Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging

Abstract

Blue-conversion, a photooxidative conversion leading to the hypsochromic shift of absorption and emission spectra, occurs in popular organic dyes under conventional laser illumination and produces severe artifacts in multicolor fluorescence imaging.Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.