Expansion microscopy visualizes the photosystem distribution in Synechocystis cells

Photosynthesis in cyanobacteria relies on light capture by photosystem I (PSI), photosystem II (PSII) and the phycobilisome (PBS). Although these complexes are generally considered to be intermixed within the thylakoid membrane, several studies have suggested the presence of PSI- or PSII/PBS-enriched microdomains that may depend on environmental conditions. Here we applied cryo-Expansion Microscopy (cryo-ExM) to dark-adapted Synechocystis sp. PCC 6803 cells and achieved nanoscale resolution of thylakoid compartments and associated protein complexes. Cells were cryofixed, rehydrated at room temperature and physically expanded in a swellable hydrogel. By expanding cells 5.5-fold, we resolved individual thylakoid compartments in intact cells using confocal microscopy. Furthermore, immunolabeling allowed simultaneous localization of PSI, PSII and PBS within the expanded thylakoid network. Overall PSI, PSII, and PBS signals showed similar spatial distributions. However, PBS was excluded from the neck region between dividing cells, while PSI and PSII were present. These results establish cryo-ExM as a powerful method for visualizing cyanobacterial thylakoid membranes and mapping the distribution of key photosynthetic complexes, thereby complementing existing approaches for dissecting the spatial organization of photosynthesis.