1. Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision
- Author
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Abraham J. Koster, Barbara van der Hoeven, Clara C. Posthuma, Aat A. Mulder, Thomas H. Sharp, Frank G. A. Faas, Maarten W. Tuijtel, and Montserrat Bárcena
- Subjects
0301 basic medicine ,Multidisciplinary ,Materials science ,media_common.quotation_subject ,lcsh:R ,lcsh:Medicine ,Uranyl acetate ,Fluorescence ,Article ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,law ,Transmission electron microscopy ,Microscopy ,Biophysics ,Contrast (vision) ,lcsh:Q ,Nanometre ,Electron microscope ,lcsh:Science ,Fiducial marker ,media_common - 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.
- Published
- 2017