Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome

Abstract

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.

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

“…for lengthy higher-resolution volume EM acquisitions) and which might require <100 µm-precise targeting. Fs lasers can be programmed to specific sample positions using coordinates from upstream volume XRM imaging (mirroring recent approaches combining XRM with automated ultramicrotome milling 14 , albeit at significantly higher packing density ( Fig. 5 )).…”

“…The use of fs lasers for targeted sample preparation in materials science is a well-established method and has been applied in a variety of different studies 12,13 . Utilisation of the fs laser for sample processing in life sciences provides a tool for targeted trimming of soft biological tissue that can complement other available mechanical tools in workflows that are highly dependent on precise specimen trimming, such as ultramicrotomy 14 or lathe implementations 2 . To interrogate the ultrastructure at nanometre scale, tissue specimens of dimensions reaching several cubic millimetres need to be contrasted with heavy metals and embedded in resin [15][16][17][18][19][20][21][22] .…”

Emergent mechanical control of vascular morphogenesis

“…A low-resolution scan was captured at 40 kV/3 W with a 15-s exposure and with a pixel size of 3 μm. The data were exported as tiff, and the region of interest, on a transverse section of the upper part of the mesenchyme, was identified and targeted in each block using the Crosshair plugin in Fiji ( 81 ). Sections (70 nm) were cut using a Leica UC7 ultramicrotome (Leica Microsystems, Vienna, Austria) and picked up on Formvar-coated copper 1 mm–by–2 mm slot grids (Gilder Grids Ltd., Grantham, UK).…”

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

“…14,15 The use of fs lasers for targeted sample preparation in materials science is a well-established method and has been applied in a variety of different studies. 1,16 Utilization of the fs laser for sample processing in life sciences provides a tool for targeted trimming of soft biological tissue that can complement other available mechanical tools in workflows that are highly dependent on precise specimen trimming, such as ultramicrotomy 17 or lathe implementations. 6 To interrogate the ultrastructure at nanometer scale, tissue specimens of dimensions reaching several cubic millimeters need to be contrasted with heavy metals and embedded in resin.…”

“…This is particularly pertinent when small volumes need to be extracted and separated individually for downstream imaging (e.g., for lengthy higher-resolution volume EM acquisitions) and which might require <100 lm-precise targeting. Fs lasers can be programmed to specific sample positions using coordinates from upstream volume LXRT or SXRT imaging (mirroring recent approaches combining LXRT with automated ultramicrotome milling, 17 albeit at significantly FIG. 3.…”