title: 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.
abstract: 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.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Shuho
authors.initials: S
authors.lastname: Nozue
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Mukuno
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Tsuda
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shiina
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Masahide
authors.initials: M
authors.lastname: Terazima
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Electronic address: kumazaki@kuchem.kyoto-u.ac.jp.
authors.forename: Shigeichi
authors.initials: S
authors.lastname: Kumazaki
mesh_terms: D000460:Chlorophyta / Q000378:metabolism*; D003198:Computer Simulation; D000458:Cyanobacteria / Q000378:metabolism*; D007834:Lasers; D008856:Microscopy, Fluorescence; D045331:Photosystem I Protein Complex / Q000502:physiology; D045332:Photosystem II Protein Complex / Q000502:physiology; D020524:Thylakoids / Q000378:metabolism*
chemical_list: D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex
keywords: Chlorophyll fluorescence; Fluorescence lifetime imaging microscopy; Green algae; Heterocystous cyanobacteria; Photosystem I; Photosystem II