Differences

This shows you the differences between two versions of the page.

--- glacios_alignment [2022/11/18 12:59] – duranda
+++ glacios_alignment [2023/11/01 20:18] (current) – external edit 127.0.0.1
@@ Line 7: / Line 7: @@
   * Camera gain reference
   * Direct alignment
-  * Objective Astigmatism / Coma-free alignment
+  * Objective Astigmatism / Coma-free alignment (OPTIONAL for screening!)
-==== Gain reference of the K2 camera ===
+==== Gain reference of the K2 camera ====
-This is done in Digital Micrograph on the K2 server.
+This is done in Digital Micrograph (DM) on the K2 server.
+  - Go to an empty region on the sample
+  - Go to high mag (e.g. 45000x)
+  - Insert 150µm C2 aperture
+  - Go to spot size 2
+  - Check on the FluScreen that beam is centered
+  - Digital Micrograph > Camera > Prepare Gain Reference
+  - Perform the gain in Linear mode first
+  - Adjust Beam Intensity to match the number of count on the camera
+  - Proceed to Counting mode
+  - Press 'Yes' to aquire the dark frame reference image (take twice 3 min 30)
+  - At the end of the dark ref update, you will be prompt to lower the dose: insert 30µm C2 aperture, go to spot size 6, adjust C2 lens to 39.6%)
+  - Lift the Fluscreen and aquire Counting Gain Reference Image (you DON'T need to match the number given by DM)
-==== Direct alignment ===
+==== Direct alignment ====
-This is done in microscope interface.
+This is done in the microscope user interface.
+=== Prepare the microscope ===
-  - Insert calibration specimen (position 1 in the autoloader)
+  - Insert the calibration specimen (position 1 in the autoloader)
   - Find an unbroken square
   - Bring sample to eucentric high
   - Press Eucentric Focus on the right control panel
-  -
+  - Set the microscope to the imaging conditions you want to use (e.g. mag 45000x, Spot size 6, C2 aperture 30µm)
+  - Perform direct alignment in microprobe and nanoprobe as follow:
+=== Microprobe ===
+  - Switch the condenser system to microprobe
+  - Open Direct Alignment panel
+  - **DO NOT TOUCH GUN TILT/SHIFT**
+  - Perform Beam Tilt Pivot point X alignment
+  - Perform Beam Tilt Pivot point Y alignment
+  - Check C2 aperture centering
+  - Perform Rotation Center alignment
-Open the Direct alignment tab
+=== Nanoprobe ===
+  - Switch the condenser system to nanoprobe
+  - Open Direct Alignment panel
+  - Again, **DO NOT TOUCH GUN TILT/SHIFT**
+  - Redo Beam Tilt Pivot point X alignment
+  - Redo Beam Tilt Pivot point Y alignment
+  - Check C2 aperture centering
+  - Redo Rotation Center alignment
-DO NOT TOUCH GUN TILT/SHIFT
+Eventually, recenter the beam
+==== Objective Astigmatism / Coma-free alignment ====
+This is done in SerialEM. You need to have a sample inserted to do it. Re-use the calibration specimen or a carbon Quantifoil (not a gold grid!). Use the Imaging state you will use for data collection (e.g. mag 45.000kx, spot 6, C2 aperture 30 µm, C2 lens 39.6%)
+  - Insert and center Objective Aperture
+  - In SerialEM, Focus/Tune > Correct Astigmatism by CTF
+  - In SerialEM, Focus/Tune > Coma-free by CTF