Bäuerlein FJB et al. (2021)
A common difficulty in cryo-FIB/ET is to visualize and subsequently target appropriate tomographic regions in low magnification lamella overviews. The contrast of these images is dominated by curtaining artifacts and thickness variations, arising from the different milling efficiency on different cellular components and the limited focus depth of the ion beam, respectively. Increasing image contrast makes these artifacts even more pronounced. This is especially detrimental for analyzing cryo-FIB lamellae of tissues, given their rich biological complexity. To address this issue, we developed an algorithm termed LisC (“Lamella in silico Clearing”) to remove obscuring features from lamella overviews.
The algorithm was coded as Macro for execution in the FIJI image processing package.
Recommended TEM Acquisition parameters:
pixel size ∼3 nm (≃ Magnification 4800x) acquired with a defocus of -20um.
The lines of the curtaining artifacts need to be roughly horizontally oriented - if necessary the lamella should be rotated.
Install the FIJI image processing package (https://fiji.sc).
Start FIJI and install the Macro under the menu:
Plugins > Macros > Install
select the „Lisc_Algorithm.ijm“ file.
To run the macro, simply open the lamella overview, that should be filtered and start the macro under:
Plugins > Macros > Lisc_Algorithm.
The only obligatory input is the pixel size in nm of the lamella overview. The High-pass filter threshold is set to a preset of 5000 nm.