EM Set-up Protocol
- If Serial EM is already running from a previous capture:
- Check to see if prior capture went to completion. Hit OK.
- Close Serial EM.
- Close navigator. Do not save.
- Pull out specimen rod to safety position. Flip pump switch to AIR.
- Remove rod, place in holder and place on bench.
- 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.
- Check HT (should be at 80). Turn ON if necessary. Check filament beam current (should be @ 40 – 55).
- If first start of the day, allow beam to warm up and stabilize for 30 mins.
- Open Serial EM.
- Raise screen (to protect sample in next steps).
- Hit low mag button and go to 150X.
- Turn aperture dial to red dot.
- Lower screen and turn on filament. Wait until fully on (bright green).
- Examine section for damage or large dirt. If found, STOP and leave for Kevin.
- Locate center of tissue (move if necessary).
- Cook tissue:
- Center the beam and condense beam to inner brackets. Make sure you are at 150X!
- Change spot size to 1. Cook for 7 minutes using Burn Wobble (see below).
- Run > Macro 1: Burn wobble.
Bonus: this will start a timer – expand the Log dialogue box to watch the time.
- To stop cooking after the 7 minutes, change spot size to 3.
- Find center spot – Image – Screen Capture – Paste in Paint – Save to desktop: RC2_Sec#_150X.bmp
- Raise screen (to prevent damage to section in next step).
- Return aperture to first white dot (aperture 1; largest white dot on dial).
- Hit mag 1 button.
- Lower screen and lower mag to 600X while spreading beam.
- Find center spot – Image – Screen Capture – Paste in Paint – Save to desktop: RC2_Sec#_600X.bmp
- Go to 2000X.
- Recenter BOTH aperture and beam.
- Find center spot. Place center point in SerialEM.
- Image – Screen Capture – Paste in Paint – Save to desktop: Volume_Sec#_2000X.bmp
- Open Navigator.
- Navigator > Montage and grids > Add circular polygon (or draw polygon for non-connectome volumes) > Enter desired radius (125 for RC2, 45 RPC2).
- Go to 5000X.
- Recenter beam.
- In Acquire dialogue box:
- New file item > Montage > Fit to polygon > OK > OK.
- Create new folder for section number > open folder and save: Sec#.idoc for connectome volumes Bloc#_Grid#.idoc
- Lower screen and narrow the beam to 2 clicks inside the inner brackets.
- Lid on.
- Calibration > Image shift (make sure you are at5000X and spot size 3!!).
- See if calibration says: “THIS IS GOOD”. If not, redo calibration until it does.
- If message pops up: “Do you want to modify all other old measured…” > select NO.
- Navigator > Acquire at points > OK.
- Watch for a moment to ensure capture is proceeding.
- SerialEM will automatically capture the montage. Indicated by status bar stating “capturing X of total # of tiles”.
- Open Notepad. Type the following data on separate lines:
- Any new changes made in settings
- Any relevant observations regarding the capture process or data quality, Examples: “Building demolition next door” or “First capture after transition to copper grids”
- 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
- 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
- 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
- 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 #.