SerialEM Screening Standard Operating Procedure

1. Purpose

Successfully screen grids with SerialEM

2. Definitions:

2.1 SerialEM is a data collection software from UC Boulder

2.2 TEM UI is the microscope user interface located on the microscope PC

2.3 Autoloader Inventory is an automated procedure that identifies filled/empty slots in the cassette

3. Supplies & Equipment 

3.1 Microscope

3.2 SerialEM

4. Procedure

4.1. Pre-screening Software and Microscope Checks

  • TEM UI/Microscope PC:
    • Confirm Autogrids are loaded
    • Perform Autoloader Inventory
    • Confirm SerialEM server is running
    • Confirm 150µm C2 aperture is inserted
  • SerialEM PC:
    • Confirm “Low Dose Mode” is enabled
    • Verify Imaging States are returning images:
      • Open Column Valves
      • Open Navigator (Menu: Navigator > Open)
      • Press “Search” in Camera Panel
      • Center stage over gridsquare
      • Press “View”
      • Press “Autofocus”
      • Press “Record”
    • If any of the Imaging States are in error, contact center staff for assistance.

4.2. Load Screening Grid

  • You can load a screening grid either from the Microscope PC or the SerialEM PC
  • TEM UI: Autoloader OCX: Click Slot, Click Load
  • SerialEM: Menu: Script > One-Line Scripts : LoadCartridge # (1-12)

4.3. Collect Low Magnification Montage – Whole Grid Overview

  • Open Navigator window (Menu: Navigator > Open)
  • Create and save LMM.mrc (Menu: Navigator > Montaging & Grids > Setup Full Montage)
  • (optional) Find eucentric height of central gridsquare. (Menu: Tasks > Eucentricity > Rough Eucentric)
  • Start montage collection (Montage Panel: Start)

4.4. Identify Gridsquares to Screen

  • Identify at least two gridsquares to screen; one thick, one thin
  • Left-click the center of a gridsquare and in the navigator window, press “Add Marker”
    • Repeat for each gridsquare
  • In the navigator window, select a gridsquare’s marker and click “Go To XY”
  • Once the stage stops moving, acquire a View image
  • Find eucentric height
    • For AuFoil grids, center stage with partial gridbar for accuracy. (see figure X)

4.5. Screen Gridsquare and Acquire High Magnification Record Images

  • Acquire View Image
  • Center the stage over a hole (see 7.2 for procedural information)
  • Adjust Autofocus Position (First gridsquare/Once per grid)
    • Low Dose Panel: Adjust Focus Position, select Focus
    • View Image: Left-click focus area on support film between holes
    • Low Dose Panel: select None
  • Autofocus Panel: Click “Autofocus”
    • (optional) Adjust Defocus target: Menu: Focus & Tune > Set Target (0 to -5.0µm)
  • Camera Panel: click “Record”
  • Save Record Image
    • Menu: Window > Save Image Snapshot
  • Repeat on at least one hole from same gridsquare
  • Navigate to next gridsquare of interest and repeat section 4.5

4.6. Save LMM with Targets/ Screened Squares

  • Double click the LMM in Navigator window to load it
  • Save Image Snapshot

4.7. Repeat for each grid, starting at 4.2

  • Load next grid, acquire LMM, identify gridsquares, acquire and save high magnification images

4.8. Shutdown

  • After screening is complete: Close Column Valves, Load Grid 1 (Cross-Grating)
  • Close all open maps with Menu: File > Close