glacios_alignment
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| glacios_alignment [2022/11/18 10:14] – created duranda | glacios_alignment [2023/11/01 20:18] (current) – external edit 127.0.0.1 | ||
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| ===== Basic Microscope Alignment ===== | ===== Basic Microscope Alignment ===== | ||
| - | THe microscope should be roughly aligned when you arrive. | + | 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 ' | ||
| + | - 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/ | ||
| + | - 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/ | ||
| + | - 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 | ||
| - | ==== Title 2 ==== | ||
glacios_alignment.1668766466.txt.gz · Last modified: (external edit)