TITLE: Cryo-FIB Milling protocol for mammalian cells
AUTHORS: Josh Hutchings, Tamar Basiashvili, Elizabeth Villa

[DESCRIPTION]
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). 

[STEPS]
SECTION: 1. Before you start
1. Check the status of the high-pressure LN2 tank. If empty change it. Set the regulator valve to 80psi. 
SECTION: 3. SEM and FIB Overview images
10. 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. 
SECTION: 3. SEM and FIB Overview images
11. 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. 
SECTION: 3. SEM and FIB Overview images
12. 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. 
SECTION: 4. Setting up MAPS project
13. 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. 
SECTION: 4. Setting up MAPS project
14. In the SEM lower the magnification to 59x. Open the latest version of MAPS. 
SECTION: 4. Setting up MAPS project
15. Start a new project in MAPS. 
SECTION: 5. Selecting Lamellae sites
18. In MAPS select cells that you would like to mill. Add lamellae sites and rank them in order of preference. 
SECTION: 5. Selecting Lamellae sites
19. Find the eucentric height for each lamellae site. 

SECTION: 6. Pre-Milling preparation
20. Sputter coat the grids. 
SECTION: 6. Pre-Milling preparation
21. 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. 
SECTION: 6. Pre-Milling preparation
22. Deposit PT on the grids.
SECTION: 6. Pre-Milling preparation
23. Move back to the mapping position, take an overview, save the image, and note the contrast change. 
SECTION: 7. Manual Cryo-FIB Milling
24. 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.  
SECTION: 7. Manual Cryo-FIB Milling
25. In MAPS, drive to eucentric position. Check focus and centering in SEM. (~1200x, 2kV) 
SECTION: 7. Manual Cryo-FIB Milling
26. 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. 
SECTION: 7. Manual Cryo-FIB Milling
27. Load a rectangle pattern preset and position at 10pA. 
 

SECTION: 7. Manual Cryo-FIB Milling
28. Approximate milling routine: 

 

SECTION: 7. Manual Cryo-FIB Milling
29. 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. 
SECTION: 7. Manual Cryo-FIB Milling
28.8. Repeat the above steps for all desired lamella.
SECTION: 7. Manual Cryo-FIB Milling
30. 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.
SECTION: 7. Finishing up
31. Retrieve grids from the microscope and store them.  

SECTION: 7. Finishing up
32. Set the flow of the heat exchanger to 10mg/s and remove the heat exchanger from the dewer. 
SECTION: 7. Finishing up
33. Update your notes and sample information. Update personal spreadsheets/logs with information about the session. 
SECTION: 5. Selecting Lamellae sites
19.1. Drive to the eucentric position and select calculate.
SECTION: 5. Selecting Lamellae sites
19.2. Check SEM centering at medium magnification and focus accordingly 

SECTION: 5. Selecting Lamellae sites
19.3. Tilt step of 10o, click the LEFT arrow, update SEM and recenter by double-clicking on the desired feature. Repeat until stage angle = 5o.  
SECTION: 5. Selecting Lamellae sites
19.4. Once centered at 5o stage tilt, click to calculate eucentric position. 
SECTION: 5. Selecting Lamellae sites
19.5. Repeat the above steps for each lamella site.
SECTION: 7. Manual Cryo-FIB Milling
28.1. 0.5 nA, 12 um X-width, 3um separation
SECTION: 7. Manual Cryo-FIB Milling
28.2. 0.3 nA, 11.5 um X-width, 1.5 um separation

SECTION: 7. Manual Cryo-FIB Milling
28.3. 0.1 nA, 11 um X-width, 750 nm separation
SECTION: 7. Manual Cryo-FIB Milling
28.4. 50 pA, 10.5 um X-width, 400 nm separation
SECTION: 7. Manual Cryo-FIB Milling
28.5. 30 pA, 10 um X-width, 200 nm separation
SECTION: 7. Manual Cryo-FIB Milling
28.6. 30 pA, 0.5’ under-tilt, 10 um X-width
SECTION: 7. Manual Cryo-FIB Milling
28.7. Mill under-side from lamella back. 30 pA, 0.5’ over-tilt, 10 um X-width, mill top-side from lamella back.
SECTION: 1. Before you start
4. Retrieve the grids intended for milling. 
SECTION: 1. Before you start
2. Fill 4L dewer with clean liquid nitrogen (LN2) for sample loading. 
SECTION: 1. Before you start
3. Fill the heat exchanger tank with liquid nitrogen. 
SECTION: 1. Before you start
3.1. Once filled place a heat exchanger rod into the tank. Set the flow rate to 190-210 and start cooling down the stage.
SECTION: 2. Sample loading
6. Prepare the loading station. Slowly add clean liquid nitrogen to the basin and wait till the LN2 equilibrates.  
SECTION: 2. Sample loading
6.1. Load the grids of interest into the sample shuttle. Have grids oriented so that the milling notch is facing upwards.  
SECTION: 2. Sample loading
7. Once loaded, top-up LN2 in the loading station and maintain to keep the open grid box cold. Leave the base lid on. 
SECTION: 2. Sample loading
7.1. Close the shuttle and place it in the loading position. 
SECTION: 2. Sample loading
8. Insert the shuttle into the microscope using the sample transfer rod. 
SECTION: 3. SEM and FIB Overview images
9. On the microscope set the scan rotation to 180o for both SEM and FIB view. 
SECTION: 1. Before you start
5. Create a directory on your computer to save the images from the session. 
SECTION: 4. Setting up MAPS project
16. Take a quick SEM snapshot with MAPS. 
SECTION: 4. Setting up MAPS project
17. Add a 4x4 tileset and position it over the middle of the snapshot. Set the focus to Fixed and click RUN. 
SECTION: 6. Pre-Milling preparation
20.1. 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. 
SECTION: 6. Pre-Milling preparation
22.1.  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.