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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.

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.

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).

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.

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...

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.

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.

Primary photochemistry in photosystem I (PS I) reaction center complex from Acaryochloris marina that uses chlorophyll d instead of chlorophyll a has been studied with a femtosecond spectroscopy. Upon excitation at 630 nm, almost full excitation equilibration among antenna chlorophylls and 40% of the excitation quenching by the reaction center are completed with time constants of 0.6(+/-0.1) and 4.9(+/-0.6) ps, respectively. The rise and decay of the primary charge-separated state proceed with apparent time constants of 7.2(+/-0.9) and 50(+/-10) ps, suggesting the reduction of the primary electron acceptor chlorophyll (A(0)) and its reoxidation by phylloquinone (A(1)), respectively.