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.

Super-resolution imaging based on single-molecule localization has been developed for more than a decade. These techniques can break through diffraction limit of fluorescent microscopy and initially improve the resolution by an order of magnitude to ~20 nm, by introducing photoactivatable/photoswitching probes and centroid fitting method. As the demand of biological research, the localization precision of single-molecules was further improved by several state-of-the-art methods in the past several years. This review focuses on the latest developed techniques which have greatly improved the performance of single-molecule localization microscopy, from measurement principle to hardware design. These methods are essential for the study of nanostructures and biomacromolecule dynamics inside of cells.