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Recently, reversibly switchable fluorescent proteins (RSFPs) have become a new branch of the green fluorescent protein (GFP) like family. RSFPs may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths; the key structural event of this switch is a cis / trans isomerization of the chromophore which is accompanied by changes in the protonation state of the chromophore. These proteins may be applied in sub-diffraction resolution microscopy as well as in novel protein tracking schemes or even as data storage elements.Consequently, the creation of novel RSFPs with unique characteristics for the respective applications is of great importance. To this end, the combination of rational and random mutagenesis together with an automated screening system allowed us to create several new RSFPs. These include rsCherryRev, Padron and bsDronpa. rsCherryRev, the first red fluorescent monomeric RSFP, was successfully applied in a live cell sub-diffraction resolution microscopy experiment to visualize the movement of the endoplasmic reticulum. Padron, a green RSFP with reversed switching behavior, in combination with the RSFP rsFastLime allowed for multilabel single detection color microscopy, while bsDronpa, a Dronpa variant with a broad excitation spectrum, was used for two-color sub-diffraction resolution microscopy.