Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Abstract

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozenhydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

In situ structural analysis reveals membrane shape transitions during autophagosome formation

“…Thus, eGFP-Ede1 marks autophagic structures independent of Atg8 overexpression. The starved cells were subjected to a correlative focused ion beam (FIB)-milling and cryo-ET workflow 20,21 : after plunge-freezing, cryo-fluorescence stacks were recorded. Fiducial-based 3D correlation with FIB/SEM and TEM images targeted structures of interest during lamella milling and tilt-series acquisition (Extended Data Fig.…”

“…Target sites corresponding to Atg8 puncta or Ede1 END cargo were 3D-correlated to SEM/IB images in the FIB/SEM microscope (FIB Scios and Aquilos, Thermo Fisher Scientific) using the 3D-Correlation Toolbox (3DCT) 20 . Lamellae were milled in correlated sites as described in a previously published protocol 21 . In a few cases (e.g Extended Data Fig.…”

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

“…2D SEM and FIB views of the squares (with the field of view large enough to include sufficient external microspheres and features as registration points) that contained the region of interest (ROI) were collected at the eucentric height and milling angle. 3D coordinate transform between the 3D Z-stack of the Y5 channel (nanoparticles, emission λ = 680 nm) and the 2D FIB view was conducted through the optimized rigid body 3D transformation algorithm available in the 3DCT package using external 200 nm FluoSpheres (n = 4 to 10) as registration points 36 . The transformed coordinates (X, Y, Z) were then imported into CorRelator to fine tune the deviations in X and Y coordinates introduced by the Z transformation in 3DCT, using the closed-form best-fitting least-square solution.…”

ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study

“…Then, the mini-gate valve was manually opened to allow the cryo-holder to be inserted into ELI-TriScope. After waiting 5 minutes to recover the high vacuum of the chamber and confirming that the tip of the cryo-holder was far from the pole pieces of the electron and ion columns, the cryo-holder was tilted to 30 degrees right in front of the GIS 48 .…”

“…All figures were created using ChimeraX 74 , and all movies were generated in Imaris 9.8.0. experimental procedure of the in situ cryo-ET study is described (left), including steps from cell culture to plunge freezing, cryo-FM imaging, cryo-FIB imaging and correlation, cryo-FIB milling, production of a cryo-lamella and cryo-ET data collection. The reported cryo-CLEM workflows from cryo-FM imaging to cryo-FIB milling can be classified into nonintegrated workflows 29,30,35 and integrated workflows 39,48 . In a nonintegrated workflow, the cryo-FM and cryo-FIB steps are carried out individually and sequentially.…”