Cryo-fib Milling
Cryo-fib Milling - Related Videos
09:06
Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
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Cited by 2 •
2021
We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.
08:29
Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells
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Cited by 2 •
2025
This workflow enables lamella production targeting fluorescently labeled biological structures that are small (<1 μm in axial extent) and rare (1 copy per cell) using a cryogenic tri-coincident imaging platform. This platform integrates fluorescence microscopy, focused ion beam milling, and scanning electron microscopy at a single focal position and enables simultaneous fluorescence microscopy while milling.
09:16
FIBS-enabled Noninvasive Metabolic Profiling
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2014
A description of how to calibrate Förster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented. The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.
08:20
Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
0 Views •
Cited by 17 •
2021
Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.
08:10
Precision Milling of Carbon Nanotube Forests Using Low Pressure Scanning Electron Microscopy
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2017
Low pressure scanning electron microscopy in a water vapor ambient is used to machine nanoscale to microscale features in carbon nanotube forests.
FAQs
The focused ion beam removes material by sputtering, meaning energetic ions displace atoms or particles from a selected surface. Repeating this removal layer by layer exposes progressively deeper regions while maintaining control over the milling location. This approach allows researchers to access internal interfaces, microstructures, or defects without preparing the entire specimen by conventional methods.
Cryogenic conditions help preserve frozen, hydrated, or temperature-sensitive specimens while material is removed. Keeping the sample at low temperature limits structural changes that could occur during preparation and helps retain features closer to their original state. This preservation is especially important when interpreting internal organization, interfaces, or microstructures that may be altered by warming.
Unlike preparation approaches that may distort or obscure internal features, Cryo-fib Milling combines localized material removal with low-temperature preservation. The method provides direct access to selected subsurface regions while limiting changes in the specimen. As a result, researchers can examine structures that might be difficult to interpret after preparation at ordinary temperatures or with less targeted removal.
In engineering and materials research, the technique can expose interfaces, microstructures, defects, and other complex internal features. These targets often determine how a material or component is organized and may be hidden beneath the surface. Controlled access makes the method useful for investigating relationships between internal structure and observed material behavior, while preserving temperature-sensitive regions during preparation.
A specimen is maintained at cryogenic temperature while a selected region is exposed to the focused ion beam. The beam then removes that region incrementally through layer-by-layer sputtering, producing access to deeper internal material. The resulting prepared area can support cross-sectional examination, high-resolution imaging, or sequential analysis for three-dimensional reconstruction.
Prepared specimens can provide cross-sectional views of internal organization and support high-resolution imaging of features that are not accessible at the surface. Repeated access through successive layers can also contribute to three-dimensional reconstruction. In engineering studies, these outcomes help characterize complex microstructures, interfaces, and defects while reducing the risk that preparation has concealed or altered them.