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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.

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.

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).

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.

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.