Abstract
Microscopy seeks to simultaneously maximize optical resolution, contrast, speed, volume size, and probe tolerability, which is possible by combining different complementary imaging techniques with their specific strengths. Here, we show how to combine three physical concepts to increase resolution and contrast in light-sheet microscopy by making the effective light-sheet thinner through phase shaping, fluorophores-switching, and dynamic blocking of fluorescence. This shape-switch-block principle is realized by illumination with two holographically shaped, sectioned Bessel beams. Second, by switching off fluorophores in the proximity of the excitation center using continuous-wave stimulated emission depletion (STED). And third, by blocking fluorescence outside the switching region by confocal line detection. Thereby, we reduce the light-sheet thickness by 35%, achieving an isotropic resolution with beads in a 300 × 70 × 50 µm³ volume. Without STED, we obtain 0.37 µm resolution in cell clusters at improved sectioning and penetration depth. The shape-switch-block concept promises high potential, also for other microscopy techniques.
Improved optical slicing by stimulated emission depletion light sheet microscopy
