Whole-Cell Glycerol-free Fluorescence Cryo-Spectroscopy Reveals Electronic Excitation Dynamics in the Diatom Cyclotella meneghiniana.

Diatoms possess a unique photosynthetic apparatus that adapts to constant, extreme changes in light intensity, enabling efficient light harvesting and photoprotection. Fluorescence spectroscopy studies at cryogenic temperatures can reveal details of the electronic excitation dynamics in these evolutionarily successful organisms. Whole-cell studies are highly valuable since they allow investigation of the photosynthetic machinery rather than separate complexes affected by isolation. In this study, we propose an approach that enables cryogenic fluorescence analysis of photosynthetic processes in the diatom Cyclotella meneghiniana, frozen in an intact state at 16-130 K. We show the drastic effects on cell viability caused by traditionally used glycerol-containing cryo-media, including rapid cellular damage (with about 36% of cells losing membrane integrity immediately upon medium exchange, reaching >99% within 3.5 h), thylakoid membrane disruption, likely detachment of fucoxanthin chlorophyll proteins (FCPs) from photosystem II (PSII) cores, and possible FCP aggregation. By omitting glycerol, we provide whole-cell cryo-measurements of the photosynthetic apparatus in cells frozen in an intact state and demonstrate the retention of some function (e.g., activation of alternative electron transfer) of the photosynthetic complexes in damaged cells, which, however, differ from those in intact cells. We identified specific features of intact whole-cell temperature-dependent excitation dynamics, including a gradual change in the relative contributions of CP47 (690 nm) and CP43 (685 nm) emissions, slowed charge separation and energy transfer within PSII cores at low temperatures, and a monotonic increase in PSI-FCP emission at 700 nm with decreasing temperature. Our results indicate that cryo-spectroscopy data obtained in glycerol-containing media should be interpreted with caution: cell viability must be ensured before measurements, and pseudofunctioning of the photosynthetic machinery in damaged cells should be considered. This approach provides a reliable framework for investigating intact photosynthetic cells and offers new insights into the complexity of excitation dynamics.