FIG. 3.2 STED microscopy principles and comparison with confocal images. (a) A schematic comparison of a conventional scanning microscope (left) and a STED microscope (right). (b) Spatial conditions. The maximum and the “zero” of the excitation and depletion focal intensity distributions, respectively, should coincide. AU, arbitrary units. (c) Temporal conditions. All stimulating photons should act when fluorophores are in the singlet excited state S₁, and fluorescence must be registered after the stimulating photon's action. Experimental time sequences are shown for the excitation, the depletion, and the fluorescence signal detection for gated pulsed STED (gP-STED), pulsed STED (P-STED), gated CW-STED (gCW-STED), and CW-STED microscopy. The time delay T_g characterizes the time-gated detection. The pulse width T_STED and the repetition rate 1/T_rep characterize the pulsed STED beam. (d) Spectral conditions. All the photons of the STED beam should promote s.e.m. and not be absorbed. Normalized ground-state absorption and fluorescence emission spectra juxtaposed with the excited-state absorption of eGFP81 (left). The Jablonski diagram indicates the transitions relevant for STED microscopy, including the photobleaching (bl) pathways (right). The dashed lines depict unwanted transitions. (e) Dual-color 3-D confocal (left) and pulsed 3-D STED (right) axial views (x,z) of an immunolabeled fixed cell. (f) Dual-color confocal (left) and pulsed STED (right) images of the immunolabeled subunits in amphibian NPC21. (g) Four-color confocal (left) and pulsed STED (right) images of an immunolabeled fixed cell ( Winter et al., 2017 ). (h) Single-color confocal (top), gated CW-STED (middle) and deconvolved gated CW-STED (bottom) images of immunolabeled fixed cell. Scale bars, 1 µm. Adapted from Vicidomini, G. et al. , Nat. Methods 15 (3), 173–182 (2018). Copyright 2018 Nature Publishing Group. More about this image found in STED microscopy principles and comparison with confocal images. (a) A schem...

FIG. 5.6 STED microscopy breaks the diffraction limit with patterned illumination. (a) The schematic shows the configuration of STED microscopy. (b) STED beam depletes the emission induced by the excitation beam to reduce the effective fluorescent area. More about this image found in STED microscopy breaks the diffraction limit with patterned illumination. (...

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...