Avoiding photoblueing-induced artefacts in confocal and STED microscopy

Confocal and stimulated emission depletion (STED) superresolution microscopy are scanning microscopy techniques widely used in life sciences. Unfortunately, commonly used laser excitation powers can induce photochemical reactions of fluorescent dyes. A particularly concerning reaction is photoblueing, i.e. the photoconversion of dyes to molecules showing a blue-shifted spectrum, which can introduce artefacts in quantitative microscopy. Here, we use a combination of advanced microscopy techniques, such as spectral STED, fluorescence correlation spectroscopy and fluorescence lifetime imaging to 177haracterize photoblueing of popular organic dyes and its dependency on the excitation and depletion lasers. We report that some widely used fluorescent dyes undergo a significant blue shift upon laser irradiation in confocal measurements, an effect further amplified when in STED mode. Spectral shifts are not only dyedependent, but also greatly affected by common modifications such as antibody conjugation. We precisely quantify the number of molecules that undergo photoblueing which is as high as 25% for some popular dyes. Even if the brightness of photoblued dyes is significantly lower than that of the original dyes, we show that they can lead to biased estimations of membrane biophysical parameters in live cell experiments when using lipid packing sensors. Finally, we demonstrate that artifacts can be circumvented by using exchangeable dyes and we also identify STED-compatible dyes showing negligible photoblueing tendency.