Photoblueing of organic dyes can cause artifacts in super-resolution microscopy.

Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented. Commonly used organic dyes can photoconvert to blue-shifted fluorescent species, especially under intense illumination. The mechanism of this process and how to avoid unwanted artifacts in super-resolution microscopy are explored here.