Scytonemin redox status in a filamentous cyanobacterium visualized by an excitation-laser-line-scanning spontaneous Raman scattering spectral microscope.

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.

Growth-phase dependent morphological alteration in higher plant thylakoid is accompanied by changes in both photodamage and repair rates.

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.

Spectral microscopic imaging of heterocysts and vegetative cells in two filamentous cyanobacteria based on spontaneous Raman scattering and photoluminescence by 976 nm excitation.

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.

Comparative study of thylakoid membranes in terminal heterocysts and vegetative cells from two cyanobacteria, Rivularia M-261 and Anabaena variabilis, by fluorescence and absorption spectral microscopy.

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.

Characterization of thylakoid membrane in a heterocystous cyanobacterium and green alga with dual-detector fluorescence lifetime imaging microscopy with a systematic change of incident laser power.

Fluorescence Lifetime Imaging Microscopy (FLIM) has been applied to plants, algae and cyanobacteria, in which excitation laser conditions affect the chlorophyll fluorescence lifetime due to several mechanisms. However, the dependence of FLIM data on input laser power has not been quantitatively explained by absolute excitation probabilities under actual imaging conditions. In an effort to distinguish between photosystem I and photosystem II (PSI and PSII) in microscopic images, we have obtained dependence of FLIM data on input laser power from a filamentous cyanobacterium Anabaena variabilis and single cellular green alga Parachlorella kessleri. Nitrogen-fixing cells in A. variabilis, heterocysts, are mostly visualized as cells in which short-lived fluorescence (≤0.1 ns) characteristic of PSI is predominant. The other cells in A. variabilis (vegetative cells) and P. kessleri cells show a transition in the status of PSII from an open state with the maximal charge separation rate at a weak excitation limit to a closed state in which charge separation is temporarily prohibited by previous excitation(s) at a relatively high laser power. This transition is successfully reproduced by a computer simulation with a high fidelity to the actual imaging conditions. More details in the fluorescence from heterocysts were examined to assess possible functions of PSII in the anaerobic environment inside the heterocysts for the nitrogen-fixing enzyme, nitrogenase. Photochemically active PSII:PSI ratio in heterocysts is tentatively estimated to be typically below our detection limit or at most about 5% in limited heterocysts in comparison with that in vegetative cells.

Changes in antenna sizes of photosystems during state transitions in granal and stroma-exposed thylakoid membrane of intact chloroplasts in Arabidopsis mesophyll protoplasts.

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.

Transformation of thylakoid membranes during differentiation from vegetative cell into heterocyst visualized by microscopic spectral imaging.

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.

Anti-Stokes fluorescence spectra of chloroplasts in Parachlorella kessleri and maize at room temperature as characterized by near-infrared continuous-wave laser fluorescence microscopy and absorption microscopy.

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.

Selective excitation of photosystems in chloroplasts inside plant leaves observed by near-infrared laser-based fluorescence spectral microscopy.

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.

A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120.

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 mum 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.

Energy equilibration and primary charge separation in chlorophyll d-based photosystem I reaction center isolated from Acaryochloris marina.

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.