TITLE: Grid patterning protocol for Cryo-FIB/ET workflow
AUTHORS: Farhaz Shaikh, Josh Hutchings, Tamar Basiashvili, Elizabeth Villa

[DESCRIPTION]
Cryo-Electron Tomography combined with Cryo-Focused Ion Beam milling provides a novel way to study the structure of proteins and the architecture of organelles in situ. Electron microscopy grid preparation is a key step toward a successful Cryo-FIB/ET workflow execution. Here we provide an optimized protocol to generate photo-micropatterns on the grids to enhance cellular deposition and accessibility for Cryo-FIB milling. 

[STEPS]
SECTION: 1. Passivation
1. Place a PDMS stencil (14 mm with 2x2 4 mm wells)into a 35 mm confocal dish (MatTek P35G-1.5-20-C). 

SECTION: 2. Preparing Microscope
7. Switch on a light source, microscope, microscope light, Primo (button on the back and key to the right), PC, then PCO camera – in that order.
SECTION: 2. Preparing Microscope
9. Open Micromanager. Select Leonardo from plug-ins menu.

SECTION: 1. Passivation
2. Glow-discharge both sides of the grids—standard settings suffice (20 mA, 1 min hold, 3-sec hold). Discharge gold side up first. Then discharge carbon film side up.
SECTION: 1. Passivation
3. While grids are discharging you can place 1 uL of water in the center of each of the PDMS stencil sections. This is intended to keep grids in place.

SECTION: 1. Passivation
5. After grids are discharged, add the grid to the center of each of the PDMS wells. Make sure the carbon film side is up. 

SECTION: 1. Passivation
4. Get PLL-PEG (100 ul of 1ug/ml) from the -20 freezer. Dilute two fold to working concentration with 100 ul of HEPES buffer (10 mM, pH 7.4).
SECTION: 1. Passivation
6. Apply 10 uL of the PLL-PEG solution onto each grid. Start a timer for 1 hour. After 1 hour, you can begin patterning. 60 min 


SECTION: 2. Preparing Microscope
8. Switch objective to 20x lens with correction collar (S plan Fluor20x), set filter cube #6, and set condenser turret to #7.
SECTION: 2. Preparing Microscope
10. We recommend using a glass slide with yellow highlighter to verify and focus the Primo laser. The laser can be toggled on/off in Leonardo when autoscale is on.
SECTION: 3. Patterning
11. Remove PLL-PEG solution from the grid.
SECTION: 3. Patterning
12. Wash with 10 uL of DPBS four times, try not to touch the grid. During each wash, pipette up and down four times.

SECTION: 3. Patterning
13.  Blot extra liquid around grids. 
SECTION: 3. Patterning
14. Add 1 uL of PLPP onto the grid.
SECTION: 3. Patterning
15. Place the dish onto the microscope stage, drive stage to grid center and
focus on the foil giving the sharpest view of holes in the foil mesh.

SECTION: 3. Patterning
16. Set contrast by pressing and unpressing autoscale.
SECTION: 3. Patterning
17. Automatic Patterning: Load TEM wizard and specify settings. 
Select "Add Grid"
Select "Automatic execution"
Select "Lock"
Select "Rotate"
Select "Finish
 
SECTION: 3. Patterning
18. Manual Patterning: 
Select "MicroPatterning"
Select desired pattern
Set desired ration and angle 
Set dose to 1000
Select "Lock"
Select "Play" 
SECTION: 4. Finishing up
19. Wash the grids with 10ul of DPBS four times, try  not to touch the grid. During each wash, pipette up and down four times. 
SECTION: 4. Finishing up
20. Leave 10ul of DPBS on the grids.  
SECTION: 4. Finishing up
21. Repeat the patterning steps for each grid you have.