===== Basic Microscope Alignment =====
The microscope should be roughly aligned when you arrive.

__//**There is no need to do a complete alignment every session, if the microscope behaves strangely, ask for help!**//__

You should perform only two or three steps:
  * Camera gain reference
  * Direct alignment
  * Objective Astigmatism / Coma-free alignment (OPTIONAL for screening!)

==== Gain reference of the K2 camera ====
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 ====
This is done in the microscope user interface.

=== Prepare the microscope ===
  - 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

=== 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

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