYLLS OF GREEN ALGAE*

Single-photon timing measurements on flowing samples of Chlorella vulgaris and Chlamydomonas reinhardtii at low excitation intensities at room temperature indicate two main kinetic components of the fluorescence at open reaction centers (F₀) of photosystem II with lifetimes of approx. 130 and 500 ps and relative yields of about 30 and 70%. Closing the reaction centers progressively by preincubation of the algae with increasing concentrations of 3-(3′,4′-dichlorophenyl)-l,l-dimethylurea (DCMU) and hydroxylamine gave rise to a slow component with a lifetime increasing from 1.4 to 2.2 ns (F_max) The yield of the slow component increased to 65-68% of the total fluorescence yield in parallel to a decrease in the yield of the fast component to a value close to zero at the f_max-level. The 130 ps lifetime of the fast component remained unchanged. The middle component showed an increase of its lifetime from 500 to 1100 ps and of its yield by a factor of 1.5. Spacing of the ps laser pulses by 12 μs allowed us to resolve a new long-lived fluorescence component of very small amplitude which is ascribed to a small amount of chlorophyll not connected to functional antennae. The opposite dependence of the yield of the fast and the slow component on the state of the reaction centers at almost constant lifetimes is consistent with a mechanism of energy conversion in largely separately functioning photosystem II units. Yields and lifetimes of these two components are in agreement with the high quantum yield of photosynthesis. The lower lifetime limit of 1.4 ns of the slow component is assigned to the average transfer time of an excited state from a closed to a neighboring open reaction center and the increase in the lifetime to 2.2 ns is evidence for a limited energy transfer between photosystems II. Relative effects of changing the excitation wavelength from 630 to 652 nm on the relative fluorescence yields of the kinetic components were studied at the fluorescence wavelengths 682, 703 and 730 nm. Our data indicate that (i) the middle component has its fluorescence maximum at shorter wavelength than the fast component and (ii) that the antennae chlorophylls giving rise to the middle component are preferentially excited by 652 nm light. It is concluded that the middle component originates from the light-harvesting chlorophyll alb protein complexes and the major portion of the fast component from the chlorophyll a antennae of open photosystem II reaction centers.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

PICOSECOND RESOLUTION OF INDOLE ANISOTROPY DECAYS AND SPECTRAL RELAXATION BY 2 GHz FREQUENCY-DOMAIN FLUOROMETRY

Abstract— We used frequency-domain fluorescence spectroscopy to measure rotational diffusion and time-resolved emission spectra of indole in methanol on the picosecond timescale. The indole emission was quenched by acrylamide to allow measurements to the instrumental limit of 2 GHz and to eliminate emission from the longer-lived indole molecules, which can no longer provide information on the picosecond (ps) processes. The resolution was adequate to measure rotational correlation times as short as 8 ps at 80†C, and spectral relaxation times as short as 16 ps at 5†C.     

PICOSECOND FLUORESCENCE SPECTROSCOPY ON INCORPORATION PROCESSES OF HEMATOPORPHYRIN DERIVATIVE INTO MALIGNANT TUMOR CELLS in vitro

Abstract— By using a highly sensitive streak-camera technique, we investigate incorporation processes of HpD into malignant tumor m-KSA cells in vitro. The picosecond decays of the total fluorescence spectra, the wavelength-resolved fluorescence decays and the time-resolved fluorescence spectra from HpD in the cells are measured as a function of the incubation time. The results show that the aggregate component of HpD which has a fast fluorescence lifetime of 100 ps and a red-shifted band of ∼ 660 nm selectively accumulates more and more in the cells with the increase of the incubation time.     

APPARATUS FOR RECORDING LIGHT FLASH INDUCED MEMBRANE VOLTAGE TRANSIENTS WITH 10 ns RESOLUTION

Abstract— Apparatus for recording voltage waveforms resulting from flash illumination of bilayer membranes with sorbed dyes or pigments are described. Time constants of light-induced molecular events which displace electric charge in membranes can be taken directly from these waveforms. The waveforms induced across bilayer membranes by a carbocyanine dye and by chlorophyllin illustrate the apparatus performance. The risetime of the chlorophyllin induced photo-voltages are ˜10ns.