Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; Collaboration for Joint PhD Degree Between EMBL and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany
Wei Guan
Francis Crick Institute, London, United Kingdom
Alfons Riedinger
Electronic Workshop, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
Vera Stankova
Electronic Workshop, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; Electron Microscopy Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
Volume electron microscopy (EM) is a time-consuming process – often requiring weeks or months of continuous acquisition for large samples. In order to compare the ultrastructure of a number of individuals or conditions, acquisition times must therefore be reduced. For resin-embedded samples, one solution is to selectively target smaller regions of interest by trimming with an ultramicrotome. This is a difficult and labour-intensive process, requiring manual positioning of the diamond knife and sample, and much time and training to master. Here, we have developed a semi-automated workflow for targeting with a modified ultramicrotome. We adapted two recent commercial systems to add motors for each rotational axis (and also each translational axis for one system), allowing precise and automated movement. We also developed a user-friendly software to convert X-ray images of resin-embedded samples into angles and cutting depths for the ultramicrotome. This is provided as an open-source Fiji plugin called Crosshair. This workflow is demonstrated by targeting regions of interest in a series of Platynereis dumerilii samples.
