Recent advances in correlative cryo-light and electron microscopy

Abstract

Correlative light and electron microscopy (CLEM) pipelines serve to integrate the imaging modalities of fluorescence light microscopy (FLM) and cryogenic electron microscopy (cryo-EM) to produce contextually relevant high-resolution structural snapshots of biological systems. Innovations in sample preparation, instrumentation, imaging, and data processing have advanced the field of cryo-EM. This review focuses on prior work and recent developments in the field of cryo- EM that support further integration of technologies for correlative microscopy workflows.

Figure 1.. Correlative cryo-ET workflow.

The cryo-CLEM process starts with fluorescent labeling of the biological target. Live-cell FLM prior to freezing is routinely applied for RT-FLM-TEM correlation (blue path) while assessing sample quality. Plunge-freezing or high-pressure freezing (HPF) are used to vitrify the labeled sample. The frozen sample can be imaged under LN₂ via various FLM modalities equipped with cryo-stages to obtain cryo-FLM data. Using cryo-FLM done either externally or via an integrated FLM modules, 3D correlative cryo-FIB milling is used to remove unwanted materials to expose fluorescent ROI accessible to TEM. Alternatively, 2D cryo-FLM-TEM could be performed when thinning is not required (green path). This may be an iterative procedure where the sample could go back and forth between FLM and FIB process. In the end, cryo-FLM data of the final thin lamella is obtained. On-the-fly rough and fine correlations are performed to correlate the lamella FLM and cryo-ET acquisition. Post acquisition correlation of the lamella cryo-FLM or cell cryo-FLM (prior to milling) can be used to provide novel structural insights.

Figure 2.. Cryo-FLM-CorRelator-cryo-EM workflow to guide multi-channel fluorescent ROI acquisition.

(A-B) Raw (A) and small volume computational clearing (SVCC)-processed (B) cryo-FLM image of HeLa cells expressing GFP-labeled actin (green signal) and infected by respiratory syncytial virus (RSV, red signal) with the viral Fusion (F) glycoprotein native-immunolabeled (pink signal). (C) Cryo-TEM image of the same cells in A and B. (D) Superimposition of B and C through the CorRelator transformation. Hole centroids noted in blue. (E) SerialEM screenshot of real-time CorRelator-SerialEM correlation that guides the ROI acquisition. (F) High-pass filtered cryo-EM image of the red boxed and neighboring region in D. (G) Tomographic slice of ROI (yellow asterisk in F and E) captures the viral particle scission process from the cell edge. White arrowheads indicate viral ribonucleoprotein complex (RNP), black arrowheads note actin cytoskeleton corresponding to the boxed green signal in D and E, and red arrowhead for the narrowing of the RSV filament during scission. Scale bars = 20 μm in A-B, 10 μm in C-D, F. 500 nm in G.