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UV radiation, desiccation, and starvation induce some cyanobacteria to produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although the accumulation of scytonemin is recognizable as dark sheaths through optical microscopes, it has been nontrivial to identify its redox status and obtain its subcellular distribution in response to physiological conditions. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of non-UV-induced scytonemins with distinct redox statuses in a filamentous cyanobacterium with a single nitrogen-fixing cell at the basal end. Cellular differentiations and scytonemin redox conditions were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The molecular imaging results give insights into not only secretion mechanisms of the sunscreen pigment but also interdependence between photosynthesis, nitrogen fixation, and redox homeostasis in one of the simplest forms of multicellular organisms.

Some cyanobacteria produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although scytonemin-containing dark sheaths are recognizable through optical microscopes and its redox changes have been known for decades, there has been no report to obtain images of both oxidized and reduced scytonemins at subcellular resolution. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of both the oxidized and reduced scytonemins in a filamentous cyanobacterium. The redox changes of scytonemin were supported by comparison in the Raman spectra between the cyanobacterial cells, solid-state scytonemin and quantum chemical normal mode analysis. Distributions of carotenoids, phycobilins, and the two redox forms of scytonemin were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The redox differentiation of scytonemin will advance our understanding of the redox homeostasis and secretion mechanisms of individual cyanobacteria as well as microscopic chemical environments in various microbial communities. The line-scanning Raman microscopy based on the 1064 nm excitation is thus a promising tool for exploring hitherto unreported Raman spectral features and distribution of nonfluorescent molecules embedded below nontransparent layers for visible light, while avoiding interference by autofluorescence.

Thylakoid membranes of young leaves consist of grana and stroma lamellae (stroma-grana [SG] structure). The SG thylakoid is gradually converted into isolated grana (IG), almost lacking the stroma lamellae during growth. This morphological alteration was found to cause a reduction in maximum photosynthetic rate and an enhancement of photoinhibition in photosystem II (PSII). In situ microspectrometric measurements of chlorophyll fluorescence in individual chloroplasts suggested an increase of the PSII/PSI ratio in IG thylakoids of mature leaves. Western blot analysis of isolated IG thylakoids showed relative increases in some PSII components, including the core protein (D1) and light-harvesting components CP24 and Lhcb2. Notably, a nonphotochemical quenching-related factor in the PSII supercomplex, PsbS, decreased by 40%. Changes in the high light response of PSII were detected through parameters of pulse-amplitude modulation fluorometry. Chlorophyll fluorescence lifetime indicated an increase of fluorescence quantum yield in IG. A minimal photodamage-repair rate analysis on a lincomycin treatment of the leaves indicated that repair rate constant of IG is slower than that of SG, while photodamage rate of IG is higher than that of SG. These results suggest that IG thylakoids are relatively sensitive to high light, which is not only due to a higher photodamage rate caused by some rearrangements of PS complexes, but also to the retarded PSII repair that may result from the lack of stroma lamellae. The IG thylakoids found among many plant species thus seem to be an adaptive form to low light environments, although their physiological roles still remain unclear.

Photosynthetic pigment-protein complexes are highly concentrated in thylakoid membranes of chloroplasts and cyanobacteria that emit strong autofluorescence (mainly 600–800 nm). In Raman scattering microscopy that enables imaging of pigment concentrations of thylakoid membranes, near infrared laser excitation at 1064 nm or visible laser excitation at 488–532 nm has been often employed in order to avoid the autofluorescence. Here we explored a new approach to Raman imaging of thylakoid membranes by using excitation wavelength of 976 nm. Two types of differentiated cells, heterocysts and vegetative cells, in two diazotrophic filamentous cyanobacteria, Anabaena variabilis, and Rivularia M-261, were characterized. Relative Raman scattering intensities of phycobilisomes of the heterocyst in comparison with the nearest vegetative cells of Rivularia remained at a significantly higher level than those of A. variabilis. It was also found that the 976 nm excitation induces photoluminescence around 1017–1175 nm from the two cyanobacteria, green alga (Parachlorella kessleri) and plant (Arabidopsis thaliana). We propose that this photoluminescence can be used as an index of concentration of chlorophyll a that has relatively small Raman scattering cross-sections. The Rivularia heterocysts that we analyzed were clearly classified into at least two subgroups based on the Chla-associated photoluminescence and carotenoid Raman bands, indicating two physiologically distinct states in the development or aging of the terminal heterocyst.

Microscopic spectral properties of a laser dye oxazine 1 in ethanol were characterized in an effort to understand anti-Stokes fluorescence upon one-photon absorption of near infrared continuous wave laser. Comparison between three excitation modes, 808 nm subpicosecond pulsed laser, 785 nm continuous wave laser, and 488 nm continuous wave laser enabled us to estimate absolute one-photon absorption-based excitation probability of singlet excited state of oxazine 1 in ethanol at 785 and 808 nm.

Heterocyst is a nitrogen-fixing cell differentiated from a cell for oxygen-evolving photosynthesis (vegetative cell) in some filamentous cyanobacteria when fixed nitrogen (e.g., ammonia and nitrate) is limited. Heterocysts appear at multiple separated positions in a single filament with an interval of 10-20 cells in some genera (including Anabaena variabilis). In other genera, a single heterocyst appears only at the basal terminal in a filament (including Rivularia M-261). Such morphological diversity may necessitate different properties of heterocysts. However, possible differences in heterocysts have largely remained unexplored due to the minority of heterocysts among major vegetative cells. Here, we have applied spectroscopic microscopy to Rivularia and A. variabilis to analyze their thylakoid membranes in individual cells. Absorption and fluorescence spectral imaging enabled us to estimate concentrations and interconnections of key photosynthetic components like photosystem I (PSI), photosystem II (PSII) and subunits of light-harvesting phycobilisome including phycocyanin (PC). The concentration of PC in heterocysts of Rivularia is far higher than that of A. variabilis. Fluorescence quantum yield of PC in Rivularia heterocysts was found to be virtually the same as those in its vegetative cells, while fluorescence quantum yield of PC in A. variabilis heterocysts was enhanced in comparison with its vegetative cells. PSI concentration in the thylakoid membranes of heterocysts seems to remain nearly the same as those of the vegetative cells in both the species. The average stoichiometric ratio between PSI monomer and PC hexamer in Rivularia heterocysts is estimated to be about 1:1.

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.

Microscopic autofluorescence spectral imaging of chloroplasts in maize mesophyll cells using near-infrared laser excitation has previously shown that a photosystem I spectral component exhibits an intensity similar to that of photosystem II at ~294 K when a continuous-wave laser at 800-820 nm is used. To establish the generality of this phenomenon, chloroplasts in Parachlorella kessleri cells (P. kessleri) were studied. A continuous-wave laser at 785 nm promoted photosystem-I-specific fluorescence in P. kessleri chloroplasts. The difference in chlorophyll fluorescence peak wavelengths between P. kessleri and maize correlated well with those observed at cryogenic temperatures. To further clarify the nature of anti-Stokes fluorescence, we studied chloroplasts in acetone-treated P. kessleri cells (in a medium containing 15% (v/v) acetone) by microscopic fluorescence and absorption spectra on a cell-by-cell basis. A continuous-wave laser at 785 nm led to significant fluorescence from acetone-treated cells, which was attributed to aggregation of chlorophylls. Anti-Stokes fluorescence spectral imaging thus seems to be effective for detection of lowest-energy trap states that are only weakly fluorescent.

Comparison of the steady-state FTIR absorption spectra of coumarin-102 (C-102) in tetrachloroethylene with added aniline of various concentrations, in neat aniline and in neat N,N-dimethylaniline (...

A brief review is given on how ultrafast laser spectroscopy has been recently applied to the research of photosynthetic reaction center, which achieves the most essential energy conversion from light to chemical (physiological) energy. Photosystem I reaction center (PSIRC) is one of the several types of photosynthetic reaction center, and it is working in plants and some photosynthetic bacteria. Light-illuminated PSIRC generates the highest reducing capability among all biological systems, which is necessary for the reduction of carbon dioxide, the most controversial greenhouse effect gas. The understanding on PSIRC has been relatively delayed due to its stoichiometric and spectroscopic complexity. Thanks to the advance of ultrafast laser spectroscopy and the finely determined crystal structure in 2001, very detailed picture is being rapidly constructed on the mechanism of the electronic excitation transfer and the primary charge separation processes among the constituent chlorophylls in PSIRC. Key words: Ultrafast laser spectroscopy, photosynthesis, electron transfer, electronic excitation transfer, chlorophyll １．はじめに 地球上の全生物は植物や光合成細菌で行われる光合成に依存している。光合成反応の本質は、太陽光を吸収した色素分子が電子励起され、その電子励起エネルギーが電荷移動のエネルギーに変換されることである。この変換を行うのが光合成反応中心と呼ばれる色素とタンパク質の複合体である。このエネルギー変換の量子効率はほとんど１００％であって、人工的光合成には未到達の高みにある。 植物の緑色（反射スペクトル）は主にクロロフィル分子（Chl, Fig. 1）の吸収スペクトルによって与えられている。反応中心では, ある特別な状態におかれたChlの電子励起状態から電荷分離は開始される。Chl が有機溶媒中で孤立している時に示す最低励起一重項状態（S

Excitation-wavelength dependence of the transient absorbance changes of photosystem I reaction center particles with a reduced number of antenna chlorophylls (16 chlorophylls/primary electron donor chlorophyll (P700)) hasbeen studied in an effort to understand the energy equilibration among the chlorophylls in the electron-transfer system. The photobleaching and stimulated-emission signals in the Q y band region of the chlorophylls upon the preferential excitation of chlorophyll spectral forms at the blue edge (662 nm) and red edge (697 nm) of the Q y band are analyzed. In the case of the red-edge excitation, spectral equilibration proceeds with a time constant of 0.34 (′0.07) ps, which is attributable to the energy equilibration between P700 and neighboring chlorophylls absorbing around 686 nm in the electron-transfer system. This equilibration seems to precede the fastest phase of the primary charge separation (apparent time constant of 0.8 ps) reported previously (Kumazaki et al. J. Phys. Chem. B 2001, 105, 1093). A slow decay of the excited states because of the slow phase of the primary charge separation proceeds with a time constant of 7.2 (′0.6) ps. In the case of the blue-edge excitation, vibrational relaxation and downhill energy transfer proceed with a time constant of 0.38 (′0.08) ps, which are followed by a slow downhill energy transfer from residual antenna chlorophylls to the electron-transfer system. Even with the slow energy transfer from the residual antenna chlorophylls to the electron-transfer system, the overall primary charge separation is completed with a time constant of 10 (′0.7) ps. These interpretations are in part supported by control experiments on chlorophyll a in ethanol under equivalent optical conditions. Implications of these results for understanding the primary processes in more intact photosystem I are discussed.

Spectral sensitization and supersensitization of the silver bromide nanocrystals were studied by the femtosecond fluorescence up-conversion technique. An annihilation-free fluorescence from J-aggregates of the Dye 1 (3,3‘-disulfopropyl-5,5‘-dichloro-9-ethylthiacarbocyanine) adsorbed on the silver bromide nanocrystals (edge-to-edge distance from 40 to 900 nm) of different shapes (cubic and octahedral) was measured. Very fast nonexponential fluorescence decays were observed with a fast component from 0.4 to 2.5 ps, and an average decay time from 1.1 to 5.5 ps, depending on the type and size of AgBr grains. The average fluorescence decay time is several times longer on the cubic grains than that on the octahedral grains. Upon addition of a supersensitizer (SS) (3,3‘-disulfopropyl-9-ethyl-4,5,4‘,5‘-dibenzothiacarbocyanine), which is coadsorbed on the surface of silver bromide grains, the fluorescence decay became several times faster with a mean decay time of 0.60−1.3 ps. Different models of supersensitizatio...

The ultrafast excitation-energy transfer of the J-aggregates on the octahedral AgBr microcrystal (0.2 μm ) is studied by time-resolved fluorescence spectroscopy. A rise component is observed with a time constant of about 0.1 ps. The fluorescence-anisotropy decays are biphasic with two time constants of ∼0.1 and 2–7 ps. The short time constant is almost independent of the dye coverage, while the second one becomes slower at higher dye coverages. An ultrafast excitation transfer (0.1 ps) is suggested to take place between epitaxially arranged J-aggregates on the {1 1 1} surface prior to the primary electron injection to AgBr (0.7 ps).

In chloroplasts of plants and algae, state transition is an important regulatory mechanism to maintain the excitation balance between PSI and PSII in the thylakoid membrane. Light-harvesting complex II (LHCII) plays a key role as the regulated energy distributor between PSI and PSII. It is widely accepted that LHCII, which is bound to PSII localized mainly in the granal thylakoid, migrates to bind with PSI localized mainly in the stroma-exposed thylakoid under preferential excitation of PSII. The phenomena have been extensively characterized by many methods. However, the exchange of LHCII between PSII and PSI has not been directly observed in vivo at physiological temperatures. Herein we applied fluorescence spectromicroscopy to Arabidopsis mesophyll protoplasts in order to observe in vivo changes in fluorescence spectra of granal and stromal thylakoid regions during the state transition. The microscopic fluorescence spectra obtained from a few sections with different depths were decomposed into PSI and PSII spectra and self-absorption effects were removed. We were able to determine amplitude changes of PSI and PSII in fluorescence spectra solely due to state transition. Subdomain analysis of granal and stromal thylakoid regions clarified variant behaviors in the different regions.

Radiationless deactivation of 2-piperidinoanthraquinone (2PAQ) in its excited singlet state induced by an intermolecular hydrogen bond interaction was studied by picosecond and femtosecond transien...

Femtosecond transient absorption spectroscopy was applied to the photosystem I reaction center particles containing only 12−13 chlorophylls per primary electron donor chlorophyll (P700), which were prepared from spinach. Upon preferential excitation of P700, an absorption band due to the excited state of P700 was newly observed in the 730−750 nm region. The primary charge separation dynamics at ≈280 K were revealed by the rise of the absorption band of the radical pair of chlorophylls. The rise was biphasic: 0.8 ps (50%) and 9 ps (50%). The 0.8 ps component is the fastest phase of the charge separation ever observed in photosystem I reaction center. On the basis of the observed dynamics and a kinetic model of excitation migration, the rate constant of the charge separation between P700 and the nearest-acceptor chlorophyll is predicted to be (0.5−0.8 ps)-1.

CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.

The primary processes in a photosystem I reaction center were studied by fluorescence up-conversion with a subpicosecond time resolution at room temperature. The samples were P700(primary donor chlorophyll)-enriched particles which retained ≈14 chlorophylls per P700. Upon selective excitation of P700 at 701 nm at ≈5 °C, anisotropy of the fluorescence at 749 nm decayed from ≈0.3 to ≈0.15 with a time constant of 1 ps. The dynamic depolarization is attributed to electronic excitation equilibration between P700 and the surrounding chlorophylls. In the isotropic fluorescence kinetics, at least two decaying components of 2.2 ps (≈35%) and 15 ps (≈55%) were found. The fast and slow components indicate the charge separation before and after full equilibration of excitation energy, respectively. A kinetic model calculation based on the above results suggests that the intrinsic rate constant of the primary electron transfer from P700* is > 0.25 ps-1.

The rate of the electron transfer reaction from the reduced primary electron acceptor chlorophyll a (A0-) to the secondary acceptor quinone (Q) was measured by picosecond−nanosecond laser spectroscopy at 280 K in the photosynthetic reaction center (RC) complex of plant photosystem I (PS I). The free energy change (ΔG0) of the reaction was varied between −1.1 and +0.2 eV by the reconstitution of 13 different quinone/quinonoid compounds after the extraction of the intrinsic phylloquinone. Phylloquinone and its natural analog menaquinone, both of which show a ΔG0 value of −0.34 eV, gave the highest rate constant (k) of (23 ps)-1. Analysis of log k versus ΔG0 plot according to the quantum mechanical electron transfer theory gave the total reorganization energy (λtotal) of 0.30 eV and the electronic coupling (V) of 14 cm-1. The natural system is shown to be highly optimized to give a ΔG0= −λtotal condition. The λtotal value is smaller and the V value is larger than those estimated in the corresponding reaction...

Transient absorption spectroscopy has been used to study sub-picosecond energy transfer processes in isolated photosystem II (PS II) reaction centers. As reported previously [Durrant, J. R.; et al. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11632−11636], using long wavelength (694 nm) excitation, spectral evolution of the isotropic Qy band bleach/stimulated emission is dominated by energy transfer processes with a 100 ± 50 fs time constant. In contrast, depolarization of this signal occurs with a time constant of 400 ± 30 fs, from an initial anisotropy of ∼0.4 to a value of ∼0.15 at 1.5 ps. This decay of the anisotropy is attributed to energy transfer between at least two degenerate states contributing to reaction center absorption circa 680 nm, with these states having approximately orthogonal transition dipoles. The transient anisotropy barely changes between 1.5 and 60 ps, indicating that under these excitation conditions equilibration of the excitation energy between reaction center excited states occurs...

Rates of sequential electron transfer reactions from the primary electron donor chlorophyll dimer (P700) to the electron acceptor chlorophyll a-686 (A[sub 0]) and to the secondary acceptor quinone (Q[sub [phi]]) are measured by picosecond absorption spectroscopy in spinach photosystem I (PS I) particles. In the particles, 85-90% of antenna chlorophylls are extracted and the intrinsic phylloquinone (Q[sub [phi]]) is removed and replaced by different quinones. (1) The excited single state of P700, monitored by the absorbance change at 695 nm, appeared with a time constant shorter than 1 ps after excitation with a 605 nm, 1 ps pulse at an intensity of 0.8 or 1.5 photons/reaction center. Indication for the absorbance change of the chlorophylls, which may function as the intermediate electron acceptor between P700 and A[sub 0] or between A[sub 0] and Q[sub [phi]], was not obtained. 45 refs., 5 figs., 1 tab.

We have investigated the ultrafast intermolecular electron transfer (ET) from an electron-donating solvent (aniline (AN) or N, N -dimethylaniline (DMA)) to an excited dye molecule (oxazines (Nile blue and oxazine 1) or coumarins). A non-exponential time dependence was observed in AN and can be explained by solvent reorientation and nuclear motion of the reactants. However, in DMA, a single exponential process was observed for Nile blue (160 fs) and oxazine 1 (280 fs), which can be explained by assuming that the rate of ET is limited mainly by ultrafast nuclear motion. A clear substituent effect on intermolecular ET was observed for the 7-aminocoumarins. When the alkyl chain on the 7-amino group is extended and a hexagonal ring with the benzene moiety is formed, the rate of ET is reduced by three orders of magnitude. This effect can be explained by a change in the free energy difference of the reaction and by the vibrational motion of the amino group.

We have measured the picosecond pump—probe spectra of BIC J aggregates using a spectral continuum for the probe. The excitation pulses were intense (1015−2×1016 photons/cm2) and created many excitons per aggregate. The observed dynamics are intensity dependent and reflect exciton—exciton annihilation processes. In addition, the zero-time spectrum is also intensity dependent, probably due to a reduction of k space at high exciton concentrations.

In this study, we produced selective images of photosystems in plant chloroplasts in situ. We used a spectroimaging microscope, equipped with a near-infrared (NIR) laser that provided light at wavelengths mainly between 800 and 830 nm, to analyze chlorophyll autofluorescence spectra and images from chloroplasts in leaves of Zea mays at room temperature. Femtosecond laser excitation of chloroplasts in mesophyll cells revealed a spectral shape that was attributable to PSII and its antenna in the centers of grana spots. We found that a continuous wave emitted by the NIR laser at a wavelength as long as 820 nm induced chlorophyll autofluorescence with a high contribution from PSI through a one-photon absorption mechanism. A spectral shape attributable to PSI and its antenna was thus obtained using continuous wave laser excitation of chloroplasts in bundle sheath cells. These highly pure spectra of photosystems were utilized for spectral decomposition at every intrachloroplast space to show distributions of PSI and PSII and their associated antenna. A new methodology using an NIR laser to detect the PSI/PSII ratio in single chloroplasts in leaves at room temperature is described.

We have constructed a laser-line-scanning semiconfocal multiphoton fluorescence microscope with a capability of simultaneous broadband spectral acquisition from a whole linear region. It was applied to the study of the thylakoid membrane of chloroplasts in a plant, Zea mays and Chlorella, all of which were studied at room temperature. In the case of Zea mays, we have determined spectral regions by which photosystem I and II including their associated antenna complexes are almost exclusively probed. The photosystem II distribution showed locally intense spots on a sub micrometer scale, resembling typical images of grana as reported by electron microscopy. The photosystem I fluorescence was rather homogeneously distributed and its image is in good agreement with bright-field images of the same chloroplast. This supports a model in which photosystem I is distributed on both stroma thylakoid and stroma-exposed thylakoid surrounding the granal thylakoid. It is thus shown that simultaneous detection of both photosystem I and photosystem II fluorescence images is possible at room temperature. Spectral images of Chlorella chloroplasts were also compared with those in Zea mays in order to understand different features of the thylakoid membranes.

The purified photosystem I (PS I) reaction center complex of a cyanobacterium Acaryochloris marina contained 88 Chl d: 1.1 Chl a: 19 carotenoids: 2.0 phylloquinone. Amino acid sequences of PsaA and PsaB polypeptides were almost homologous to those in the other cyanobacteria. The ligands for A0 was Met698A and that for A0′ was Leu688B but not Met that is conserved in all the other PS I. Laser excitation induced the 10-ps bleach and the 40- ps recovery at 680 nm of Chl a-type pigment in parallel with the 49-ps bleach of the Chl d-dimer P740 at 740 nm. The results indicate that A0 is Chl a-680 ligated by Met698A and that the PsaB branch with Leu688B is inactive for the electron transfer. The PS I of A. marina, thus, is the unique asymmetric type I reaction center with the unidirectional electron transfer pathway only through PsaA branch.

Summary We describe the construction and characterization of a laser-line-scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near-infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi-photon–induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed-loop controlled sample scanning stage and a piezo-driven objective actuator. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39–0.40, 0.33 and 0.56–0.59 μm for the X (lateral axis along the line-scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub-cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub-cellular domains. The efficiency of the new line-scanning spectromicroscope was estimated in comparison with our own point-by-point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line-scanned region, as expected.

Spectral sensitization and supersensitization of silver bromide nanocrystals are studied by the femtosecond fluorescence up-conversion technique. Fluorescence from J-aggregates of cyanine dyes adsorbed on AgBr nanocrystals (40-900 nm) with different shapes (cubic or octahedral) is measured. Fast non-exponential fluorescence decays are observed with a fast component ranging from 400 fs to 2.5 ps depending on the type and size of the crystals. The rates of electron injection from J-aggregate dye to the conduction band of AgBr at various conditions are determined. The increase of the electron injection rate with the increase of the grain size is observed and explained by the space charge theory. Upon addition of a super-sensitizer (SS), which is different cyanine dye co-adsorbed on the surface of silver bromide grains, the fluorescence decay becomes several times faster. The results are analyzed in the framework of the “hole-trapping” supersensitization model. The effective hole trapping (electron transfer from SS to the excited J-aggregate) rate constant is found to be independent of the grain size and equal to ∼0.60 ps -1 . By fluorescence anisotropy measurement, the ultrafast energy transfer of the J-aggregates on the octahedral crystal (200 nm) is studied. The anisotropy decays are biphasic with two time constants of ∼100 fs and a few picoseconds, the short time constant being assigned to energy transfer between epitaxially arranged J-aggregates on the {111} surface.

Deuterium isotope effect on the solvation dynamics is observed in the system of aniline (AN) as a solvent for the first time by the dynamic Stokes shift method. Perdeuterated AN (AN–d7) or amino deuterated AN (AN–d2) shows slower solvation dynamics than normal AN. Deuterium effect on the solvation of N,N–dimethylaniline (DMA) is also studied and there is no isotope effect on the solvation process. The differences between AN and DMA are proposed to be related to the presence and absence of the intermolecular hydrogen bondings.

CooA, which is a transcriptional regulator heme protein allosterically triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme in the Soret band is detected at approximately 1979 cm(-1), which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 and 290 ps, caused by the rebinding of the CO to the heme. About 98% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than those in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees on the basis of the anisotropy measurements. A shift of the bleached mid-IR spectrum of the bound CO is detected and has a characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in an active conformation and in an intermediate one, which is on the way toward an inactive conformation. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.

The primary charge separation in photosystem I reaction center of plant at room temperature is found to occur intrinsically on a subpicosecond time scale, which is faster than that of purple bacterial reaction center.

The most recent X-ray analysis of the crystal structure of Photosystem I (PS I) has shown six chlorophyll a molecules (Chl) in the electron transfer system [1]. There seem to be two pathways of the electron transfer: P700→A→A0 and P700→A’→A0’. It is not yet clarified which pathway is used nor how A and/or A’ are involved in the electron transfer. This situation is analogous to those of purple bacterial reaction centers (RC). One unique feature of PS I RC is that there are two “connecting” Chls which structurally link core antenna Chls to the electron transfer system [1]. Main excitation transfer pathway might be like: core antenna→ “connecting” Chl→A0→A→P700 and core antenna→ “connecting” Chl’→A0’→A’→P700. It is thus important to characterize the spectral properties and the excitation transfer processes associated with the “connecting” Chls, A, A’, A0 and A0’. This is, however, an extremely difficult task, because there are ≈ 100 core antenna Chls which mask the excitation transfer steps.

Transient absorption spectroscopy has been used to study sub-picosecond energy transfer processes in isolated photosystem II (PS II) reaction centres. As reported previously [1], using long wavelength (694 nm) excitation, spectral evolution of the isotropic Qy band bleach / stimulated emission is dominated by energy transfer processes with a 100 ± 50 fs time constant. In contrast, depolarisation of this signal occurs with a time constant of 400 ± 30 fs, from an initial anisotropy of ~0.4 to a value of ~0.15 at 1.5 ps. This is illustrated in figure 1 which shows the isotropic absorption and anisotropy transients at a probe wavelength of 683 nm. This decay of the anisotropy is attributed to energy transfer between at least two degenerate states contributing to reaction centre absorption circa 680 nm, with these states having approximately orthogonal transition dipoles. The transient anisotropy barely changes between 1.5 ps and 60 ps, indicating that under these excitation conditions equilibration of the excitation energy between reaction centre excited states occurs on a sub-picosecond timescale. These excited state equilibration processes preceed charge separation, which occurs primarily with a 21 ps time constant [3]. Transient data collected for pheophytin Qx absorption bands indicate that pheophytin molecules are included in the 100 fs equilibration process.

The ultrafast dynamics of electron relays in the Photosystem I (PS I) reaction center (RC) is of particular interest because the amino acid sequences of the polypeptides show almost no homology to those of PS II-purple-bacterial type RC’s. Electron carriers and energy levels of PS I RC are also different from those of the latter type of RC’s. However, studies in Photosystem I have been scarce due to its large number of antenna chlorophylls (more than 100) per RC and to resultant spectral congestion. Even though the presence of the primary electron acceptor chlorophyll (A0) has been suggested by a recent X-ray crystallographic work [1], spectroscopic detection of sequential reduction and reoxidation of A0 has never been successful.