Fotobleaching

Fotobleaching, more commonly spelled photobleaching, is the light-induced loss of fluorescence from a fluorophore. It typically results from chemical changes to the fluorescent molecule after absorption of photons during imaging and is a common limitation in fluorescence microscopy, flow cytometry, and related techniques that rely on fluorescent labels. In some cases, partial reversible dark states can occur, but true bleaching generally implies permanent loss of signal.

The underlying mechanism involves the excited fluorophore transitioning to reactive states. After absorbing light, a fluorophore may enter a triplet state or react with molecular oxygen to form reactive species such as singlet oxygen or free radicals. These species can modify or destroy the chromophore, leading to diminished or extinguished fluorescence. Bleaching susceptibility varies by dye, excitation wavelength, environment, and presence of quenchers or antioxidants.

Implications for experiments include reduced signal over time and potential biases in quantitative measurements of fluorescence intensity or localization. Bleaching can be a challenge in live-cell imaging but is also exploited in methods like fluorescence recovery after photobleaching (FRAP) to study molecular mobility and dynamics. Bleaching rates are influenced by fluorophore properties, illumination conditions, oxygen levels, and sample composition.

Prevention and mitigation strategies focus on preserving signal. These include choosing more photostable fluorophores, using the lowest feasible illumination intensity and exposure time, employing anti-bleaching additives or oxygen-scavenging systems, optimizing imaging media, and applying computational corrections during data analysis. Understanding a fluorophore’s bleaching behavior aids in experimental design and interpretation of time-resolved fluorescence data.