Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Abstract

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

“…To achieve this resolution improvement, several challenges must be addressed when adapting SRM for cryosamples . Vitrified samples must be kept below 133 K at all times to avoid devitrification and crystallization of the vitreous water in the sample, which causes severe structural damage to the near‐native sample and impedes subsequent cryoEM imaging . Also, many SRM techniques depend on nonlinear effects of fluorophores that become apparent when illuminated with high intensity light .…”

“…Also, many SRM techniques depend on nonlinear effects of fluorophores that become apparent when illuminated with high intensity light . However, intense illumination can cause devitrification . We have systematically determined the illumination intensity that is sufficient to perform cryoSRM without any sample devitrification (Calibration Protocol #2 in the Supporting Information), and without using cryoprotectants to maintain the native environment of the biological specimens .…”

“…However, the resolution gap between cryoFM and cryoEM is higher than that of RT‐CLEM; the optical resolution of cryoFM is considerably lower than that achievable with RT‐FM due to the long working‐distance objective lenses with a lower numerical aperture (NA), limiting the resolution of cryoFM to roughly 400 nm, while cryoEM can achieve sub‐nanometer resolution . This resolution gap hinders accurate correlation, and hence interpretability, of cryoCLEM …”

“…By repeating the process of imaging different individual fluorophores and precise localization, a super‐resolved image can be reconstructed by combining the localizations found in all images. SMLM has been successfully adapted for various RT‐CLEM workflows, and extended to cryoCLEM . We recently described super‐resolution cryoCLEM (SR‐cryoCLEM) on biological samples using genetically encoded fluorescent proteins and readily available equipment .…”

“…SMLM has been successfully adapted for various RT‐CLEM workflows, and extended to cryoCLEM . We recently described super‐resolution cryoCLEM (SR‐cryoCLEM) on biological samples using genetically encoded fluorescent proteins and readily available equipment . In the protocol described herein, we explain how we perform SR‐cryoCLEM on vitrified intact mammalian cells, without compromising successive cryoEM imaging ( Figure and Supporting Information).…”