Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy.

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.