Crosshair: semiautomated targeting for electron microscopy

Abstract text (incl. figure legends and references)

Volume EM brings new insights on cells and tissues ultrastructure in 3D. However the techniques allowing volume EM are low throughput. Increasing the throughput can be achieved, for instance, via targeting approaches that enable replicates and multiplication of observations.

Here we are presenting a newly developed workflow to target structures of interest within a resin-embedded block. The block has to be first trimmed and subsequently imaged by means of X-rays with a microCT machine. The X-ray tomogram generated is a 3D map, from which we can calculate the location of a specific structures of interest using the Fiji plugin called Crosshair (https://github.com/K-Meech/crosshair). The targeting of the structures of interest is then achieved by the use of a motorized ultramicrotome designed by the EMBL electronic and mechanical workshops that allows precise angular movements.

An example of the application of this workflow to find Plasmodium falciparum-infected red blood cells in bioengineered 3D microvessels (that mimic the brain blood barrier) is also shown.

Reference:

Kimberly Meechan, Wei Guan, Alfons Riedinger, Vera Stankova, Azumi Yoshimura, Rosa Pipitone, Arthur Milberger, Helmuth Schaar, Inés Romero-Brey, Rachel Templin, Christopher J Peddie, Nicole L Schieber, Martin L Jones, Lucy Collinson and Yannick Schwab. Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome. https://doi.org/10.1101/2022.06.02.491151. Accepted to be published in eLife.