Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome
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Evaluation Summary:
Meechan et al. present a systematic approach to semi-automate an ultramicrotome operation for targeting a specific plane aided by x-ray tomography measurements. It is a fundamental work of great interest to any users of using electron microscopy (EM), particularly when targeting the imaging of thin sections in a select region of interest by ultramicrotomy, or when targeting volume EM of select sample regions. The manuscript documents with exceptional detail a workflow including both microtome modifications and software adaptations for semi-automated targeting of structures with micrometer precision, resulting in a faster and more accurate orientation of the image acquisition planes for volume electron microscopy, a task that has traditionally been difficult and time-consuming. Therefore, this work will reduce sample preparation labor and, critically, facilitate the comparison of the ultrastructure of multiple samples. The method is based on X-ray imaging acquisition prior to any sectioning and proposes a solution for the two instruments commercially available in the field, and by transparently sharing all the data, hardware, and software, and by describing every detail of the workflow, this fundamental method can be readily adopted by any practitioner, enabling its wide application - it is a key step in the field regarding speed-up, accuracy, and reproducibility in electron microscopy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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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.
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Evaluation Summary:
Meechan et al. present a systematic approach to semi-automate an ultramicrotome operation for targeting a specific plane aided by x-ray tomography measurements. It is a fundamental work of great interest to any users of using electron microscopy (EM), particularly when targeting the imaging of thin sections in a select region of interest by ultramicrotomy, or when targeting volume EM of select sample regions. The manuscript documents with exceptional detail a workflow including both microtome modifications and software adaptations for semi-automated targeting of structures with micrometer precision, resulting in a faster and more accurate orientation of the image acquisition planes for volume electron microscopy, a task that has traditionally been difficult and time-consuming. Therefore, this work will reduce sample …
Evaluation Summary:
Meechan et al. present a systematic approach to semi-automate an ultramicrotome operation for targeting a specific plane aided by x-ray tomography measurements. It is a fundamental work of great interest to any users of using electron microscopy (EM), particularly when targeting the imaging of thin sections in a select region of interest by ultramicrotomy, or when targeting volume EM of select sample regions. The manuscript documents with exceptional detail a workflow including both microtome modifications and software adaptations for semi-automated targeting of structures with micrometer precision, resulting in a faster and more accurate orientation of the image acquisition planes for volume electron microscopy, a task that has traditionally been difficult and time-consuming. Therefore, this work will reduce sample preparation labor and, critically, facilitate the comparison of the ultrastructure of multiple samples. The method is based on X-ray imaging acquisition prior to any sectioning and proposes a solution for the two instruments commercially available in the field, and by transparently sharing all the data, hardware, and software, and by describing every detail of the workflow, this fundamental method can be readily adopted by any practitioner, enabling its wide application - it is a key step in the field regarding speed-up, accuracy, and reproducibility in electron microscopy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors of this paper are offering the electron microscopy community an affordable tool to semi-automatize some of the most challenging and time-consuming steps to target a region of interest in a sample prepared for electron microscopy. This article is sharing in total transparency all their work and the immense development efforts put in by the authors in terms of finance, manpower, software, and hardware development. A huge effort has been done to make all the parts of the workflow accessible. The way to add the hardware to the existing ultramicrotomes is clearly explained and documented. The hardware to be purchased and adapted is also clearly documented. All the software needed is open-source, the code fully documented and the implementation documented. A critical assessment of the performances is …
Reviewer #1 (Public Review):
The authors of this paper are offering the electron microscopy community an affordable tool to semi-automatize some of the most challenging and time-consuming steps to target a region of interest in a sample prepared for electron microscopy. This article is sharing in total transparency all their work and the immense development efforts put in by the authors in terms of finance, manpower, software, and hardware development. A huge effort has been done to make all the parts of the workflow accessible. The way to add the hardware to the existing ultramicrotomes is clearly explained and documented. The hardware to be purchased and adapted is also clearly documented. All the software needed is open-source, the code fully documented and the implementation documented. A critical assessment of the performances is shown for the two main and only suppliers of ultramicrotomes. The reproducibility of the approach has been quantified on numerous samples in a fair and systematic way. The limits and ways for improvements are openly and clearly discussed at the end of the article. All the process is documented by clear and didactic figures helping the readers to put the equations in context.
The implementation of this solution by laboratories will still be a substantial investment but the impact on the research can be so crucial that it can motivate groups to make the effort. The generosity of the authors to share all the data and the fact that nothing is hidden or prevents anybody to adapt this solution is exceptional and should be encouraged.
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Reviewer #2 (Public Review):
For volume Electron Microscopy pipelines, an essential step is to target a specific plane within a resin specimen using an ultramicrotome. Up till now, this can only be done manually. Such a skill set requires months if not years of training and practice to master. This method paper is a pioneer work that offers a first step towards fully automated targeting of structures within resin blocks. It is well structured to document the targeting workflow in EM sample preparation, especially the video tutorial for the UC7 pipeline is professionally produced.
Meechan et al. first described the workflow and techniques used to automate two models of ultramicrotomes (Leica EM UC7 and RMC PowerTome PC), which involve control of the rotations in three orthogonal axes via individual motors. They then integrated Crosshair, …
Reviewer #2 (Public Review):
For volume Electron Microscopy pipelines, an essential step is to target a specific plane within a resin specimen using an ultramicrotome. Up till now, this can only be done manually. Such a skill set requires months if not years of training and practice to master. This method paper is a pioneer work that offers a first step towards fully automated targeting of structures within resin blocks. It is well structured to document the targeting workflow in EM sample preparation, especially the video tutorial for the UC7 pipeline is professionally produced.
Meechan et al. first described the workflow and techniques used to automate two models of ultramicrotomes (Leica EM UC7 and RMC PowerTome PC), which involve control of the rotations in three orthogonal axes via individual motors. They then integrated Crosshair, a Fiji plugin that provides visualization of x-ray tomography measurements and calculates operating parameters for automation. The authors accessed the accuracy of the method through ten test runs using two ultramicrotome platforms, and reported reasonable results: the Leica system had a mean angular error of 1.1 degrees, mean absolute solution distance error of 4.5 microns, and a mean point to plane distance error of 4.8 microns, while the RMC system had a mean angular error of 0.7 degrees, mean absolute solution distance error of 1.6 microns, and a mean point to plane distance error of 1.3 microns. Overall the descriptions of hardware and software implementations are clear and comprehensive to allow for replication by instrumentation experts.
Although this method lowers the barrier for newcomers who start 3D targeting using an ultramicrotome, experienced users who have good 3D thinking and are familiar with ultramicrotomes might find such an approach yielding lower throughput, particularly when trimming one single plane. As the authors pointed out in the Discussion, trimming the four block sides to target a specific feature is essential for FIB-SEM applications. Hence, it would be very important to extend the workflow to demonstrate the additional targeting of four block sides, at least for the RMC system which has automated translational movements.
Readers should also be aware of the limitations of this targeting method. For example, during coarse trimming, the distance from the block surface to the target plane often exceeds one ultramicrotome feed length (~ 200 microns). Therefore, distance target accuracy will be affected by the reset of the cutting feed. Furthermore, such a reset inevitably undermines this semi-automatic approach thus turning it into a more manual process.
Additionally, due to the data requirement of x-ray tomography imaging and the hardware/software implementation complexity, the wide adoption of this method might be hindered by lacking access to x-ray capability and instrumentation expertise in traditional EM facilities.
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Reviewer #3 (Public Review):
Meechan et al. describe a technical modification of a standard ultramicrotome that allows, in combination with software solutions provided, both, the precise orientation and the depth of the cutting plane according to sample features pre-defined by X-ray imaging. Accurate targeting of specific structures in heavy-metal¬-impregnated volume EM samples is challenging and time-consuming and good reproducibility across samples is difficult. Since the applications for volume EM are rapidly increasing during the last years, improved workflows can have an important impact in the field.
A great strength of the workflow described here is the easy access to the required components. Once X-ray data acquisition at a micron-resolution has been achieved, no further expensive, sophisticated equipment is required for its …
Reviewer #3 (Public Review):
Meechan et al. describe a technical modification of a standard ultramicrotome that allows, in combination with software solutions provided, both, the precise orientation and the depth of the cutting plane according to sample features pre-defined by X-ray imaging. Accurate targeting of specific structures in heavy-metal¬-impregnated volume EM samples is challenging and time-consuming and good reproducibility across samples is difficult. Since the applications for volume EM are rapidly increasing during the last years, improved workflows can have an important impact in the field.
A great strength of the workflow described here is the easy access to the required components. Once X-ray data acquisition at a micron-resolution has been achieved, no further expensive, sophisticated equipment is required for its application. Motors and controllers are assembled from common electronics or mechanical parts. The microtomes used are standard microtomes as they are available in most electron microscopy laboratories. No major modification to the microtome is required. However, a statement on whether a dedicated microtome is recommended, or how fast the system can be disassembled would have been useful.
The comparative data collection on two different microtome setups, regarding both microtome brand and users, provides a big credit to the study. The design and calibration steps for the microtome motorization are well documented. The success of reaching the targeting plane with an average of below 2 microns in the RMC setup is an amazing result when considering cellular dimensions, and even the 4.5-micron precision achieved on the Leica system is in the range of a single cell.
In this regard, however, the correlation of the targeting precision with user skills remains an open question that has not been addressed. Prior to the automated cutting, the initial manual alignment of the block surface to the knife is of crucial importance (as stated as a potential explanation for differences in the RMC and Leica setup performance). A comparison of the precision reached by different users on one setup could have further completed the study.Pre-selection of the precise cutting orientation can challenge the users' 3D imagination. Here, the authors have modified modules of existing software solutions (mostly Fiji plugins) for the visualization of the X-ray data and presumptive cutting views. The resulting Crosshair Fiji plugin can be used on a standard computer and is provided with detailed and clear documentation. The implementation within a standard software (Fiji) with existing modules, will ease the use of this plugin.
The choice of Platynereis larvae for targeting the imaging plane allows very clear visualization of the whole procedure. Both the general workflow as well as the specific cases of 10 test samples are well-illustrated by this example tissue. In the future, this proof of principle documented here for the simple larvae should be further validated by a structure embedded in the context of a dense tissue, which can be more challenging.
Further applications will reveal whether this semi-automated workflow can be expanded to correlative light and electron microscopy, with or instead of X-ray imaging. A rapid, precise trimming of fluorescent structures will be of great impact on the volume EM community. For the correlation between X-ray and EM data, the workflow documented by the authors here is already offering an elegant improvement to the time-consuming sample approach with a standard setup.
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