Uniform distribution of photosystems in dark-adapted Synechocystis cells

Photosynthesis in cyanobacteria relies on light capture by photosystem I (PSI), photosystem II (PSII) and the phycobilisome (PBS). While these complexes are thought to be intermixed within the thylakoid membrane, there is also evidence for PSI-enriched and PSII-PBS-enriched microdomains, and their spatial organization is still debated. This organization may further depend on environmental conditions. To study it, a range of methods are available. Cryo-electron tomography offers the highest resolution but is limited in throughput, while super-resolution fluorescence techniques such as Airyscan and structured illumination microscopy provide improved resolution but cannot resolve individual thylakoid membranes. To expand this toolbox, we applied cryo-Expansion Microscopy (cryo-ExM) to dark-adapted Synechocystis sp. PCC 6803 cells. Cells were cryofixed, rehydrated at room temperature and physically expanded in a swellable hydrogel. By expanding cells 5.5-fold, we resolved individual thylakoid membranes in intact cells using confocal microscopy. Immunostaining further allowed simultaneous localization of PSI, PSII and PBS within the expanded thylakoid network. Quantitative analysis of fluorescence covariance revealed a high degree of colocalization among PSI, PSII and PBS, providing no evidence for microdomains under dark-adapted conditions. PBS was excluded only from the neck region between dividing cells, while PSI, PSII and PBS were otherwise distributed throughout the thylakoid membrane. Together, these results establish cryo-ExM as a powerful method for visualizing individual cyanobacterial thylakoid membranes and mapping the distribution of key photosynthetic complexes, thereby complementing existing approaches for dissecting the spatial organization of photosynthesis.