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Cryo-FIB
Sculpting with Ions now prepares frozen cells for cryo-electron tomography.
Live: https://advanced-em.github.io/FIB-view/?mode=cryo
New
- Cryo stage (−196 to −100 °C) with a 45° pre-tilted shuttle, copper cooling braids and an anticontaminator, shown in the 3D instrument with their own instrument cards.
- Two frozen specimens: yeast cells plunge-frozen in vitreous ice on a holey-carbon gold grid, and high-pressure-frozen yeast for cryo slice & view (SE contrast of walls, nuclei, vacuoles and lipid in ice).
- Cryo lamella mode (key
\): sputter coat, cold GIS organometallic Pt, milling angle (stage tilt − 7°), micro-expansion joints, milling passes at the current and target you choose, and a final coat. The true slab is tracked: thickness and t/λ, the damaged layer on each face (Ga⁺ ~42 nm, Ar⁺ ~31 nm, Xe⁺ ~18 nm at 30 kV), devitrification from beam heating or a warm stage, ice contamination growing in real time, bending without relief cuts, and a cryo-ET verdict. - Physics: vitreous ice and frozen cells mill ~4× faster than silicon and charge above ~1.6 kV until coated; the cold GIS layer mills ~10× slower than ice; Ga⁺ 30 keV reaches ~52 nm in ice (binary-collision Monte Carlo).
- Training task Cryo lamella: 200 nm for cryo-ET with the true thickness hidden, and two experiments: A cryo lamella from a frozen cell and Gallium vs argon in ice.
- Voice and AI: "cryo lamella", "sputter coat", "milling angle 12", "mill to 800 nm at 300 pA", "anticontaminator on"; the analysis
cryo_quality. - Python:
fib.cryo(...)and new settings (stage_temp_C,anticontaminator,milling_angle_deg,relief_cuts); examplecryo_lamella.py(Ga vs Ar vs Xe). - MCP:
cryo_lamella(headless recipe → true result) andexhibit_cryo(drive a live exhibit). - Guide: SOP-03 Cryo lamella for cryo-ET (12 pages, attached), with figures and numbers computed by the exhibit.
Tests
npm test (physics, 13 modes × 7 specimens, training tasks incl. cryo, cryo failure modes, grammar), the MCP tests and the Python tests pass.