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.
Keywords: STORM; dSTORM; direct stochastic optical reconstruction microscopy; fluorescent probes; multicolor; organic dyes; single molecule localization; super-resolution microscopy.
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