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

Maarten W. Tuijtel,Thomas H. Sharp,Frank G. A. Faas,Abraham J. Koster,Stefan Jakobs

doi:10.1038/s41598-018-37728-8

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.

Highlights

Cryo electron microscopy provides in-situ structural information of biological samples at high resolution, but it is essential that samples are fixed in their near-native state by vitrification
Devitrification invariably started as a region of crystalline ice in the centre of the grid squares, which increased in size with increasing illumination intensity (Fig. 1d–f)
We described the application of single-molecule localisation microscopy (SMLM) on cryosamples to increase the interpretability of cryoCLEM, and addressed several challenges

Summary

Introduction

Cryo electron microscopy (cryoEM) provides in-situ structural information of biological samples at high resolution, but it is essential that samples are fixed in their near-native state by vitrification. Several workflows have previously been developed to combine SR microscopy and EM23–26 Combining these techniques is challenging, as the fixation and staining protocols for EM are often not compatible with fluorescence[21,27]. These methods suffer from the use of heavy-metal staining instead of directly imaging biomaterial, as is done with cryoEM. The carbon support film can be replaced by more transparent materials, such as plastics like formvar[30] These materials have a high fluorescent background and require ultraclean fabrication to be compatible with cryoFLM30. Structural studies have shown that while the chromophore inside Padron can isomerise between the cis and trans state at 100 K while crystallised, for other FPs, such as Dronpa, mTFP0.7 and IrisFP, isomerisation when vitrified is prohibited by several amino acid residues blocking this reorganisation[42]

Methods

Results

Conclusion