Hyperspectral fluorescence imaging and fluorescence lifetime imaging of heterocystous cyanobacteria Anabaena variabilis and Rivularia M-261

Shuho Nozuea, Shinji Fukudaa, Koto Tamamizua, Akira Mukunoa, Yumi Tsudaa, Takashi Shiinab, Masahide Terazimaa, Mitsunori Katayamac, Shigeichi Kumazakia
aGraduate School of Science, Kyoto University, Japan; bGraduate School of Life and Environmental Science, Kyoto Prefectual University, Japan; cDepartment of Civil Engineering College of Industrial Technology, Nihon University, Japan

Heterocyst is a nitrogen-fixing cell differentiated from a vegetative cell in filamentous cyanobacteria. The differences in the cellular properties under physiological conditions should be ideally studied on a cell-by-cell basis by a microspectroscopy. We have aimed at understanding the differences between heterocyst and vegetative cells of both Anabaena variabilis and Rivularia M-261, which leads to a systematic comparison in the heterocyst functions between the two species. Broadband fluorescence spectral imaging simultaneously yielded 128 color images with a 2 nm wavelength resolution [1]. The spectral imaging was performed with 808 nm 2-photon excitation, 785 nm 1-photon excitation (anti-Stokes fluorescence), and 488 nm 1-photon excitation modes [2].

Fluorescence lifetime imaging microscopy with a systematic change in scanning laser power of 404 nm 1-photon excitation enabled us to differentiate cells with photosystem II (PSII) from those free of PSI[3]. As easily expected based on past works, most heterocysts in both the species were visualized as cells in which short-lived fluorescence (< 0.1 ns) characteristic of PSI is predominant. We have succeeded to quantitatively reproduce the dependence of fluorescence lifetime on the absolute laser power in the case of vegetative cells[3]. Acquisition of both the photosystem I-rich anti-Stokes fluorescence spectra and 2-photon-excitation-induced fluorescence spectra from identical cells have strongly suggested the following situation. Heterocysts in Rivularia generally contain a far greater amount of phycobilisome and/or the energy transfer efficiency from the phycobilisome to photosystem I is far lower than those in Anabaena variabilis.


[1] S. Kumazaki et al. 2007. Journal of Microscopy, 228: 240 - 254.
[2] S. Kumazaki et al. 2013. Plant Physiology, 161:1321-1333
[3] S. Nozue et al. 2016. Biochimica et Biophysica Acta - Bioenergetics, 1857:46-59