EM Set-up Protocol

1. If Serial EM is already running from a previous capture:
   1. Check to see if prior capture went to completion. Hit OK.
   2. Close Serial EM.
   3. Close navigator. Do not save.
2. Pull out specimen rod to safety position. Flip pump switch to AIR.
3. Remove rod, place in holder and place on bench.
4. Insert new rod and flip pump switch to PUMP. Do NOT turn yet. Let pump down to green values. Then, twist rod and fully insert, gently.
5. Check HT (should be at 80). Turn ON if necessary. Check filament beam current (should be @ 40 – 55).
6. If first start of the day, allow beam to warm up and stabilize for 30 mins.
7. Open Serial EM.
8. Raise screen (to protect sample in next steps).
9. Hit low mag button and go to 150X.
10. Turn aperture dial to red dot.
11. Lower screen and turn on filament. Wait until fully on (bright green).
12. Examine section for damage or large dirt. If found, STOP and leave for Kevin.
13. Locate center of tissue (move if necessary).
14. Cook tissue:
    1. Center the beam and condense beam to inner brackets. Make sure you are at 150X!
    2. Change spot size to 1. Cook for 7 minutes using Burn Wobble (see below).
    3. Run > Macro 1: Burn wobble.

Bonus: this will start a timer – expand the Log dialogue box to watch the time.

3. To stop cooking after the 7 minutes, change spot size to 3.

15. Find center spot – Image – Screen Capture – Paste in Paint – Save to desktop: RC2_Sec#_150X.bmp
16. Raise screen (to prevent damage to section in next step).
17. Return aperture to first white dot (aperture 1; largest white dot on dial).
18. Hit mag 1 button.
19. Lower screen and lower mag to 600X while spreading beam.
20. Find center spot – Image – Screen Capture – Paste in Paint – Save to desktop: RC2_Sec#_600X.bmp
21. Go to 2000X.
22. Recenter BOTH aperture and beam.
23. Find center spot. Place center point in SerialEM.
24. Image – Screen Capture – Paste in Paint – Save to desktop: Volume_Sec#_2000X.bmp
25. Open Navigator.
26. Navigator > Montage and grids > Add circular polygon (or draw polygon for non-connectome volumes) > Enter desired radius (125 for RC2, 45 RPC2).
27. Go to 5000X.
28. Recenter beam.
29. In Acquire dialogue box:
    1. New file item > Montage > Fit to polygon > OK > OK.
    2. Create new folder for section number > open folder and save: Sec#.idoc for connectome volumes Bloc#_Grid#.idoc
30. Lower screen and narrow the beam to 2 clicks inside the inner brackets.
31. Lid on.
32. Calibration > Image shift (make sure you are at5000X and spot size 3!!).
    1. See if calibration says: “THIS IS GOOD”. If not, redo calibration until it does.
    2. If message pops up: “Do you want to modify all other old measured…” > select NO.
33. Navigator > Acquire at points > OK.
34. Watch for a moment to ensure capture is proceeding.
35. SerialEM will automatically capture the montage. Indicated by status bar stating “capturing X of total # of tiles”.
36. Open Notepad. Type the following data on separate lines:
    1. TEM#
    2. Bloc#
    3. Grid#
    4. Investigator
    5. Operator
    6. Any new changes made in settings
    7. Any relevant observations regarding the capture process or data quality, Examples: “Building demolition next door” or “First capture after transition to copper grids”
    8. Save as Sec# notes.txt for connectome volumes or Bloc#_Grid#notes.txt for other captures (Be aware the notes.txt remains with the data and can be seen online with the published data.) In the same folder as the capture
37. Once capture is complete, copy the notes.txt file and the 150X, 600X, and 2000X screen captures and the capture data into a folder labeled
    1. For Connectome captures: with the section number and upload to volume folder on Dropbox (e.g RC2, RC3, RPC2, etc).in a subfolder titled either TEM1 or TEM2 depending on which scope it was captured
    2. For Non connectome projects: label capture folder Bloc#_Grid# and put it in in a subfolder titled either TEM1 or TEM2 depending on which scope it was captured inside a folder named Investigator_ExperimentalSeriesName on DROPBOX.

*If capture is a redo, add “redo” to file name after section #.