Cryo-correlative light and electron microscopy (cryo-CLEM) is a powerful technique that combines fluorescence imaging for specific localization with electron microscopy for detailed structural analysis, enabling high-resolution exploration of synaptic structures in neurons. In this study, we employed a cryo-CLEM approach using three independent alignment markers to precisely correlate electron microscopy (EM) images with light microscopy (LM) images of neuronal synapses under cryogenic conditions. This methodology revealed a distinctive pattern of electron densities in the synaptic clefts. Additionally, we were able to capture high-resolution images of presynaptic vesicles in various states, underscoring the potential of cryo-CLEM in advancing synaptic research.

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.