Advances in Cellular Imaging by Cryo-3D Correlative Light and Electron Tomography

Dr Herman Fung

Postdoctoral Fellow, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Advances in cryo-electron tomography (cryo-ET), including cryo-focused ion beam (FIB) milling for the thinning of cells and new cryo-TEM instrumentation, allow the visualization of molecular sociology inside cells in near-native states.

However, a major challenge remains in the localization of structures of interest.

Cryo-correlative light and electron microscopy (CLEM) provides a way to localize structures during sample preparation, imaging, and data analysis through registration with fluorescence data.

Using a prototype cryo-confocal fluorescence microscope developed in collaboration with Leica Microsystems, researchers at EMBL have established a 3D CLEM-guided imaging workflow, benchmarked by localizing lipid droplets inside human cells. [1]

Watch the webinar to see how the researchers used this workflow to localize nuclear pore complexes undergoing turnover in yeast cells. [2]

You will also discover new functionalities in the registration software 3D correlation toolbox (3DCT), [3] and see the open-source GUI-based software, SerialFIB, which was developed for automated and customizable cryo-FIB operations to overcome the labor- and time-intensiveness of current gallium-based FIB milling approaches. [1]

References
1. Klumpe, S, et al. (2021) A modular platform for automated cryo-FIB workflows. Elife 10; e70506.
2. Allegretti, M, et al. (2020) In-cell architecture of the nuclear pore and snapshots of its turnover. Nature 586; 796–800.
3. Arnold J, et al. (2016) Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. Biophysical Journal 110; 860–869.

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