The light-harvesting reactions of photosynthesis take place on the thylakoid membrane inside chloroplasts. The thylakoid membrane is folded into appressed membranes, the grana, and nonappressed membranes that interconnect the grana, the stroma lamellae. This folding is essential for the correct functioning of photosynthesis. Electron microscopy and atomic force microscopy are commonly used to study the thylakoid membrane, but these techniques have limitations in visualizing a complete chloroplast and its organization. To overcome this limitation, we applied expansion microscopy (ExM) on isolated chloroplasts. ExM is a technique that involves physically expanding a sample in a swellable hydrogel to enhance the spatial resolution of fluorescence microscopy. Using all-protein staining, we visualized the 3D structure of spinach (Spinacia oleracea) thylakoids in detail. We were able to resolve stroma lamellae that were 60 nm apart and observe their helical wrapping around the grana. Furthermore, we accurately measured the dimensions of grana from top views of chloroplasts, which allow for precise determination of the granum diameter. Our results demonstrate that ExM is a fast and reliable technique for studying thylakoid organization in great detail.
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