Redefining the photo-stability of common fluorophores with triplet state quenchers: mechanistic insights and recent updates.

Light microscopy can offer certain advantages over electron microscopy in terms of acquiring detailed insights into the biological/intra-cellular milieu. In recent years, with the development of new fluorescence imaging technologies, it has become extremely important to assess the role of designing appropriate fluorophores in acquiring desired biological information without encountering any untoward hitches. Over the years, external fluorophores have been prevalently used in fluorescence microscopy and single-molecule fluorescence microscopy-based studies. Photostable fluorogenic probes with high extinction coefficients and quantum yields, exhibiting minimum autofluorescence and photobleaching properties, are preferred in single-molecule microscopy as they can tolerate long-term laser exposure. Therefore, the development of triplet state quenchers and/or any other suitable new strategy to ensure the photo-stability of the fluorophores during long-term live cell imaging exercises is highly anticipated. In this feature article, various strategies for stabilizing fluorophores, including the mechanisms of TSQ-induced stabilization, have been thoroughly reviewed considering contemporary literature reports and applications.