Figure 1.

Principles of super-resolution single-molecule active control microscopy. (a) A hypothetical arrangement of fluorescent molecules, i.e. a labeled “super-structure” (here: outline of “La Paloma de la Paz” (The Dove of Peace) by P. Picasso, 1961). (b) In conventional fluorescence microscopy, all molecules emit simultaneously, so their diffraction-limited images overlap on the detector (camera) and information about the underlying structure is irretrievably lost. (c) Addition of on-off control, toggling any one single-molecule emitter between a dark and a fluorescent state. (d) If individual sparse subsets of single molecules that are spatially separated further than the diffraction limit are made to emit, their positions may be extracted in a time-sequential manner by finding the center position of a mathematical fit of the single-molecule images. (e) From the list of localized molecules, a super-resolution reconstruction is assembled in a post-processing step. Note that if the majority of molecules is detected at least once, the resolution is then governed by the fidelity of the localization estimate of individual localizations. This precision is shown by the blue circles which, for reasonable signal-to-background of single-molecule detection, are dramatically smaller than the extent of the diffraction-limited image given by the microscope PSF. Scale bar: 250 nm. (f) The pixelated images of single-molecule emissions in a 2D imaging experiment are typically (g) fit by a Gaussian function with variable center coordinates (x,y) to extract (h) single-molecule position estimates. (i) Illustration of the inherent trade-off between spatial and temporal resolution when imaging a dynamic process: Cartoon view based on a general membrane fusion scenario, e.g. SNARE-mediated [87], evolving from a membrane stalk between a vesicle (top membrane) and the plasma membrane (bottom membrane) to a resulting fusion pore. If temporal resolution is prioritized, two or more reconstructions can be obtained, however a lower and possibly insufficient number of position samplings in the reconstructions – possibly also at worse localization precision – leave details unresolved. By contrast, collecting many positions while the structure is changing leads to time-averaging over the acquisition, and a similar loss of information.