Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.
Keywords: Correlative light and electron microscopy; GFP; High pressure freezing; In-resin fluorescence; SMLM; Single-molecule localization microscopy; Super-resolution microscopy; Tannic acid; Transmission electron microscopy.