We would like to thank Dr. Sam Hanlon, Evelyn Brown, and Margaret Gondo for their excellent technical assistance. This research was supported in part by National Institutes of Health (NIH) R01 EY-018239 and P30 EY007551 (National Eye Institute), in part by the Lion&#39;s Foundation for Sight, and in part by NIH 1R15 HD084262-01 (National Institute of Child Health &#38; Human Development).

The authors have nothing to disclose.

The purpose of this methods paper is to highlight the tissue preparation and imaging methodology that has allowed our lab to reliably capture high-resolution serial electron microscopy images, and to point out critical steps that lead to this outcome as well as potential pitfalls that can occur when conducting SBF-SEM imaging. Success using this protocol requires proper fixation of tissue, impregnation of heavy metals into the sample, modifications of the embedding resin to reduce charging, as well as an understanding of the microscope and imaging settings used to collect images. The maxim, &#34;quality in, quality out&#34; is an appropriate axiom for SBF-SEM imaging. As the goal of SBF-SEM often is the appreciation or quantification of ultrastructural detail, extra care must be given to fixation strategy in order to ensure that tissue distortion does not occur. If tissue becomes distorted at any point in the preparation of samples (i.e., undergoes swelling, shrinking, or disruption of cellular morphology), then tissue reconstruction and quantization will not yield accurate data. Furthermore, the use of incorrect imaging settings can lead to loss of data that cannot be recaptured as SBF-SEM imaging is a destructive process. Additionally, care must be used when loading a tissue sample as the delicate diamond knife can be damaged by hasty or incorrect sample preparation. This can result in chips or breaks in the knife, which can leave visible scratch marks in images (Figure 10C). The diamond knife can also be damaged by calcified structures, hard granules, or accidentally embedded glass (e.g., from reagent ampules).
While the majority of SBF-SEM literature to date uses beam acceleration voltages in the range of 1 to 3 kV alongside pixel dwell times closer to 1-5 &#181;s/px (Figure 11)45,46,47,48,49, the current protocol uses acceleration voltages of 7-12 kV and a pixel dwell time of 12 &#181;s/px for serial imaging and 32 &#181;s/px for imaging regions of interest (Figures 12 &#38; 13). These settings, coupled with a slice thickness of 100-200 nm allows for high-quality and high resolution imaging of a wide range of biological tissue. Increased acceleration voltage allows for an increase in contrast, resolution, as well as signal-to-noise ratio. Increased dwell time further increases resolution and signal-to-noise ratio, while increased slice thickness leads to decreased charging on the block surface during sectioning and combats beam-induced damage in subsequent images14. While this imaging method may differ from convention, the images and datasets produced speak for themselves. If we had to speculate on the reason for this success, it is possible that it is a result of our unique combination of high kV values, longer pixel dwell times, and block preparation. Increasing imaging kV results in an increased interaction volume between the electron beam and the sample. This interaction volume is both deeper as well as wider resulting in a theoretical increase in the number of electrons detected that originate from deeper within the sample block, or from a wider cross section of tissue as the spot size teardrop increases in diameter. As SBF-SEM is interested in the surface detail of the block, this results in a theoretical decrease in signal-to-noise ratio. However, the increase in kV also pushes electrons deeper into the sample where they are less likely to escape the block and contribute to the electrons collected by the detector. With the added benefit of an increased signal via longer pixel dwell times and higher beam intensity, it is possible that this imaging method results in a greater increase in signal from the sample surface in relation to noise originating within the interaction volume. Additionally, the increased sample conductivity introduced with carbon black as well as silver and gold coating helps to ameliorate charge buildup which now occurs deeper within the block and further from the block-face. Indeed, Figure 11, Figure 12, Figure 13 show that as kV is increased sample charging begins to diminish as it is potentially pushed deeper into the block. Samples imaged at low magnification can be captured with adequate contrast using the conventional settings, however these images often lack detail upon close inspection. Our data clearly show that when using relatively high magnification where the goal is cellular detail, increasing the conventional settings can produce exceptional results. The 2020 article by P. Goggin, et al provides a useful table outlining the effect of changing imaging settings on final image quality, and is a helpful reference to consult if optimizing the protocol for novel tissues becomes necessary14. The 100-200 nm slice thickness recommended in this protocol has the added benefit of allowing the collection of large SBF-SEM data sets at a rapid rate. While collecting images at 12&#181;s/px for example, imaging through a 100 &#181;m depth at 2048x2048 px requires ~14 hours while sectioning at 100nm/section but would require ~56 hours if sectioned at 25nm/section. While x,y resolution remains unchanged as a result of section thickness, not accounting for the added ability to image using higher kV values and pixel dwell times that come with larger sections, it is important to note that the resolution along the z-axis does suffer. The loss of z-resolution is an important consideration and should be contemplated when deciding how tissue should be oriented in the resin block and in relation to the imaging plane, and has the potential to preclude the study of smaller cell features or interactions (e.g., synaptic invaginations or intracellular features on the scale of tens of nanometers). However, in addition to rapid imaging time, this protocol has additional added benefits in that it rapidly produces ideal datasets for stereological analysis as well as the study of rare biological events or cells. Larger section thickness can also aid in manual 3D reconstruction, as a 100 &#181;m region sectioned at 100 nm/section would require manual segmentation of 1,000 images while the same region sectioned at 25 nm/section would require manual segmentation of 4,000 images.
SBF-SEM has the benefit of generating large datasets in a relatively short period. While data analysis can be performed using quantitative methods such as stereology, which will be discussed below, it can often be informative to create 3D reconstructions via image segmentation. An image stack created using SBF-SEM can be thought of as a collection of voxels, while segmentation is the process of assigning these voxels to user-defined objects thereby creating 3D representations of tissue structures. These reconstructions often provide a heretofore unseen perspective on tissue ultrastructure and cell-cell interaction (Figure 5,&#160;Figure 6,&#160;Figure 7, Figure 8,&#160;Figure 9). Furthermore, once reconstructions have been created it is possible to use data inherent in the reconstructions to extract a wealth of information from segmented tissue. Parameters ranging from surface area, volume, length and distance, as well as angular data are all readily available once a reconstruction has been created50,51. While this can be incredibly useful, especially when paired with videos and images pulled from reconstructed data sets, the time required for manual segmentation is an important consideration when attempting to extrapolate data from SBF-SEM datasets. There are currently a host of both free and purchasable software available for the manual and semi-manual segmentation of SBF-SEM image stacks. One free option for reconstruction software is the image processing package Fiji for ImageJ, an open source image processing program, which contains a segmentation editor plugin that allows for manual segmentation52,53. Additionally, the software Reconstruct offers an alternative free segmentation option54 (Figure 8). While potentially expensive, purchasable options often contain more robust feature sets, such as semi-automated segmentation processes or movie and image creation suites. One such option was used to create the reconstructions found in Figure&#160;5,&#160;Figure 6,&#160;Figure 7&#160;and Figure 9 (Details available in Table of Materials). Additionally, tools are available for the creation, analysis, and rendering of contrast-based 3D reconstructions using virtual reality with the potential to greatly speed up the reconstruction process20,55. While not always available for all applications, a host of software tools are available for computer assisted manual segmentation which have the potential to greatly decrease the time required for segmentation56,57,58. Regardless of the software used, considerable forethought and an understanding of the question being answered, or gap in knowledge to be filled, by serial reconstructions should precede segmentation, as the process is laborious and time-intensive.
The production of 3D reconstructions comes with its own considerations. With larger data sets processing power can be a limiting factor, and so optimizing the use of system resources can be critical for maintaining a productive workflow and speeding up the reconstruction and rendering process. When rendering a 3D reconstruction, most software converts segmented image stacks into a surface comprised of interconnected triangles. If a reconstruction project is large or intricate, the rendering of these triangles can require a great deal of computing power. While working on a 3D reconstruction, it can be helpful to limit the number of triangles the reconstruction software can use to convert the segmented images into reconstructed surfaces. This can be useful for monitoring the progress of a 3D reconstruction during the segmentation process. Once segmentation is complete, the triangle limit can be removed before rendering images or videos of reconstructions. Alternatively, and if the reconstruction software allows for it, we have found success monitoring the progress of a reconstruction using volume rendering rather than surface generation. Volume rendering, while not as suitable for images or videos meant for publication or presentation, requires far less processing power and as such can be helpful in providing a smooth experience when reconstructing and preparing images and videos of reconstructions. Additionally, it is best practice when manually segmenting an SBF-SEM data set to define every object to be reconstructed with its own unique identifier. If a field of epithelial cells is being reconstructed for example, rather than assigning all epithelial cells to a voxel group entitled &#34;epithelium,&#34; each epithelial cell should be assigned its own moniker (i.e., Epi1, Epi2, Epi3, etc.). This affords greater freedom when the reconstruction is complete, as each cell can be either included or excluded from the final rendering, assigned different colors or transparencies, or removed or introduced individually if a video is being produced. Furthermore, this allows metrics such as surface area or volume to be collected from each reconstructed object rather than the object group as a whole.
Another incredibly powerful tool for extracting quantitative data from SBF-SEM image stacks is the practice of stereology. Stereology takes advantage the inherent mathematical relationships between three-dimensional objects and their two-dimensional representations (i.e., electron micrographs). SBF-SEM data sets are ideal for the application of stereology, as this method for extracting 3D information from large datasets is considerably less time- and labor-intensive when compared to segmented reconstruction. Stereology generally consists of applying geometric grids to random, uniformly sampled images and has been used extensively over the past 50 years in order to produce accurate and unbiased estimates of cell/organelle number, length, surface area, and volume21,59,60,61,62,63. While 3D reconstructions can be impressive and provide a novel perspective on biological tissues, it is often quicker, more accurate, reproducible, and conducive for large sample sizes to use a stereological approach to data extraction. While there are many papers discussing the practical application of stereology64,65,66, a number of textbooks provide useful, in-depth overviews of the methodology as well as provide a number of stereological grids which can be applied to the study of tissue ultrastructure67,68,69.
SBF-SEM is a powerful imaging method that allows for the three-dimensional appreciation of tissue ultrastructure. While the ability to create 3D datasets with SEM resolution puts previously unanswerable questions within our reach, proper tissue preparation and an understanding of SBF-SEM imaging is paramount for the success of studies that utilize this microscopy method. It is our hope that the application of this protocol to future studies will lead to greater and greater insight into the biological mysteries that surround us, and continue to push us further into the frontiers of human knowledge.

Serial block face scanning electron microscopy (SBF-SEM) was first described by Leighton in 1981 where he fashioned a scanning electron microscope augmented with an in-built microtome which could cut and image thin sections of tissue embedded in resin. Unfortunately, technical limitations restricted its use to conductive samples, as non-conductive samples such as biological tissue accumulated unacceptable levels of charging (electron buildup within the tissue sample)1. While coating the block-face between cuts with evaporated carbon reduced tissue charging, this greatly increased imaging acquisition time and image storage remained a problem as computer technology at the time was insufficient to manage the large file sizes created by the device. This methodology was revisited by Denk and Horstmann in 2004 using a SBF-SEM equipped with a variable pressure chamber2. This allowed for the introduction of water vapor to the imaging chamber which reduces charging within the sample, making imaging of non-conductive samples viable albeit with a loss of image resolution. Further improvements in tissue preparation and imaging methods now allow for imaging using high vacuum, and SBF-SEM imaging no longer relies on water vapor to dissipate charging3,4,5,6,7,8,9. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality.
SBF-SEM allows for the automated collection of thousands of serially-registered electron microscopy images, with planar resolution as small as 3-5 nm10,11. Tissue, impregnated with heavy metals and embedded in resin, is placed within a scanning electron microscope (SEM) containing an ultramicrotome fitted with a diamond knife. A flat surface is cut with the diamond knife, the knife is retracted, and the surface of the block is scanned in a raster pattern with an electron beam to create an image of tissue ultrastructure. The block is then raised a specified amount (e.g., 100 nm) in the z-axis, known as a &#34;z-step,&#34; and a new surface is cut before the process is repeated. In this way a 3-dimensional (3D) block of images is produced as the tissue is cut away. This imaging system further benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day.
While SBF-SEM imaging primarily uses backscattered electrons to form an image of the block-face, secondary electrons are generated during the imaging process12. Secondary electrons can accumulate, alongside backscattered and primary-beam electrons that do not escape the block, and produce &#34;tissue charging,&#34; which can lead to a localized electrostatic field at the block-face. This electron accumulation can distort the image or cause electrons to be ejected from the block and contribute to the signal collected by the backscatter detector, decreasing the signal-to-noise ratio13. While the level of tissue charging can be decreased by reducing the electron beam voltage or intensity, or reducing beam dwell time, this results in a diminished signal-to-noise ratio14. When an electron beam of lower voltage or intensity is used, or the beam is only allowed to dwell within each pixel space for a shorter period of time, less backscattered electrons are ejected from the tissue and captured by the electron detector resulting in a weaker signal. Denk and Horstmann dealt with this problem by introducing water vapor into the chamber, thereby reducing charge in the chamber and on the block face at the cost of image resolution. With a chamber pressure of 10-100 Pa, a portion of the electron beam is scattered contributing to image noise and a loss of resolution, however this also produces ions in the specimen chamber which neutralizes charge within the sample block2. More recent methods for neutralizing charge within the sample block use focal gas injection of nitrogen over the block-face during imaging, or introducing negative voltage to the SBF-SEM stage to decrease probe-beam-lading energy and increase signal collected6,7,15. Rather than introducing stage bias, chamber pressure or localized nitrogen injection to decrease charge buildup on the block surface, it is also possible to increase the conductivity of the resin by introducing carbon to the resin mix allowing for more aggressive imaging settings16. The following general protocol is an adaptation of the Deerinck et al. protocol published in 2010 and covers modifications to tissue preparation and imaging methodologies we found useful for minimizing tissue charging while maintaining high resolution image acquisition3,17,18,19. While the previously mentioned protocol focused on tissue processing and heavy metal impregnation, this protocol provides insight into the imaging, data analysis, and reconstruction workflow inherent to SBF-SEM studies. In our laboratory, this protocol has been successfully and reproducibly applied to a wide variety of tissues including cornea and anterior segment structures, eyelid, lacrimal and harderian gland, retina and optic nerve, heart, lung and airway, kidney, liver, cremaster muscle, and cerebral cortex/medulla, and in a variety of species including mouse, rat, rabbit, guinea pig, fish, monolayer and stratified cell cultures, pig, non-human primate, as well as human20,21,22,23. While small changes may be worthwhile for specific tissues and applications, this general protocol has proven highly reproducible and useful in the context of our core imaging facility.

<table><tbody><tr><td>1/16 x 3/8 Aluminum Rivets</td><td>Industrial Rivet &amp;amp; Fastener Co.</td><td>6N37RFLAP/1100</td><td>Used as specimen pins.</td></tr><tr><td>2.5mm Flathead Screwdriver</td><td>Wiha Quality Tools</td><td>27225</td><td></td></tr><tr><td>Acetone</td><td>Electron Microscopy Sciences</td><td>RT 10000</td><td>Used to dilute silver paint.</td></tr><tr><td>Aspartic Acid</td><td>Sigma-Aldrich</td><td>A8949</td><td></td></tr><tr><td>Calcium Chloride</td><td>FisherScientific</td><td>C79-500</td><td></td></tr><tr><td>Conductive Silver Paint</td><td>Ted Pella</td><td>16062</td><td></td></tr><tr><td>Denton Desk-II Vacuum Sputtering Device equipped with standard gold foil target</td><td>Denton Vacuum</td><td>N/A</td><td>This is the gold-sputtering device used by the authors, alternates are acceptable.</td></tr><tr><td>Double-edged Razors</td><td>Fisher Scientific</td><td>50-949-411</td><td></td></tr><tr><td>Embed 812</td><td>Electron Microscopy Sciences</td><td>14120</td><td></td></tr><tr><td>Gatan 3View2 mounted in a Tescan Mira3 Field emission SEM</td><td>Gatan &amp;amp; Tescan</td><td>N/A</td><td>This is the SBF-SEM device used by the authors, alternates are acceptable.</td></tr><tr><td>Glass Shell Vials, 0.5 DRAM (1.8 ml)</td><td>Electron Microscopy Sciences</td><td>72630-05</td><td></td></tr><tr><td>Gluteraldehyde</td><td>Electron Microscopy Sciences</td><td>16320</td><td></td></tr><tr><td>Gorilla Super Glue - Impact Tough</td><td>NA</td><td>NA</td><td>Refered to as cyanoacrylate glue in text.</td></tr><tr><td>Ketjen Black</td><td>HM Royal</td><td>EC-600JD</td><td>Refered to as carbon black in text.</td></tr><tr><td>KOH</td><td>FisherScientific</td><td>18-605-593</td><td></td></tr><tr><td>Lead Nitrate</td><td>Fisher Scientific</td><td>L62-100</td><td></td></tr><tr><td>Microwave</td><td>Pelco</td><td>BioWave Pro</td><td>This is the microwave used by the authors, alternates are acceptable.</td></tr><tr><td>Osmium Tetroxide</td><td>Sigma-Aldrich</td><td>201030</td><td></td></tr><tr><td>Potassium Ferrocyanide</td><td>Sigma-Aldrich</td><td>P9387</td><td></td></tr><tr><td>Silicone Embedding Mold</td><td>Ted Pella</td><td>10504</td><td></td></tr><tr><td>Sodium Cacodylate Trihydrate</td><td>Electron Microscopy Sciences</td><td>12300</td><td></td></tr><tr><td>Samco Transfer Pipette</td><td>ThermoFisher Scientific</td><td>202</td><td>Used to make specimen pin storage tubes.</td></tr><tr><td>Swiss Pattern Needle Files</td><td>Electron Microscopy Sciences</td><td>62115</td><td></td></tr><tr><td>Thiocarbohydrazide</td><td>Sigma-Aldrich</td><td>223220</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Polysciences, Inc.</td><td>21447-25</td><td></td></tr><tr><td>&lt;strong&gt;Reconstruction Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amira Software</td><td>Thermo Scientific</td><td>N/A</td><td>Used to create the reconstructions found in figures 5-7 and 9.</td></tr><tr><td>Fiji (Fiji is Just ImageJ)</td><td>ImageJ.net</td><td>N/A</td><td>TrakEM2 can be added to Fiji to asist in manual segmentation.</td></tr><tr><td>Microscopy Image Browser (MIB)</td><td>University of Helsinki, Institute of Biotechnology</td><td>N/A</td><td></td></tr><tr><td>Reconstuct Software</td><td>Neural Systems Lab</td><td>N/A</td><td></td></tr><tr><td>SuRVoS Workbench</td><td>Diamond Light Source &amp;amp; The University of Nottingham</td><td>N/A</td><td></td></tr><tr><td>SyGlass</td><td>IstoVisio, Inc.</td><td>N/A</td><td>Allows for reconstruction in virtual reality and histogram-based reconstruction methods.</td></tr></tbody></table>

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, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Watch this Scientific Journal Video about Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples at JoVE.com

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

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

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, ...

serial-block-face-scanning-electron-microscopy-sbf-sem-biological

Nanyang Technological University

Research

JoVE Journal

Biology

7.5K Views.  University of Houston. Seriële blok-gezicht scanning elektronenmicroscopie biedt een ongekend beeld van driedimensionale weefsel ultra structuren, en kan een groot aantal vragen beantwoorden die voorheen onmogelijk of buitengewoon moeilijk na te streven. Deze techniek maakt de snelle en reproduceerbare productie van driedimensionale datasets mogelijk met minimale weefsellading en artefacten. En wat belangrijk is, het maakt het mogelijk om dagelijks duizenden afbeeldingen automatisch vast te leggen.Standaard...

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

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Developmental Biology

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Neuroscience

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

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 also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

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

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. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

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

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.

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 ...

SEM Imaging of Biological Samples

Source: <a href="https://www-jove-com.remotexs.ntu.edu.sg/author/Peiman_Shahbeigi-Roodposhti">Peiman Shahbeigi-Roodposhti</a> and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
A scanning electron microscope (SEM) is an instrument that uses an electron beam to nondestructively image and characterize conductive materials in a vacuum. As an analogy, an electron beam is to the SEM as light is to the optical microscope. The difference is that the electron microscope yields images of much higher resolution and magnification. The best optical microscopes typically have a resolution down to 200 nm, whereas SEMs usually claim a resolution of 0.5 nm. This is due to the fact that optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. Conversely, the SEM uses an energized electron beam, which as a wavelength of 1 nm. This characteristic makes them very dependable tools for the study of nano and microstructures. Electron microscopes also enable the study of biological samples with feature sizes too small for optical microscopy.
This demonstration provides an introduction to sample preparation and initial image acquisition of biological samples using a scanning electron microscope. In this case, a collagen-hydroxyapatite (HA) cellular scaffold will be studied. The vacuum environment of the SEM and the induced charging by the electron beam on non-conductive samples (such as organic matter) creates challenges that will be addressed in the preparation. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to give the participant more information on SEM to determine if this microscopy module is the best fit for a type of biological sample.

Scanning Electron Microscopy: Operations and Sample Preparation

Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
A scanning ...

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 Imaging of Neuronal Mitochondria Using a Serial Block-Face Scanning Electron Microscope

Source:&#160;Mukherjee, K.,&#160;et. al. Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.&#160;J. Vis. Exp. (2016)This ...

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

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