Fig. 2a–d.

Principles of super-resolution microscopy techniques. a Left: Scheme of six filaments decorated with fluorophores (represented by large icons for visibility) and grouped into three pairs at simulated distances of 50, 100, and 150 nm; scale 200 nm. Right: A typical image of this structure obtained by conventional fluorescence microscopy is limited by the diffraction of light. b i, top: For STED, the structure is scanned by a subdiffraction excitation spot obtained by combining an excitation laser (green) with a, by phase-modulation shaped, depletion laser (red). After scanning the entire structure (i, bottom), and without performing any further post-processing steps (ii), an image is reconstructed (iii). c In SIM, fluorophores are excited by a series of regularly spaced illumination patterns of known frequency, orientation, and phase which modulate the fluorophore emissions. This results in visible low-frequency Moiré patterns that are dependent on the structure imaged (i). By analyzing the images for their spatial frequencies, an enlarged frequency space is obtained (ii), and a subdiffraction image is reconstructed (iii). d In SMLM, the fluorescence is modulated by photoswitching between “off” and “on” states. Most of the fluorophores are forced to reside in a dark off state; only a small subset of spatially separated fluorophores in the on state is allowed to emit fluorescence at a given time. After sequentially imaging thousands of subsets of fluorophores (i), the nanometer-precise fluorophore positions can be extracted from the diffraction-limited individual emissions (ii), and an image is reconstructed (iii). The three super-resolved images labeled (iii) visualize typical resolutions obtained by the methods: on the order of 50 nm (STED), 100 nm (SIM), and 20 nm (SMLM); scale 200 nm