title: Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.
abstract: A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39μm along the lateral direction and 1.3μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki aza Aoba, Aoba-ku, Sendai 980-8578, Japan. Electronic address: shibata@m.tohoku.ac.jp.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Shibata
authors.affiliation: Division of Material Science (Physics), Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Katoh
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki aza Aoba, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Chiba
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki aza Aoba, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Namie
authors.affiliation: Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
authors.forename: Norikazu
authors.initials: N
authors.lastname: Ohnishi
authors.affiliation: Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Minagawa
authors.affiliation: Division of Material Science (Physics), Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Hanayo
authors.initials: H
authors.lastname: Nakanishi
authors.affiliation: Division of Material Science (Physics), Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Noguchi
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki aza Aoba, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukumura
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010788:Photosynthesis; D045331:Photosystem I Protein Complex / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000378:metabolism
chemical_list: D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex
keywords: Chlamydomonas reinhardtii; Fluorescence blinking; Lateral heterogeneity of photosystem; Photosystem I; Single-molecule spectroscopy