Fig. 1

Imaging principle of super-resolution microscopy techniques such as stimulated emission depletion microscopy (STED), structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). STED: During excitation, two laser beams—a laser beam producing a fluorescent spot with a diffraction-limited size (green) and a second laser beam depleting a donut-shaped area around the excited spot (magenta)—generate a sub-diffraction-limit fluorescent point for detection (solid red). The excitation/depletion spot is then scanned across the sample to generate the final image. SIM: Patterned excitations are first performed with multiple shifts and rotations. The partial fluorescent images from these excitation patterns are then detected and combined using an algorithm to reconstruct the final image of the sample. SMLM: In one implementation, a subset of photoswitchable fluorophores will turn on and off each time the sample is excited. During this process, only a sparse subset of the fluorophores—those at the on-state—are detected. After repeating the excitation-detection cycle many times, an increasing number of the single fluorescence points are captured. The centers of these single fluorescence points are then computationally determined and combined to reconstruct the final image of the sample