Single-molecule fluorescence microscopy

Fluorescence measurements resolving individual labelled molecules or molecular events, allowing heterogeneous dynamics to be distinguished from population averages.

Uncertainty in an estimated emitter position. It can be much smaller than the width of its point spread function, but it does not by itself guarantee that densely packed emitters or an entire labelled structure are resolved.

Stochastic optical reconstruction microscopy reconstructs an image from the estimated positions of individual fluorescent labels that are observed in sparse subsets over many frames. The experiment repeatedly switches labels between detectable and dark states, fits isolated point spread functions, and combines the localizations.

This separates emitters in time instead of requiring all neighboring labels to be spatially resolved in one exposure. Localization precision depends on detected photons and background; drift, incomplete labeling and simultaneous overlapping emitters constrain final optical resolution. In the original demonstration, reversibly switched dye labels enabled repeated sparse localization.^[ref]