perfectem 0.9.5

Installation

Requirements:

• python >= 3.8

• matplotlib, mrcfile, numpy, scipy

• SerialEM Python module will be compiled during installation unless already present

py -m pip install perfectem

pip install perfectem

py -m pip install matplotlib mrcfile numpy scipy perfectem --no-index --find-links .
py -m pip install -e <perfectem_source_directory>

pip install matplotlib mrcfile numpy scipy perfectem --no-index --find-links .
pip install -e <perfectem_source_directory>

Available scripts

• AFIS validation

  • Specification (Krios): coma < 750 nm, astigmatism < 10 nm for 5 um shift. Glacios: coma < 1200 nm, astigmatism < 15 nm for 6 um shift

  • Description: Measure residual beam tilt and astigmatism at different image shift positions while EPU is open.

• Atlas realignment

  • Specification: none

  • Description: Compare the shift and rotation between two atlases acquired when reloading the same grid.

• Magnification anisotropy

  • Specification: <1%

  • Description: Acquire a defocus series and plot astigmatism versus defocus. Calculate anisotropy by estimating deviation from linear behaviour.

• C2 Fresnel fringes

  • Specification: on FFI system there should be <5 fringes at 96 kx in nanoprobe close to focus

  • Description: Take a picture of a flood beam to see if the fringes from C2 aperture extend all the way to the center (non-FFI systems).

• Eucentricity check

  • Specification: <2 um in X/Y, <4 um defocus (Krios G2, G3, G3i)

  • Description: Estimate X,Y and defocus offset while tilting the stage.

• Gain reference check

  • Specification: none

  • Description: Take a picture of a flood beam and check the auto-correlation image.

• Gold diffraction

  • Specification: none

  • Description: Take a high magnification image of Au-Pt and check the diffraction spots up to 1 A in all directions.

• Information limit

  • Specification (Krios < G4): 0.14 nm at 0 tilt, 0.23 nm at 70 deg. tilt. Glacios: 0.23 nm at 0 tilt, 0.34 nm at 70 deg. tilt.

  • Description: Take two images with a small image shift (0.12 nm), add them together and calculate FFT. You should observe Young fringes.

• Point resolution

  • Specification (Krios): 0.2 nm at 73 nm defocus. Glacios: 0.24 nm at 82 nm defocus.

  • Description: Take a high-resolution image on carbon (Pt-Ir grid recommended) at extended 1.2 Scherzer defocus. The first CTF ring defines the point resolution.

• Stage drift

  • Specification: 0.5 nm/min (but TFS does the test in a very different way)

  • Description: From a starting position move 1 um in each direction and measure drift until it is below threshold (1 A/s).

• Thon rings

  • Specification (Krios): rings visible beyond 0.33 nm at -1 um defocus. Glacios: rings visible beyond 0.37 nm at -2 um defocus.

  • Description: Take a high-resolution image on carbon and fit CTF rings as far as you can. Calculate a radial average from one quadrant.

• Tilt axis offset

  • Specification: <1 um

  • Description: Estimate the tilt axis offset optimized for movement along the z-axis during tilting

Running scripts

The scripts have been tested only on TFS Titan Krios and Glacios microscopes. All tests except maybe Point resolution (which needs a Pt-Ir grid) require a cross-grating grid (e.g. AGS106L Diffraction grating replica with latex spheres) inserted and the eucentric height adjusted. Also, it is assumed that the microscope is already well aligned.

First, have a look at config.py: edit microscopes dictionary and individual parameters for each test. Make sure SerialEM is open. To start the program, simply type in the Windows CMD / Linux console:

perfectem

If you prefer clicking buttons over console, you can create a desktop script PerfectEM.bat that contains one line:

perfectem-gui

PS. The simple GUI requires Python built with tkinter support.