Vorschau

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Abstract

The concept of RESOLFT was introduced to break the resolution limit in fluorescence microscopy, which is set by the physical phenomenon of diffraction. It enables the separation of fluorophores inside a focal volume by driving reversible optical transitions between two discernible states. The family of reversibly switchable fluorescent proteins (RSFPs) with long-lived on- and off-states allows for RESOLFT imaging at low light levels. Up to now, RESOLFT involving RSFPs as fluorescent markers has been exclusively demonstrated to enhance the resolution in the lateral dimension. In this work, a novel RSFP-based RESOLFT microscope is presented, that, for the first time, breaks the diffraction barrier in the axial direction by switching fluorophores in the volume of a light-sheet. It is realized with the optical arrangement of a selective plane illumination microscope (SPIM), that illuminates only a thin section of a sample perpendicular to the detection axis and thus reduces the overall light exposure in volume recordings. The symbiotic combination of RSFP-based RESOLFT and SPIM, the so called RESOLFT-SPIM nanoscope, offers highly parallelized, fast imaging of living biological specimens with low light doses and sub-diffraction axial resolution. Compared to the diffraction-limited SPIM analogue an improvement in axial resolution by more than a factor of 12 is demonstrated.