FIG. 3.3 RESOLFT microscopy architecture and comparison with confocal microscopy. (a) Schematic of the RESOLFT microscope. The Gaussian-shaped read-out (or excitation) beam and the doughnut-shaped OFF-switching beam—both having the same wavelength (blue)—are combined with a polarized beam splitter. Successively the two beams are combined with the Gaussian-shaped probe (or ON-switching) beam (magenta) with a dichroic mirror (DM). All co-aligned beams are deflected by the galvanometer mirrors (GMs) and focused by the objective lens (OL) on the sample. Fluorescence (green) is collected by the same objective lens, descanned by the GMs, filtered by the DM, and recorded by a photodetector. (b) Similar to STED microscopy also in RESOLFT microscopy a specific and well-determined temporal beam sequence is necessary to achieve subdiffraction imaging. However, this temporal alignment enables a completely different precision, i.e., microsecond instead of picosecond range. For each sample position (i.e., scanning position or pixel), first the probe beam activates all the fluorescent protein in a well-defined diffraction-limited region, successively the OFF-switching beam de-activates all the fluorescent protein in the periphery of the probed region, leaving only a tiny region close to the center active. Finally, the read-out beam excites this residual region whose fluorescence is registered by the photodetector. This sequence is iterated across the whole sample. Synchronization between the beam temporal sequence, the scanning, and the fluorescence detection can efficiently be implemented by an FPGA-based data acquisition system. (c) Side-by-side comparison of confocal (top) and RESOLFT image of vimentin-rsEGFP2. Scale bar, 1 µm. Adapted from Dreier, J. et al. , Nat. Commun. 10 , 556 (2019). Copyright 2019 Authors. More about this image found in RESOLFT microscopy architecture and comparison with confocal microscopy. (a...