National Institutes of Health/Office of the Director
1DP2GM123494-01
United States
National Science Foundation (NSF, United States)
ECCS-1542148
United States
Citation
Journal: Nat Protoc / Year: 2020 Title: Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Authors: Felix R Wagner / Reika Watanabe / Ruud Schampers / Digvijay Singh / Hans Persoon / Miroslava Schaffer / Peter Fruhstorfer / Jürgen Plitzko / Elizabeth Villa / Abstract: Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron ...Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.
Organism: Homo sapiens (human) / Organ: bone / Tissue: epithelial cell
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Experimental details
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Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
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Sample preparation
Buffer
pH: 7
Grid
Model: Quantifoil R2/1 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE-PROPANE
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 90 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Scios. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.
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Electron microscopy
Microscope
FEI POLARA 300
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
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Image processing
Final reconstruction
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.28 Å / Software - Name: eTomo / Number images used: 66
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