Abstract
Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.
Highlights
Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information
Collection of cryo-sections on silicon wafers facilitates Correlative light and electron microscopy methods (CLEM). 100 nm-thick Tokuyasu ultrathin kidney cryo-sections were collected on a 7 × 7 mm silicon wafer (Figs 1a and 2a)
This latter step protects the tissue from drying artefacts[16] and avoids the need of using solvents and embedding in resin, which contribute to major problems in tissue extraction and shrinkage
Summary
Collection of cryo-sections on silicon wafers facilitates CLEM. 100 nm-thick Tokuyasu ultrathin kidney cryo-sections were collected on a 7 × 7 mm silicon wafer (Figs 1a and 2a). The flat silicon surface provides stability, facilitates the handling of the sections compared to EM grids, reduces the deformations in the tissue caused by drying or exposure to a vacuum, and minimizes folds and breaks[20] that are often found in sections collected on formvar film-coated grids[21] (Fig. 2b) This conductive support prevents charging artefacts and is ideal for scanning electron microscopy (SEM) imaging[21]. Super-resolution microscopy with SEM allows a quicker overview signal detection and identification of rare events (Fig. 3c) This method can be applied to multiple labelling approaches and in other tissues and cells (Supplementary Figs 2 and 3)
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