The ability to switch fluorophore emission on and off, both temporally and spatially, has also led to the emergence of super-resolution imaging techniques, in which the spatial resolution is now approaching the molecular scale. This makes it possible to identify specific species in complex mixtures and optically noisy environments, such as living cells, as long as the surrounding objects are non- (or weakly) fluorescent at the combination of illumination and detection wavelengths. (8) These experiments benefit from the specific advantage of fluorescence: the spectral separation between illumination and emission profiles, which enables efficient background suppression. Single-molecule experiments in the form of single-emitter fluorescence detection and imaging have since become routine. (5−7) These single-molecule experiments were performed under cryogenic conditions with specifically selected chromophores to maximize the interaction strength between the incident light and the molecules of interest. In the context of sensitivity, light microscopy arguably reached its ultimate limit more than 30 years ago with the detection of individual ions and molecules using absorption and fluorescence as contrast mechanisms.
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