Time-Space-Resolved Fluorescence Spectroscopy in Chlamydomonas Cells under Light-Harvesting Regulation

We report here a technical advancement that enables time-resolved fluorescence spectroscopy in spatially resolved domains of a living cell at low temperatures. The technique is based on a combination of a self-developed cryo-confocal microscope and a streak camera. An instrumental response time of 24 ps was achieved. This technique was applied to reveal the light-harvesting dynamics in local domains within single Chlamydomonas reinhardtii cells. Organisms performing oxygenic photosynthesis have evolved a regulation mechanism, called state transitions (ST), that maintains the excitation balance between PSI and PSII. Cells were induced either to state1, where LHCII is primarily bound to PSII, or state2, where a fraction of LHCII has moved and is bound to PSI. The observation was made at 80 K, where PSI and PSII show clearly separated fluorescence emission bands. We separately analyzed fluorescence rise dynamics in the wavelength regions of 685-693 nm assigned to PSII and 705-724 nm assigned to PSI in both the PSI-rich and PSII-rich domains. The observed rising fluorescence dynamics reflect the excitation energy flow from the antenna to the core parts of these PSs. The time constant of the fluorescence rise of the PSII emission was slower in the PSII-rich than in the PSI-rich domain in both state1 and state2, whereas that of the PSI emission was slower for the PSI-rich than for the PSII-rich domain in state2. Based on these results, we concluded that the intracellular inhomogeneity in the PSII/PSI fluorescence ratio comes partially from the heterogeneity of the antenna sizes of the PSs.