The cryogenic focused ion beam-scanning electron microscope (cryo-FIB-SEM) is a semi-automated instrument that provides pseudo-three-dimensional imaging of thick samples with ~10-nm resolution or thinning of samples for subsequent imaging on Krios cryo-TEM at nanometer to angstrom resolution. These capabilities broadly support EMSL’s fundamental research in Earth systems.
Supporting the Structural Biology Integrated Research Platform, the Aquilos 2 cryo-FIB-SEM can thin samples to make them compatible with imaging on the Krios cryogenic transmission electron microscope (Cryo-TEM) instrument. Thinning can be performed by lamella generation of samples directly on the grid or by site-selective lift-out, where a region of a sample is plucked out of a tissue or thick sample and then moved and attached to a TEM grid for thinning and downstream imaging. By thinning samples to ~300 to 500 nm in thickness, this approach makes it possible to perform high-resolution electron tomography and sub-tomogram averaging to visualize the architecture of cells or micro-electron diffraction from thick crystals.
Supporting the Rhizosphere Function Integrated Research Platform, the Aquilos 2 cryo-FIB-SEM can perform similar thinning of thick samples such as plant-microbe interactions. Additionally, it can perform cryogenic slice-and-view imaging to visualize the ultrastructure of individual cells and their interactions within a tissue or complex biosystem community.
Aquilos 2 Cryo Focused Ion Beam-Scanning Electron Microscope
- Semi-automated cryogenic batch lamella generation workflow
- Semi-automated cryogenic slice-and-view workflow
- Cryogenic site-selective lift-out workflow
Leica EM-GP vitrification system
- Vitrification of samples < 10 µm in thickness
- Samples adhered to standard 3-mm diameter TEM grid
Leica HPM100 system
- High-pressure freezing and vitrification of samples < 200 µm in thickness
- Samples frozen in copper hats with dimensions of 1.5 mm diameter and 200 µm height
- Semi-automated micro-electron diffraction and cryo-electron tomography of thinned samples with nanometer to Å resolution
Tips for success
- Compatible science samples include
- Biosystems normal and environmentally stressed conditions
- Unicellular and filamentous organisms, cell cultures, and biofilms
- Plant-microbe-fungi interactions under different environmental conditions
- Host-viral/phage interactions
- Properly vitrify the sample to preserve ultrastructure (plunge freezing for samples less than 10 µm thick, high pressure freezing of samples less than 200 µm thick).
- Ensure the feature of interest is strongly characteristic and identifiable. Biological contrast in vitreous ice can be minimal and requires having a clear method for targeting the sample and regions of interest.
- Cell environments in standard growth or culture conditions are preserved until the moment of plunge-freezing, which occurs on a millisecond timescale.
- Typical dimensions of a lamella thinned or lift-out sample field-of-view is around 5 µm x 10–20 µm x 0.3–0.5 µm. Note that the downstream field-of-view imaged on the Krios will be smaller.
- Typical dimensions for a slice-and-view reconstructed field-of-view is around 12–24 µm x 10–20 µm x 10–50 µm.