Dual-colour super-resolution cryoCLEM in mammalian cells using the fluorescent proteins rsTagRFP and rsEGFP2

• Dual-colour SRcryoCLEM has been limited by a lack of suitable fluorescent labels. • We introduce rsTagRFP as a cryo-compatible reversibly switchable red fluorescent protein. • rsTagRFP and rsEGFP2 allow simultaneous imaging of two distinct targets with 30 nm precision. Correlating super-resolution fluorescence light microscopy with cryo-electron tomography (SRcryoCLEM) is a feasible way of targeting specific proteins of interest for high-resolution cryo-electron tomography (cryoET) imaging within cells. Among different approaches for performing super-resolution fluorescence microscopy on cryogenically preserved samples, cryo-single molecule localization microscopy (cryoSMLM) offers one of the highest imaging resolutions. Thus far, applications of cryoSMLM in SRcryoCLEM have been limited to targeting a single protein structure at a time, as the available palette of cryo-compatible reversibly photoswitchable fluorescent proteins, required for cryoSMLM imaging, is severely limited. Here, we present rsTagRFP and rsEGFP2 as a compatible pair of red and green fluorescent labels that enables dual-colour cryoSMLM, and thus dual-target SRcryoCLEM, in mammalian cells. We demonstrate the simultaneous targeting and identification of two separate structures, MAP2-decorated microtubules and vimentin intermediate filaments, with 30 nm accuracy and within the same cell.