Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

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.

SUBMITTER: Tuijtel MW 

PROVIDER: S-EPMC5585351 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature