Aug 07, 2023
Version 1
Cryo-FIB Milling protocol for mammalian cells V.1
This protocol is a draft, published without a DOI.
- Josh Hutchings1,
- Tamar Basiashvili2,
- Elizabeth Villa1,3
- 1Biological Sciences, UCSD;
- 2Chemistry and Biochemistry, UCSD;
- 3HHMI
Protocol Citation: Josh Hutchings, Tamar Basiashvili, Elizabeth Villa 2023. Cryo-FIB Milling protocol for mammalian cells. protocols.io https://protocols.io/view/cryo-fib-milling-protocol-for-mammalian-cells-cxtyxnpw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 27, 2023
Last Modified: August 07, 2023
Protocol Integer ID: 85592
Keywords: Cryo-FIB milling, Aquilos , Cryo-EM, In situ structural biology
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000519
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Abstract
Cryo-Electron Tomography (Cryo-ET) and Cryo-Focused Ion Beam (Cryo-FIB) milling provides insight into the structure and architecture of various proteins and organelles in cells. Mammalian cells require to be thinned into sections of ~150-200nm to gain high-resolution information about the contents of the cell. Cryo-FIB milling allows us to ablate regions of cells leaving behind a thin section of cellular material called Lamella. In this protocol, we describe how to manually prepare lamellae using a dual-beam FIB/SEM microscope (Aquilos 2+ system).
Materials
- Aquilos Cryo-FIB/SEM dual beam microscope
- Liquid Nitrogen
- Cellular sample on an autogrid designated for milling
1. Before you start
1. Before you start
1h
1h
Check the status of the high-pressure LN2 tank. If empty change it. Set the regulator valve to 80psi.
Fill 4L dewer with clean liquid nitrogen (LN2) for sample loading.
Fill the heat exchanger tank with liquid nitrogen.
Once filled place a heat exchanger rod into the tank. Set the flow rate to 190-210 and start cooling down the stage.
Retrieve the grids intended for milling.
Create a directory on your computer to save the images from the session.
2. Sample loading
2. Sample loading
30m
30m
Prepare the loading station. Slowly add clean liquid nitrogen to the basin and wait till the LN2 equilibrates.
Load the grids of interest into the sample shuttle. Have grids oriented so that the milling notch is facing upwards.
Once loaded, top-up LN2 in the loading station and maintain to keep the open grid box cold. Leave the base lid on.
Close the shuttle and place it in the loading position.
Insert the shuttle into the microscope using the sample transfer rod.
3. SEM and FIB Overview images
3. SEM and FIB Overview images
30m
30m
On the microscope set the scan rotation to 180o for both SEM and FIB view.
Move the stage to the Mapping position and drop SEM magnification to the lowest possible magnification. Check the focus and take an overview image and save it with a desired name. Assess the distribution of cells in SEM overview and do a rough count of mill-able cells.
Check hydration of cells in FIB view. Move the stage to the Milling position. Set the current to 10pA, and set it to a magnification that includes most of the center of the grid (or the majority of cells). Check focus and take snapshots and save it in a designated folder.
Repeat for the second loaded grid if applicable. If you are happy with the grids and cell distribution, put away open grid boxes in the loading station.
4. Setting up MAPS project
4. Setting up MAPS project
20m
20m
For chosen grid, find focus and link focus to the stage. In the mapping position increase the magnification to 250 in the SEM view. Find a focus with the - Reduced area. Link the focus position to the stage.
In the SEM lower the magnification to 59x. Open the latest version of MAPS.
Start a new project in MAPS.
Take a quick SEM snapshot with MAPS.
Add a 4x4 tileset and position it over the middle of the snapshot. Set the focus to Fixed and click RUN.
5m
5. Selecting Lamellae sites
5. Selecting Lamellae sites
10m
10m
In MAPS select cells that you would like to mill. Add lamellae sites and rank them in order of preference.
Find the eucentric height for each lamellae site.
Drive to the eucentric position and select calculate.
Check SEM centering at medium magnification and focus accordingly
Tilt step of 10o, click the LEFT arrow, update SEM and recenter by double-clicking on the desired feature. Repeat until stage angle = 5o.
Once centered at 5o stage tilt, click to calculate eucentric position.
Repeat the above steps for each lamella site.
6. Pre-Milling preparation
6. Pre-Milling preparation
15m
15m
Sputter coat the grids.
Click on Prepare for the sputtering button. Choose conditions you would like to use for sputtering (i.e. 30mA, 0.1mbar, 30 sec). Press Run and wait. Once done recover the system.
Wake up the beam and move back to the Mapping position. Take a new lowest magnification overview, save the image, and note the contrast change.
Deposit PT on the grids.
Move to the deposition position for the chosen grid. Click on the Cryo-TEM preparation tab in xTUI. Under the Cryo-GIS deposition tab, enter the grid number and set the desired time for the deposition. (e.g. 60 seconds). Click Start to start deposition.
Move back to the mapping position, take an overview, save the image, and note the contrast change.
7. Manual Cryo-FIB Milling
7. Manual Cryo-FIB Milling
45m
45m
Go to the Milling position in xTUI and set the desired milling angle. For mammalian cells use 10-11'. Make a note of this for cryo-TEM data collection.
In MAPS, drive to eucentric position. Check focus and centering in SEM. (~1200x, 2kV)
With FIB at ~2000x at 10pA, center the same cell in FIB view with beam shift knobs and check focus. Increase the magnification to fill FOV with most of the cell.
Load a rectangle pattern preset and position at 10pA.
Note
Preset parameters used:
Width 12mm - length - 5mm
The separation between the two patterns - 3-4mm
Stress relief cuts - 500nm
Approximate milling routine:
Note
Use scan rotation to make the milling pattern and lamella parallel for more even/smoother milling.
0.5 nA, 12 um X-width, 3um separation
0.3 nA, 11.5 um X-width, 1.5 um separation
0.1 nA, 11 um X-width, 750 nm separation
50 pA, 10.5 um X-width, 400 nm separation
30 pA, 10 um X-width, 200 nm separation
30 pA, 0.5’ under-tilt, 10 um X-width
Mill under-side from lamella back. 30 pA, 0.5’ over-tilt, 10 um X-width, mill top-side from lamella back.
Repeat the above steps for all desired lamella.
Things to monitor during milling.
GIS layer at the front of the lamella. When this breaks through, becomes very difficult to mill.
Observed transparency in SEM at 2 kV. Becomes homogenous and dark at thickness < 200 nm.
Polishing: Return to each lamella and aim for <10 mins of polishing for each.
10 pA top-side only, X-width 10 um, no over/under-tilt.
7. Finishing up
7. Finishing up
15m
15m
Retrieve grids from the microscope and store them.
Set the flow of the heat exchanger to 10mg/s and remove the heat exchanger from the dewer.
Update your notes and sample information. Update personal spreadsheets/logs with information about the session.
Protocol references
Lam V, Villa E. Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography. Methods Mol Biol. 2021;2215:49-82. doi: 10.1007/978-1-0716-0966-8_3. PMID: 33367999; PMCID: PMC8836527.