Precise multicolor single molecule localization‐based microscopy (SMLM) requires bright probes with compatible photo‐chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super‐resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing‐based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3‐color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.
A major limitation of multicolor single molecule localization based super‐resolution microscopy (SMLM) is the availability of suitable photo‐switchable fluorescent dyes. By screening 28 commercially available dyes, novel dyes in different spectral regimes were identified that are well suited for dual and triple color SMLM with low crosstalk. These novel dyes are employed to image the relative nanoscale distribution of sub‐cellular components.
