Study on adaptation of single photosynthetic cells against environmental stress by using three-dimensional super-resolution microscopy with time resolution ability

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A near-infrared fs pulse was illuminated on a line in a specimen by a resonant scanning mirror oscillating at 7.9kHz, and total multiphoton-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 colors at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. 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 0.56-0.59μm for the X, Y and Z axes, respectively, at fluorescence wavelengths between 644 and 690nm. This microscope was applied to a study of the subcellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), 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 subcellular domains. Adaptation of the thylakoid membrane against light conditions, and its variation between chloroplasts and cyanobacteria, has been known for decades, but characterization based on fluorescence spectrum at a subcellular resolution has been scarce. Our newly developed spectroimaging system has also been applied to chloroplasts in tabacco, zea mays, and chlorella. Tissue dependence in the case of tobacco, daily cycle in the case of zea mays, and culture-condition dependence in the case of chlorella were found.