Fluorescent Protein Photobleaching: From Molecular Processes to Spectromicroscopy.

Fluorescent proteins (FPs) are essential tools for biological imaging, but their use is limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. The molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decrease in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs.