A family of photo-switchable fluorescent probes was developed for multicolor super-resolution imaging. These probes consist of a photo-switchable "reporter" fluorophore and an "activator" that facilitates photo-activation. By combinatorially pairing reporters and activators, distinct colors can be achieved. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, multi-color imaging of DNA model samples and mammalian cells with 20-30 nm resolution was demonstrated. This technique enables direct visualization of molecular interactions at the nanometer scale. STORM relies on detecting single fluorescent molecules and localizing them with nanometer accuracy. The development of multicolor STORM depends on the construction of bright, photoswitchable probes with distinct colors. Chromatically distinguishable photo-switchable reporters can be activated by the same activator dye. This selective activation provides a second parameter for multicolor detection. The combination of three reporters and three activators can generate up to nine distinguishable fluorescent probes, requiring only four light sources for excitation and three spectrally distinct channels for detection. The results show that chromatically distinguishable photo-switchable reporters can be activated by the same activator dye. The activation rate constants were measured for different dye pairs at various wavelengths, showing high activation specificity. The localizations within each cluster approximately follow a Gaussian distribution with a full-width-half-maximum (FWHM) of 26 ± 1 nm, 25 ± 1 nm, and 24 ± 1 nm for the three color channels, suggesting an imaging resolution of ~25 nm for 3-color STORM imaging. Applying STORM to cell imaging, microtubules and clathrin-coated pits (CCPs) were imaged. The STORM image showed a drastic improvement in the resolution of the microtubule network compared to the conventional fluorescence image. The intrinsic imaging resolution, as determined from the FWHM of point-like clusters of localizations, was 30 ± 1 nm for each of the two color channels. The green channel revealed filamentous structures as expected for microtubules, while the red channel revealed predominantly spherical structures, representing clathrin-coated pits and vesicles. The STORM image allowed the measurement of the size distribution of CCPs, which agrees quantitatively with the size distribution determined using electron microscopy. This technique may be readily expanded to include more colors using the photo-switchable activator-reporter pairs reported here, significantly enhancing our ability to visualize molecular interactions in cells and tissues.A family of photo-switchable fluorescent probes was developed for multicolor super-resolution imaging. These probes consist of a photo-switchable "reporter" fluorophore and an "activator" that facilitates photo-activation. By combinatorially pairing reporters and activators, distinct colors can be achieved. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, multi-color imaging of DNA model samples and mammalian cells with 20-30 nm resolution was demonstrated. This technique enables direct visualization of molecular interactions at the nanometer scale. STORM relies on detecting single fluorescent molecules and localizing them with nanometer accuracy. The development of multicolor STORM depends on the construction of bright, photoswitchable probes with distinct colors. Chromatically distinguishable photo-switchable reporters can be activated by the same activator dye. This selective activation provides a second parameter for multicolor detection. The combination of three reporters and three activators can generate up to nine distinguishable fluorescent probes, requiring only four light sources for excitation and three spectrally distinct channels for detection. The results show that chromatically distinguishable photo-switchable reporters can be activated by the same activator dye. The activation rate constants were measured for different dye pairs at various wavelengths, showing high activation specificity. The localizations within each cluster approximately follow a Gaussian distribution with a full-width-half-maximum (FWHM) of 26 ± 1 nm, 25 ± 1 nm, and 24 ± 1 nm for the three color channels, suggesting an imaging resolution of ~25 nm for 3-color STORM imaging. Applying STORM to cell imaging, microtubules and clathrin-coated pits (CCPs) were imaged. The STORM image showed a drastic improvement in the resolution of the microtubule network compared to the conventional fluorescence image. The intrinsic imaging resolution, as determined from the FWHM of point-like clusters of localizations, was 30 ± 1 nm for each of the two color channels. The green channel revealed filamentous structures as expected for microtubules, while the red channel revealed predominantly spherical structures, representing clathrin-coated pits and vesicles. The STORM image allowed the measurement of the size distribution of CCPs, which agrees quantitatively with the size distribution determined using electron microscopy. This technique may be readily expanded to include more colors using the photo-switchable activator-reporter pairs reported here, significantly enhancing our ability to visualize molecular interactions in cells and tissues.