Photoactivatable fluorophores for single-molecule localization microscopy of live cells

Photochemical reactions can be designed to convert either irreversibly or reversibly a nonemissive reactant into an emissive product. The irreversible disconnection of a photocleavable group from an emissive chromophore or the reversible interconversion of a photochromic component is generally exploited to implement these operating principles for fluorescence switching. In both instances, the interplay of activating radiation, to convert the nonemissive state into the emissive species, and exciting radiation, to produce fluorescence from the latter, can be exploited to switch fluorescence on in a given area of interest at a precise interval of time. Such a level of spatiotemporal control provides the opportunity to reconstruct sub-diffraction images with resolution at the nanometer level. Indeed, closely-spaced emitters can be switched on under photochemical control at distinct intervals of time and localized independently at the single-molecule level. In combination with appropriate intracellular targeting strategies, some of these photoactivatable fluorophores can be switched and localized inside live cells to permit the visualization of sub-cellular structures with a spatial resolution that would be impossible to achieve with conventional fluorophores. As a result, photoactivatable fluorophores can become invaluable probes for the implementation of super-resolution imaging schemes aimed at the elucidation of the fundamental factors controlling cellular functions at the molecular level. (topical review)