Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy.

摘要: Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with microscopy, both at room cryogenic temperatures. Fluorescence applications cryo-temperatures have typically been limited to localization tagged protein oligomers due known issues extended triplet state duration, spectral shifts, reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics behaviors that could enable more applications. We describe how dialkylcarbocanine DiD, its autoquenching by resonant energy transfer (RET), can be used distinguish fusion lipid bilayer cryo-temperatures. By adapting an established assay work under conditions, identified areas between influenza virus-like particles fluorescently labeled vesicles on cryo-EM grid. This result demonstrates localize functions in addition proteins, RET incorporated successfully into approaches. In case membrane applications, this method provides orthogonal confirmation functional independent morphological description from way bridge room-temperature kinetic assays images.