Abstract
Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (−195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.
Highlights
Fluorescence light microscopy (FLM) is highly suitable for locating and imaging specific regions of interest within a large field of view
By exploiting the increased brightness of uranyl acetate at cryogenic temperatures, we have established that uranyl acetate can be used as a dual-purpose contrast agent for both transmission electron microscopy (TEM) and cryoFLM
The equivalent staining in each imaging modality allows cell sections prepared for TEM to be imaged by fluorescence light microscopy (FLM), which makes navigation and interpretation of the samples faster and more intuitive, and enables FLM and TEM images to be aligned with nanometre precision
Summary
Fluorescence light microscopy (FLM) is highly suitable for locating and imaging specific regions of interest within a large field of view. With the high resolution and wealth of ultrastructural information available when using TEM comes a concomitant decrease in the ease of which regions of interest can be relocated Combining these advantages of FLM and TEM using correlative light electron microscopy (CLEM) allows the large field of view of FLM to be used to identify rare or transient cellular events for subsequent inspection with the high spatial resolution of TEM7, 8. Typical sample preparation for TEM includes the use of chemical fixatives and polymers that are autofluorescent, can quench the fluorescence of fluorophores, and induce changes at the ultrastructural level[18,19,20,21,22] Despite these difficulties, multiple approaches for combining light and electron microscopy have been developed[9, 23]. We show that using uranyl acetate as a dual-purpose stain can increase the accuracy and ease of correlation when compared to fluorescent beads, and apply this technique by performing multi-colour CLEM of virus-induced ultrastructural modifications
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