Breaking the diffraction limit of light sheet fluorescence microscopy by RESOLFT
Significance Light sheet fluorescence microscopy (LSFM) is an imaging modality in which a sample is illuminated from the side by a beam engineered into a wide and relatively thin "sheet." This allows highly parallelized planewise scanning of volumes with inherent optical sectioning, offering a good balance between spatial and temporal resolution with reduced photostress. Unfortunately, the axial extent of the illuminated section is ultimately limited by diffraction. Here, we show that a RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] strategy neutralizes the resolution limiting role of diffraction in LSFM. While other LS strategies exist, which run into new hard axial limits of resolution, LS RESOLFT is conceptually diffraction unlimited and can be developed toward molecular scale resolution. We present a plane scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS RESOLFT nanoscopy offers wide field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5 12 fold compared with their conventional diffraction limited LS analogs.
Proceedings of the National Academy of Sciences