Cryogenic superresolution correlative light and electron microscopy of vitreous sections.

Fluorescence microscopy and electron microscopy complement each other as the former provides labelling and localisation of specific molecules and target structures while the latter possesses excellent revolving power of fine structure in context. These two techniques can combine as correlative light and electron microscopy (CLEM) to reveal the organisation of materials within the cell. Frozen hydrated sections allow microscopic observations of cellular components in situ in a near-native state and are compatible with superresolution fluorescence microscopy and electron tomography if sufficient hardware and software support is available and a well-designed protocol is followed. The development of superresolution fluorescence microscopy greatly increases the precision of fluorescence annotation of electron tomograms. Here, we provide detailed instructions on how to perform cryogenic superresolution CLEM on vitreous sections. From fluorescence-labelled cells to high pressure freezing, cryo-ultramicrotomy, cryogenic single-molecule localisation microscopy, cryogenic electron tomography and image registration, electron tomograms with features of interest highlighted by superresolution fluorescence signals are expected to be obtained.

Cryogenic superresolution correlative light and electron microscopy on the frontier of subcellular imaging.

Electron microscopy (EM) reveals cellular ultrastructure at high definition but faces the challenges of identification of specific subcellular structures and localization of specific macromolecules, whereas fluorescence microscopy (FM) can label and localize specific molecules in cells. Correlative light and electron microscopy (CLEM) combines the advantages of both microscopic techniques. Imaging vitreous hydrated samples at cryogenic temperatures using CLEM enables observations of cellular components of interest and their cellular context in a near-native state. This cryo-CLEM approach is further strengthened by incorporation of superresolution fluorescence microscopy, which can precisely pinpoint targets on electron micrographs. Cryogenic superresolution correlative light and electron microscopy (csCLEM) is an emerging and promising imaging technique that is expected to unveil its full power in ultrastructural studies. The present review describes the logic and principles behind this technique, how the method is implemented, the prospects, and the challenges.

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy.

Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ∼13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).