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).

All animals were handled according to the guidelines described in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Vision and Ophthalmic Research and the University of Houston College of Optometry animal handling guidelines. All animal procedures were approved by the institutions in which they were handled: Mouse, rat, rabbit, guinea pig, and non-human primate procedures were approved by the University of Houston Animal Care and Use Committee, zebrafish procedures were approved...

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/16 x 3/8 Aluminum Rivets</td>
			<td>Industrial Rivet &#38; 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 &#38; 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>Reconstruction Software</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 &#38; 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.

Mouse Cornea 
This protocol has been applied extensively to the mouse cornea. Using SBF-SEM imaging a network of elastin-free microfibril bundles (EFMBs) were shown to be present within the adult mouse cornea. It was previously believed that this network was only present during embryonic and early postnatal development. SBF-SEM revealed an extensive EFMB network throughout the cornea, with individual fibers found to be 100-200 nm in diameter when measured in cross-section. It was also found that thi...

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, ...