Abstract
Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.
Highlights
Correlated light and electron microscopy (CLEM) provides its users both the specificity of fluorescence microscopy (FM) and the resolution of electron microscopy (EM), and has become an established technique with many variations (Muller-Reichert & Verkade, 2017)
A fluorophore is typically embedded in one membrane; upon fusion, the fluorophore is diluted in the expanded membrane and gives a fluorescence signal, typically dequenching
We demonstrated that the established DiD-based fusion assay (Rust et al, 2004; Gui et al, 2016) can be incorporated into a cryo-CLEM workflow to locate, target, and image membrane fusion sites
Summary
Correlated light and electron microscopy (CLEM) provides its users both the specificity of fluorescence microscopy (FM) and the resolution of electron microscopy (EM), and has become an established technique with many variations (Muller-Reichert & Verkade, 2017). CLEM has been widely applied to study membrane remodeling and virus–host cell interactions (Kopek et al, 2012; Kukulski et al, 2012a; Lebrun et al, 2014; Li et al, 2014; Martinez et al, 2014; Romero-Brey et al, 2015; Bykov et al, 2016) In these cases, the object of interest is too small or rare to be found without relying on FM, while the structure is smaller than the point spread function of the light microscope and must rely on EM for its characterization
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