Advanced fluorescence imaging technology, jointly enabled by microscopes and cutting-edge fluorescent labels, promises to visualize the position and dynamic behavior of biomolecules in cells and tissues. The emerging fluorescence imaging technologies are, however, particularly demanding in the properties of fluorescent dyes. From Stimulated Emission Depletion (STED) super-resolution imaging to single-molecule Fluorescence Resonance Energy Transfer (FRET), these new practices are essentially leveraging high-intensity illuminations to trade for higher fluorescence readout which eventually translates into spatial and temporal information. Consequently, these imaging technologies can only be realized with exceptionally robust fluorophores with bright emission and high photostability [1].

Photostability stems from the photochemical and photophysical processes of the fluorophore in its excited state. Broadly speaking, there are two key aspects to consider: first, long-term fluorescent signal stability which is dependent on the irreversible damage to the fluorophore's structure due to a cascade of photochemical reactions triggered by laser excitation, a process known as photobleaching; Second, the short-term stability of the signal, which involves the transition of the dye between its bright and dark states, a phenomenon known as photoblinking. Studies on photoblinking and photoconversion have led to the development of single-molecule localization-based super-resolution imaging [2, 3, 4]. However, our understanding of the mechanisms underlying photobleaching remains limited.

In this review, we aim to provide an overview of recently elucidated photochemical processes for two types of dyes widely used in state-of-the-art fluorescence technology: rhodamine and cyanine. In addition, we aim to highlight the interplay between dyes and their microenvironment which plays a crucial yet underappreciated role in the photostability of fluorophores.