Name: array tomography workflow for the targeted acquisition of volume information using scanning electron microscopy
Uploaded: 2021-07-15T13:00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy at JoVE.com

We want to thank the members of the EM facility of the University of Lausanne for their support during this development of different steps of the AT procedure. We would like to thank Gareth Griffiths, Marta Rodrigues, Urska Repnik, Christel Genoud, Helmut Gnaegi, Einat Zelinger, Paola Moreno-Roman, Lucy O&#39;Brien and Lindsay Lewellyn for discussions during the preparation of the manuscript and critical reading. We want to acknowledge the groups that contributed the samples used to demonstrate the different scenarios: Matthias Lutolf, Michail Nikolaev, Devanjali Dutta, Till Matzat, and Fanny Langlet.

NOTE: Sample preparation methods are described elsewhere14,30,31, and are not covered in this publication. In short, the examples shown were chemically fixed with glutaraldehyde, post-fixed with 1% OsO4, then treated with 1% aqueous uranyl acetate before embedding in the embed resin. Alternatively, samples can be prepared using high-pressure freezing, freeze substituted with 0.1% uranyl acetate in acetone, and embedded in acrylic resin. Sample blocks were prepared using a flat embedding method that permits a clear view of the sample, facilitating its orientation for the sectioning (Figure 1B).
1. Array generation procedure
<ol>
	<li>Embedded sample orientation and trimming
	<ol>
		<li>Using the binocular/microscope, identify and mark the ROI on the embedded block surface by lightly scratching the block&#39;s surface with a razor blade. This will help to orient the sample inside the ultramicrotome and will reduce the sectioning surface.</li>
		<li>Clamp the sample on the ultramicrotome holder (Figure 2A).</li>
		<li>Trim the resin around the sample, first with the razor blade (rough trimming) and continue with the diamond trimming tool (fine trimming; Figure 2A). Use knives with 20&#176; or 90&#176; edge inclination to ensure that the top and bottom surfaces of the block are parallel to the knife&#39;s cutting edge. 
		&#8203;NOTE: This step is critical for serial sectioning and acquisition of straight ribbons.</li>
		<li>Mix xylene and glue in 3:1 proportion. With an eyelash attached to a toothpick, apply this mixture to the top and the bottom edges of the trimmed block. Let it dry thoroughly.</li>
	</ol>
	</li>
	<li>Prepare the array mounting support A (i.e., silicon wafers).
	<ol>
		<li>Cut the wafer piece using a wafer cleaving tool (EMS), adjusting its size to the project goal. 2 cm x 4 cm is convenient for both light and electron microscopy observations.</li>
		<li>Clean the wafer in distilled water to get rid of the debris.</li>
		<li>Glow discharge/plasma clean the surface using standard equipment. The precise parameters will depend on the machine used. Start with the parameters for the grids discharge and empirically adjust the time. This step is crucial for a good spread of the sections on the support while sections are drying and should not be omitted.</li>
	</ol>
	</li>
	<li>Prepare the arrays mounting support B (i.e., glass coverslip)
	<ol>
		<li>If planning multiplex immuno-labeling experiments, transfer the samples on a coverslip to better detect the fluorescence signal. To improve coverslip adhesiveness, use a detailed gelatin-coating procedure is described previously9.</li>
		<li>Increase the conductivity by coating the slides with the indium-tin-oxide (ITO) or gold by evaporation. Keep the prepared coverslips in a clean environment. Glow discharge the samples as described in 1.2.3.</li>
	</ol>
	</li>
</ol>
2. Sectioning of the sample
<ol>
	<li>AT knife preparation 
	NOTE: To generate the arrays, use a modified knife (ATS), designed to facilitate the acquisition of long ribbons. Histo-Jumbo knives&#160;or similar knives conceived for the generation of the sections on large supports can serve for AT sectioning as well.
	<ol>
		<li>Attach the needle to the bottom of the knife using the foamy sticky tape and pierce it (Figure 2B). Place the ATS knife in the ultramicrotome holder at 0&#176;. Adjust the edge of the knife parallel to the block surface using the standard procedure.</li>
		<li>Bring the trimmed block to the edge of the knife in a position ready for sectioning.</li>
		<li>Place the wafer/coverslip inside the knife basin and fill it with water to the same level as the knife edge. Let the diamond edge of the knife humidify properly and, if necessary, withdraw water using the attached syringe (Figure 2C).</li>
	</ol>
	</li>
	<li>Array sectioning and transfer
	<ol>
		<li>Set the microtome to the desired cutting parameters. With the ATS knife, the range of 50-100 nm and a cutting speed of 0.6-1 mm/s are advisable. Start sectioning (Figure 2Di).</li>
		<li>Obtain a ribbon of the length that will cover a targeted z-volume and stop the collection. Depending on the block size, homogeneity of the tissue, and type of resin, the ribbon will be relatively straight (Figure 2D-ii). Many samples will not produce straight ribbons, despite the invested effort and the type of knife is used.</li>
		<li>Depending on the final goal, make one long or several short ribbons aligned side by side. A 2 cm x 4 cm support can conveniently hold 100 to 1,000 sections. The arrangement of the ribbon on the support step is a step that requires dexterity and steady hands. However, the learning curve for this skill is rapidly acquired.</li>
		<li>Detach the ribbon from the knife-edge using a clean, non-sticky tip of an eyelash glued on the toothpick (Figure 2Diii).</li>
		<li>Using the eyelash, gently move the ribbon above the center of the support medium. At this point, use chloroform or heating pen for stretching of the sections if needed. However, remember that this manipulation may induce ribbon breaking and deformation.</li>
		<li>Start draining the water by pulling the syringe. For more delicate ribbons or a slower water retraction, let water drip by detaching the syringe from the hose. When the water level is lowering to the wafer level, control the ribbon and reposition it if necessary, by gently pushing the ribbon to the center. After settling the ribbons on the wafer surface, continue draining until the remaining water is completely retracted from the basin. 
		NOTE: If not using the bottom water retraction ATS knife, carefully reduce the water from the sides of the ATS knife to not induce turbulence.</li>
		<li>Leave the sections on the support inside the bath to let dry completely. Depending on the hydrophobicity of the support and environmental humidity level, water will evaporate at a different rate (Figure 2Div). It is important to let the sample dry slowly to diminish or altogether avoid all folds on the sample.</li>
		<li>Transfer the dry sample to a tightly closed box to protect from dirt contamination and place it to a 60 &#176;C oven for at least 30 min. If required, counterstain sections using heavy metals to enhance the overall contrast.</li>
		<li>At the end of the procedure, cautiously clean the knife following the manufacturer&#39;s instructions 
		NOTE: The transfer of multiple or serial sections on a wafer (Figure 2E) compared to the transfer on a slot grid (Figure 2F) change the EM preparation experience entirely. The uninterrupted sectioning and collection of the sections on single support reduces the amount of sectioning and section collection related mistakes frequent in serial sectioning. The equivalent of 100 sections of 1000 &#181;m x 500 &#181;m collected on one wafer corresponds to 33 slot grids (Figure 2G).</li>
	</ol>
	</li>
</ol>
3. Sample observation
NOTE: This section describes the workflow steps as implemented using a commercially available software (see Table of Materials). Any image acquisition software on any SEM equipped with the correct detectors can be used, however, the specific user actions will differ and will often be more manual.
<ol>
	<li>Acquire an overview map of SEM images that reveals section locations on the wafer. A built-in optical camera image helps with defining an SEM image mosaic that covers a ribbon of sections or all sections. Create the mosaic by click-drag with the mouse right on the camera image of the sample and start the Automatic Acquisition. 
	NOTE: Very coarse imaging settings, i.e., 1-2 &#181;m pixel size and 1 &#181;s dwell time suffice. This process is illustrated in Supplementary Material (pages 2-7).</li>
	<li>Locate sections with the Section Finder Auto Detection function or locate them manually. Section positions and outlines are retrieved automatically with this software based on image matching. For illustration of this process, see Supplementary Material (pages 8 - 15).</li>
	<li>In case the overview images do not show ROIs clearly, acquire higher resolution images of sections. Use the Section Preview function to create and acquire images automatically. This process is illustrated in Supplementary Material, pages 16-18. Imaging settings must be chosen according to the nature and size of the ROI that the user searches for.
	<ol>
		<li>To find optimal settings, activate the Live Imaging in the microscope control software and navigate to one ROI. Alter imaging settings until the images show ROIs clearly, but image acquisition is not excessively long.&#160;</li>
	</ol>
	</li>
	<li>Define imaging regions 
	&#8203;NOTE: Several different strategies are proposed.
	<ol>
		<li>If only a few sections need to be imaged, use the images of sections created so far for navigating to the relevant sections and use the Zoomable Viewer that shows all acquired images in their original relative locations to look at all sections. Once a section is found that should be imaged at high resolution, create an imaging region with click-drag. Choose high resolution imaging settings and store the settings in a template. Re-use this template for further sections.</li>
		<li>To find particularly small or hard to detect rare events, use the Lateral Screening approach. Manually create an imaging region with high resolution imaging settings on every tenth section or on one section per ribbon and acquire the images. Review the images and mark sections that contain the ROI in the software or take a note.
		<ol>
			<li>Starting from sections that contain the ROI, navigate forward and backward through the section set and create high resolution imaging regions in the same relative locations for as long as the structure is still visible in the section. This can be done manually or by using the procedure described in the next steps.</li>
		</ol>
		</li>
		<li>Acquire images in more than ten consecutive sections. Click Start Position Refinement after zooming the image to increase the precision of registered section locations as described in the manual. Doing so decreases the position variability of image series. The procedure is illustrated and explained in Supplementary Material pages 19-21.</li>
		<li>Click-drag on any section to define an imaging region while holding down the Alt key and select Create Tile Set Array from the context menu that opens when the mouse button is released. The software then creates imaging regions in the same relative location in all sections that have been found or marked previously. It is possible to limit imaging to a specific range of sections with the Section Span slider. 
		NOTE: This procedure can be repeated with any number of imaging regions and therefore allows recording many small high-resolution images instead of recording a single larger image on each section.</li>
		<li>Once created, set up pixel count, pixel size, tiling layout, pixel dwell time, etc. in each image series as needed. Once imaging series are created and set up, they are all listed in a job cue.</li>
	</ol>
	</li>
	<li>Configure auto functions and start image acquisition
	<ol>
		<li>Create a separate image series for auto functions using the same method as describe in the previous step. Move the image series to a position on the section that contains high-contrast structures.</li>
		<li>Set the image series to 1024 x 884 pixels and choose a pixel size corresponding to the highest resolution used in the image series set up in the previous steps. In the list of auto functions, check Auto Focus and Auto Stigmator.</li>
		<li>Select By Section in the acquisition sequence controls and make sure the auto functions image is the first item in the list. Start the image acquisition by clicking the Run button next to the job cue. These procedures are illustrated in Supplementary Material, pages 22-23. 
		&#8203;NOTE: It is not necessary to manually pre-focus on each section. During the recording session, whenever the microscope advances to a new section, the auto functions will be executed before all other images are recorded in this section.</li>
	</ol>
	</li>
</ol>
4. Data alignment and analysis
<ol>
	<li>Data export
	<ol>
		<li>Ensure that data is saved in .tif format, so there is no need for a dedicated export function. Sort data into a folder structure that corresponds directly to the layers and elements in the Layer Tree.</li>
		<li>Once image mosaics have been recorded, use the StitchAll function to automatically stitch all tiles.</li>
	</ol>
	</li>
	<li>Stack alignment and cropping in Fiji 
	&#8203;NOTE. Many software packages (free and commercial) can be used to work with Array Tomography data. The steps below are shown with open-source program Fiji32&#160;because it is widely available and contains all the required functions.
	<ol>
		<li>Import a stack of images (or stitched images) into Fiji as a virtual stack.</li>
		<li>If contrast/brightness needs to be normalized, choose Enhanced Contrast&#8230; from the Process menu. Set Saturated Pixels to 0.1 or less, and check Process all Slices.</li>
		<li>From the Plugins menu, choose Registration | Linear Stack Alignment with SIFT.</li>
		<li>Choose Rigid or Affine from the Expected Transformation drop-down menu. Otherwise, keep the default settings. Start the alignment by clicking OK. 
		NOTE: Loading the data as Virtual Stack allows Fiji to handle stacks of any size. The output of the alignment is created in RAM; however, this can limit the maximum size of stacks that can be processed. In that case, use Register Virtual Stack Slices, which is a folder-to-folder implementation of the same registration algorithm. Once registration is complete, load the output data as a virtual stack.</li>
		<li>Crop the image stack by clicking Crop so that it contains only the ROI.</li>
		<li>Save the stack as a single .tif image or series of .tif images. 
		NOTE: Critical steps of Array Tomography are shown in Figure 3.</li>
	</ol>
	</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrasynaptic+acetylcholine+signaling+through+a+muscarinic+receptor+regulates+cell+migration.">Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration.</a> Proceedings of the National Academy of Sciences. 118 (1), e1904338118 (2021).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+digestive+tract+of+Drosophila+melanogaster.">Anatomy and physiology of the digestive tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Nikolaev, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+mini-intestines+through+scaffold-guided+organoid+morphogenesis.">Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.</a> Nature. 585 (7826), 574-578 (2020).</li><li>Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+asymmetric+stem+cell+division.">Mechanisms of asymmetric stem cell division.</a> Cell. 132 (4), 583-597 (2008).</li><li>Daniel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+septate+junctions+assembly+and+completion+of+cytokinesis+in+proliferative+epithelial+tissues.">Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues.</a> Current Biology. 28 (9), 1380-1391 (2018).</li><li>Pasquettaz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peculiar+protrusions+along+tanycyte+processes+face+diverse+neural+and+non-neural+cell+types+in+the+hypothalamic+parenchyma.">Peculiar protrusions along tanycyte processes face diverse neural and non-neural cell types in the hypothalamic parenchyma.</a> Journal of Comparative Neurology. , (2020).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> Journal of Structural Biology. 116 (1), 71-76 (1996).</li><li>Cardona, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrakEM2+software+for+neural+circuit+reconstruction.">TrakEM2 software for neural circuit reconstruction.</a> PLoS One. 7 (6), 38011 (2012).</li><li>Belevich, I., Joensuu, M., Kumar, D., Vihinen, H., Jokitalo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy+image+browser:+A+platform+for+segmentation+and+analysis+of+multidimensional+datasets.">Microscopy image browser: A platform for segmentation and analysis of multidimensional datasets.</a> PLoS Biology. 14 (1), 1002340 (2016).</li></ol>

The author Tilman Franke is an employee of Thermo Fisher that manufactures electron microscopes and piloting programs that are used in the article.

Electron microscopy gives insight into the ultrastructure of the cells and organisms, for which it is often desirable to image structures of interest in their 3-dimensional context. Despite numerous EM tactics for ultrastructural analysis, there is still no &#34;gold standard&#34; solution. The main reason is the wide variety of samples, many biological questions, which frequently require a tailored approach. The proposed AT workflow is designed to minimize the time necessary for sample processing, data acquisition, evaluation, and storage. Furthermore, the modified knife provides a helpful tool to simplify array acquisition. The compact layout of sections on wafers is convenient, both for the observation and the subsequent storage of the samples. This arrangement enables &#34;Lateral Screening&#34; of samples by horizontally moving from ribbon to ribbon and scanning only one section on each, significantly reducing the time required to localize an ROI. Proposed data acquisition scenarios facilitate the targeting of small and randomly distributed areas. Once found, AT/SEM restricts high-resolution imaging precisely to the volume of interest, whether performed manually or with the help of automatic function. AT for limited volumes can be completed manually, with the operator navigating through the sample and defining imaging regions one by one. The automated module of the software provides a flexible image acquisition strategy for imaging of small areas on large sections. The automation in this software allows recording large high-resolution images on hundreds of sections, achieving similar volumes as SBFI. Recording overview images of all sections simplifies the ROI localization and reduces the time spent at the microscope. Since the sections are not harmed during the recording of overviews and higher resolution Previews, AT/SEM permits reusing the sample to collect further data of other ROIs or at a higher resolution.
Image acquisition time is one of the most important (and most expensive) aspects of 3D EM and should therefore be considered in the experiment design. While it is unsurprising that imaging large areas takes longer than imaging small areas, it is easy to under-estimate the impact: Depending on the imaging parameters selected, the acquisition time on each section can vary from seconds to hours. Critical imaging parameters include the size of the field of view, resolution, and dwell time. Assuming a target resolution of 10nm per pixel and 1 &#181;s dwell time, imaging a field of 20 &#181;m x 20 &#181;m, 100 &#181;m x100 &#181;m, or 500 &#181;m x500 &#181;m takes 4 seconds, 100 seconds, or 2,500 s to record. We can multiply these per-section imaging times by the number of sections to estimate the time required for the complete imaging job. Long per-section imaging times can be acceptable if the number of sections is small or if microscope tool time is of no concern.
However, it is necessary to limit the recording time to an overnight job or a weekend job in most cases. An equally critical aspect of 3D EM that should be considered, is the amount and structure of the resulting image data. Recording the abovementioned imaging fields in 100 sections generates 400 mb, 10 gb, or 250 gb of image data, respectively; the 500 &#181;m x 500 &#181;m images pose the additional issue of being larger than 2 gb each. Many of the software programs used for data evaluation cannot open images of this size.
To reduce imaging time, it is important to choose pixel dwell time to meet the signal-to-noise ratio requirements for the subsequent data evaluation (e.g., reconstruction, tracing), and limit the recordings to defined ROIs. The AT extension of the software facilitates image acquisition in small areas in serial sections. The software supports manual and automatic workflows and many semi-automatic variants: imaging areas can be manually positioned and focused on each section, or the user can use the automatic section finder and position alignment features. Depending on the level of automation chosen and supported by the sample type or imaging goals, the time required for setting up image acquisition in hundreds of sections can take an entire workday (done manually) or only a few minutes. In principle, Array Tomography makes it more challenging than other 3D EM methods to acquire small ROIs; imprecise region placement on consecutive sections must be compensated by acquiring larger areas. For example, if the ROI is 20 &#181;m x 20 &#181;m in size and the section-to-section position variability of imaging fields is 10&#181;m, one needs to acquire 40 &#181;m x 40 &#181;m images in order to be sure that the ROI is fully captured in each image, on every section. Real-world image position variability ranges from 100 &#181;m to &#60;10 &#181;m depending on the availability or quality of software features for position alignment or user&#39;s patience. With this software, 10 &#181;m can be achieved without too much manual intervention in most samples.
Like any technique, the AT has several weak points that can influence successful data acquisition, and many are similar to other sectioning-based methods.Lack of homogenous distribution of empty resin versus tissue can result in curved or broken arrays. In extreme cases, sections can detach from the support (Figure 6A). Variable compression or stretching during the cutting process can create folds that can disrupt the sample in variable regions on subsequent sections (Figure 6B). Knife marks can appear on the surface of the collected sections using a damaged knife (Figure 6C). Differences in sectioning conditions can induce occasional section compression and thickness differences. Dust or dirt particles can land on a section and partially obscure the zone of interest (Figure 6D). Image acquisition can fail due to imperfect auto-contrast, auto-focus, and auto-stigmator functions. The positioning of automatically created imaging areas can be variable and may fail to capture the ROI in all sections.
Several problems can arise at the stage of stitching and alignment. The automatic stitching of mosaic tiles acquisitions can fail, for example, because of the large empty space inside the sample. Due to a drastic change in shape in 3D, image stacks can be challenging to register. Specially developed programs (e.g., IMOD, Fiji, TrackEM2, MIB, or MAPS-AT) can facilitate semi-automatic alignment32,38,39,40. More challenging sections can be manually aligned using photo editing software. Unfortunately, some datasets may be impossible to align correctly.
Large samples are challenging for TEM serial sections fitting onto grids; on the other hand, many projects do not justify long automatic acquisition using FIB/SEM or SBF-SEM. The AT is a straightforward alternative to a tedious serial section TEM where collection and manipulation of serial sections on a wafer are more straightforward than with the slot grids. Several strategies were developed to facilitate the collection of the arrays, and we share our method to expand the existing toolkit. In cases where the identification of the ROI is challenging, AT-SEM provides a fundamental advantage, with the efficient screening of the samples where organelle-scale resolution across 50 to 500 sections is required. For larger volumes, the automatic collection AT strategies can be efficiently collected if more sections are required. AT samples can be re-imaged multiple times, facilitating targeted imaging of high-resolution areas based on previously-acquired overview images. We believe that the targeted analysis and reduced oversampling by AT/SEM proposed here decreases the labor and data storage requirements. Ultimately, libraries of sections can be collected and maintained for later reuse and consultation. For volume acquisition, FIB or SBF-SEM approaches offer an excellent solution whenever the ROI is easy to identify on the block face or if large 3D volumes are needed for the analysis. However, FIB/SBF-SEM are less efficient when the high-resolution stack image has to be collected from a defined ROI in a targeted manner. To conclude, the proposed methods for screening of AT samples and the use of medium resolution overview images allow limiting image acquisition to the relevant parts of the section array. Precise aiming of imaging regions speeds up time-to-data and simplifies data evaluation.
In summary, although the concept of AT/SEM is not novel, its use is still not as widespread as its merits would suggest. Overall, it provides a complementary procedure to other existing EM methods. AT/SEM is compatible with the broadest range of sample preparation protocols and imaging workflows and can be done on any FIB/SEM or SBF-SEM microscope as an accompanying technique. In this paper, we have concentrated on AT for recording ultrastructural data from samples that are less likely to be successfully addressed by other methods. We hope that the described procedure for convenient collection of sections and considerably automated acquisition strategies will aid in the first attempts for those who have never encountered the method and will help to perfect it for those who already have some experience.

Despite the importance of EM-related techniques, the effort required to master them keeps the entire field restricted to a small number of specialists. One significant difficulty is the identification and retrieval of a Region of Interest (ROI) in the samples preserved for EM. The appearance of the same sample considerably differs when analyzed by optic microscopy and after processing for EM observation. The changes for chemically prepared samples include the anisotropic sample shrinkage after the dehydration steps (~10% in each dimension) and the loss of fluorescence when using osmium in the fixation and staining protocol (Figure 1A). For the ultrathin sectioning, the samples are embedded in epoxy or acrylic resins using different strategies (Figure 1B). For successful results of this preparation, the entire sample must be fractionated into pieces that do not exceed 1 mm x 1 mm. To meet standard Transmission Electron Microscopy (TEM) observation conditions&#39; requirements, this tiny portion of the sample is further sectioned to 50-150 nm thick slices. The resulting grayscale images show tissue organization and organelle structure of a minute fraction of the entire sample at greater detail than any other microscopy technique (Figure 1C). A typical TEM dataset provides 2D information, theoretically extrapolated to understand the processes naturally occurring in a 3D space in cells and tissues. Figure 1D presents the challenge of ultrastructural volumes acquisition: if a cube of side 1,000 &#181;m is sectioned at 50 nm thickness, 20,000 sections will be required to cover the entire volume; for a 500 &#181;m side cube, it will be 10,000 sections. To cover a 50 &#181;m x 50 &#181;m x 50 &#181;m volume, 1,000 sections &#34;only&#34; might be necessary. Obtaining this volume manually is practically impossible and extremely challenging to perform with automation. If, in addition to sample depth, we need to cover the entire surface of such hypothetical cubes, the coverage of 1 &#181;m2 surface at reasonable resolution becomes a serious logistic issue (Figure 1E). While for the extraordinary large-scale projects, such as connectomics approaches, the large number of sections is crucial, for the majority of the &#34;mundane&#34; EM projects, generating more sections required for the observation presents a significant disadvantage.
There are several methods to acquire 3D ultrastructural information: serial sectioning Transmission Electron Microscopy (TEM), TEM tomography, Array Tomography (AT), Serial Block Face Imaging Scanning Electron Microscopy (SBF-SEM), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Principal differences between these methods are the sectioning strategy and whether the image acquisition is coupled to section generation1. In serial sectioning TEM, sequential sections are collected on slot grids, TEM images are generated from these sequences and aligned2,3,4,5. In TEM tomography, tilt series from 150-300 nm sections on a grid, and when coupled to serial sectioning, provide a very high resolution, though relatively small volumes6,7,8. The AT approach uses physical sectioning with diverse manual and semi-automatic manners of sections collection on relatively large support, such as glass coverslip, silicon wafers or a special tape. For image acquisition, the support is analyzed in SEM, with diverse image acquisition strategies are available9,10,11,12,13,14,15 . For SBF-SEM, physical sectioning is achieved using a mini-microtome with a diamond knife set directly inside the SEM chamber, with the SEM image generated from the surface of the resin block16,17,18,19. For FIB-SEM, ion source removes thin layers of the sample, followed by automatic imaging of the exposed surface by the SEM20,21. TEM-tomography and the AT generate physical sections, which can be re-imaged if necessary, while FSBF-SEM and FIB-SEM eliminate the section after imaging. A recent combination of physical sections imaged by a multi-beam SEM provides a combination of methods that solves the &#34;bottleneck&#34; issue of the speed of image acquisition22. Each of these techniques has revolutionized the way EM data can be obtained and analyzed, and each approach has its practical impacts related to a given research question.
Given the nature of the preparation and the scale of the ultrastructural dimensions, it is not straightforward to predict where a specific target structure is located in the sample block (Figure1D,E). One solution for the ROI localization is recording the images from the entire block at the desired resolution from the beginning. The structures of interest can be in the acquired data volume when away from the microscope. Acquisition time and data handling associated with this strategy are problematic. It is desirable to reduce the amount of recorded data, especially if the ROIs are much smaller than the tissue block, i.e., if the objects of interest are specific types of cells (not whole organs). Different correlative light and electron microscopy techniques (CLEM) can be successful when the fluorescence is preserved and localized before or after preparation within the same sample23,24,25,26,27,28,29. Nevertheless, many cellular structures are recognizable even without fluorescence correlation, only based on the known ultrastructure. For these cases, we believe that lateral screening Array Tomography provides a balance tradeoff between the effort invested in ROI localization and the ultrastructural information quality. Using this strategy, a sub-set of sections on the wafer are screened within regular intervals, which can be established based on the ROI&#39;s size and nature. Once ROIs are found, data acquisition is set up in a continuous series of sections starting before and ending after the anchor section, collecting the relevant information in a targeted manner.
We present protocols for AT that simplify and accelerate the acquisition of regions or events of interest in numerous sections and yield better-aligned image volumes. Lateral screening and multistep acquisition produce data with very high resolution in precisely targeted regions. The procedure we describe addresses several challenges of 3D EM data acquisition, as it provides for: compatibility with a broad range of specimens without fundamentally changing the sample preparation workflow; targeted localization for sectioning and SEM acquisitions; reduced time and effort during setup; imaging of regions in multiple sections with better alignment of the resulting volumes; and a smooth stitching and alignment procedure to compile different images into a stitched mosaic picture. We chose to demonstrate the strength of our method with several samples from published and ongoing projects. We believe that this approach can significantly facilitate the generation and acquisition of targeted EM data, even for investigators with limited EM experience.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Cutting</td>
		</tr>
		<tr>
			<td>AT sectioning knife</td>
			<td>Diatome</td>
			<td>DUATS3530</td>
			<td>Diatome Jumbo knife</td>
		</tr>
		<tr>
			<td>Diamond knife for trimming 90&#176;</td>
			<td>Diatome</td>
			<td>DTB90</td>
			<td>Diatome trimming 20&#176; 
			Glass knife</td>
		</tr>
		<tr>
			<td>Pattex contact adhesive</td>
			<td>Pattex</td>
			<td>PCL3C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Ted Pella</td>
			<td>16015</td>
			<td>Resistance: 1-30 Ohms 
			Type P: (Boron) (1 primary flat) 
			Roughness: 2 nm 
			No SiO2 top coating 
			TTV: = &#60;20 &#181;m 
			Wafer is polished on one side</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica UC6</td>
			<td></td>
			<td>Alternative: Leica UC7</td>
		</tr>
		<tr>
			<td>Wafer cleaving kit</td>
			<td>EMS</td>
			<td>7642</td>
			<td>EMF, Small Sample Cleaver, CatNo. 7652</td>
		</tr>
		<tr>
			<td colspan="4">Image acquisition</td>
		</tr>
		<tr>
			<td>FESEM</td>
			<td>Thermo Fischer Helios</td>
			<td>1072419</td>
			<td>Alternatives: Zeiss, Jeol, Hitachi, TESCAN</td>
		</tr>
		<tr>
			<td>Maps 3 for SEM with Correlative Workflow &#38; Array Tomography</td>
			<td>Thermo Fisher Scientific</td>
			<td>1135932</td>
			<td>Maps provides automation of SEM imaging workflows and allows importing of 3rd party data for CLEM and navigation.</td>
		</tr>
		<tr>
			<td colspan="4">Image analysis</td>
		</tr>
		<tr>
			<td>Amira x.y</td>
			<td>Thermo Fisher Scientific</td>
			<td>1131599</td>
			<td>Amira is a 3D data visualization and analysis software with several practical functions for Array Tomography data reconstruction.</td>
		</tr>
		<tr>
			<td>Image processing</td>
			<td>Open source</td>
			<td>Fiji (http://fiji.sc/#download)</td>
			<td>IMOD, MIB (See text for refferences)</td>
		</tr>
	</tbody>
</table>

array tomography workflow for the targeted acquisition of volume information using scanning electron microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

Watch this Scientific Journal Video about Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy at JoVE.com

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Electron microscopy analysis is frequently complex. We believe that our protocol facilitates processing and acquisition of the EM data from the large sample surface and intermediate volume. Our protocol helps find target structures in serial sections.Data recording is limited to what's necessary. Compared with data acquisition in whole-tissue blocks, recording time is reduced and data analysis easier. Given the overall ease of the approach, we believe that it can be used to address not only scientific questions, but also as a diagnostic tool.To generate an array for analysis, first clamp the sample onto an ultramicrotome holder and use a razor blade to roughly trim the resin around the sample. Use a diamond trimming tool to fine-trim the sample and use trimming knives of 20-or 90-degree inclination to ensure that the top and bottom surfaces of the block are parallel to the knife's cutting edge. Mix xylene and glue in a 3:1 proportion and use an eyelash attached to a toothpick to apply the mixture to the top and bottom edges of the trimmed block.While the mixture is drying, use a wafer cleaving tool to cut a piece of wafer to the appropriate size for the analysis. Clean the wafer in distilled water to rinse away any debris and clean the surface with plasma after the wafer is dry. To prepare an array tomography knife, use foamy sticky tape to attach a needle to the bottom of a histo jumbo knife and place the knife into the ultra microtome holder at zero degrees.Adjust the edge of the knife parallel to the block surface and bring the trimmed block to the edge of the knife in a position ready for sectioning. Place the clean wafer into the knife basin and fill the basin with water to the same level as the knife edge, then, let the diamond edge of the knife humidify properly, using the attached syringe to add or withdraw water as necessary. For array sectioning, set the microtome to a cutting range of 50 to 100 nanometers and a cutting speed of 6 to 1 millimeters per second, and begin sectioning to obtain a ribbon long enough to cover a targeted Z volume.Depending on the block size, homogeneity of the tissue, and type of resin, the ribbon will be approximately straight. Use a clean, non-sticky tip of an eyelash to detach the ribbons from the knife edge. Use the eyelash to gently move the ribbon over to the center of the support medium.Chloroform or a heating pen can be used to stretch the section if necessary. After stretching, retract the syringe to start draining the water. For more delicate ribbons, or a slower water retraction, detach the syringe from the hose to let the water drip.When the water level reaches the wafer level, gently push the ribbon to the center of the basin and continue draining until all of the remaining water is completely removed from the basin. Let the wafer dry in a clean environment. Drying takes approximately 30 minutes.When the sample is completely dried, transfer the array to a tightly-closed box to protect it from dirt contamination. And place the box in a 60 degree-Celsius oven for at least 30 minutes. To acquire an overview map of SEM images that reveals section locations on the wafer, use the built-in optical camera to acquire an SEM image that covers a ribbon of sections.To create a mosaic, click and drag the camera image of the sample and start the automatic acquisition. Acquire overviews at higher resolution to find target structures. If only a few sections need to be imaged, use zoomable viewer to view acquired images in their original locations.Once a section that should be imaged at high resolution has been identified, click and drag to create an imaging region, then select High-resolution Imaging Settings and store the settings in a template To find particularly small or hard-to-detect rare events, use the high-resolution imaging settings on every 10th section or on one section per ribbon to manually create an imaging region and acquire the images. Then review the images and mark sections that contain the region of interest. To acquire more than 10 consecutive sections, use the section finder to locate all sections automatically.If the overview images do not show clear regions of interest, acquire higher resolution images of the sections and use the section preview function to create and acquire the images automatically. To determine the optimal imaging settings, activate the live imaging in the microscope control software and navigate to one region of interest, then adjust the imaging settings until the images show clear regions of interest, but without an excessively-long image acquisition, according to the manufacturer's guidelines. To optimize the positioning of the imaging regions on consecutive sections, zoom to the image of interest and click Start Position Refinement to increase the precision of registered section locations.To define an imaging region, click and drag on any section while holding down the Alt key and select Create Tile Set Array from the pop-up context menu. The software will create imaging regions in the same relative location in all of the sections that have been found or previously marked. Then set up pixel count, pixel size, tiling layout, and pixel dwell time in each image series as needed.To configure the auto function, create a separate image series for auto functions as demonstrated, and move the image series to a position on the section that contains high-contrast structures. Set the image series to 1024 x 884 pixels and select a pixel size corresponding to the highest resolution used in the image series. In the list of auto functions, check Auto focus and Auto stigmator.Select Bisection in the acquisition sequence controls and confirm that the auto function's image is the first item in the list, then click Acquire All to start the image acquisition. When all of the imaging series have been created and set up, the series will be listed in a job queue. Low-resolution acquisition can be performed manually or automatically directly on selected parts of the section or an entire section using single or mosaic imaging, followed by stitching.Images from the selected area can then be acquired using high-resolution parameters to visualize mitochondria, nuclei, and microvilli, for example, After the automatic acquisition of resolved mosaic maps has selected, several regions of interest can be cropped out or used to define additional local imaging areas within the regions. Although different specialized cell types in the Drosophila gut are randomly distributed, they can be visually distinguished after screening the images using high-resolution parameters, either from single sections or as a collection of serial images. After the alignment, the stacks can be rendered using different software solutions.Array tomography analysis allows many sequential sections to be generated on the single wafer, and to be screened using low-resolution parameters to localize the general areas of interest. These areas can be targeted for further analysis using advanced acquisition parameters. For example, mytotic divisions in the notum are not easy to localize on the ultra-structural level, as the cells are relatively large compared to the abscission zone.Using this method, however, the automatic medium-resolution overview images of the leaps of 20 to 40 sections can be used to localize the dividing cells. The array tomography procedure provides a more straightforward electron microscopy data analysis. We encourage the viewers to invest in mastering the regeneration and familiarizing themselves with the maps-related workflow In our experience, few research topics require the ultra-structural analysis of entire animals or whole organs.Our method helps with rapid localization of cells and their interaction partners in tissues.

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Biology

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...