LysoTracker Deep Red exhibits photoconversion in multi-color structured illumination microscopy

In new chemical environments or for untested combinations of illumination, unexpected changes to the fluorescent labels photophysical properties, such as photoconversion, can occur. This letter reports on photoblueing of a common cellular probe, LysoTracker Deep Red, in multi-color super-resolution structured illumination microscopy. The dye was found to exhibit a blue shift of its fluorescence spectrum as a step on the photobleaching pathway during such imaging. The observed photoblueing (emission spectrum shifted to lower wavelengths) of this probe is an important finding as many cellular assays rely on the spectral separation of this and such fluorescent cellular markers. No spectral shift of LysoTracker Deep Red was observed for longterm imaging using diffraction limited microscopy requiring significantly lower light dose. We expect that the knowledge of occurrence of photoconversion during super-resolved imaging using this popular dye will help researchers design better imaging experiments and avoid potentially erroneous interpretations in multi-color microscopy.