Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

Anindita Dasgupta, Agnes Koerfer, Boštjan Kokot, Iztok Urbančič, Christian Eggeling, Pablo Carravilla
Nature methods, June 2024; doi: 10.1038/s41592-024-02297-4 (read more here).

In brief

High laser powers in fluorescence microscopy should be used with care; besides the well-known photobleaching, the dye’s emission spectrum can unexpectedly shift towards lower wavelengths (“photoblueing”). The fluorescence signal is consequently detected in another spectral channel attributed to another dye, possibly leading to erroneous experimental conclusions. Detailed characterization of this phenomenon was done by our unique combination of a STED microscope equipped with a spectral detector.