Serial Block-face SEM
What is it? Why do we use it? All the basics you need to know.
What is serial block-face SEM?
Serial block-face scanning electron microscopy (SBF-SEM) is a volume electron microscopy technique that has rapidly risen to prominence due to its ability to produce high-resolution, large volume three-dimensional reconstructions of biological specimens. SBF-SEM facilitates the automated imaging of relatively thick specimens in successive layers. The result is a series of images that can be combined to produce a volumetric representation of the specimen, providing unparalleled insights into its microstructure.
A specimen, typically embedded in resin, is placed in an ultramicrotome, which is integrated within the SEM chamber. The diamond knife of the ultramicrotome removes ultra-thin layers from the block's surface. After each sectioning, the freshly exposed block face is imaged using a scanning electron beam. This iterative process continues until the desired volume of the specimen is captured. The acquired images can then be stacked and aligned digitally to form a three-dimensional model.
A brief history of SBF-SEM
The conceptual seeds of SBF-SEM can be traced back to the 1980s, with the work of Steve Leighton and Alan Kuzirian working at the National Institutes of Health and the Woods Hole Marine Biology Laboratory. Their visionary idea revolved around developing a device to serial sectioning resin-embedded specimen block using a diamond knife inside an SEM chamber (1). In 1981, they were granted a patent for their "Method for Three-Dimensional Microscopy" which laid out the foundational principles of SBF-SEM (2).
Fast forward to the early 2000s, a significant contribution came from Winfried Denk and Heinz Horstmann, who are often credited with implementing the first functional model of SBF-SEM for study neuronal tissues. Their 2004 publication in PLOS Biology presented a system where an ultramicrotome was integrated into the vacuum chamber of an SEM. This configuration allowed for automated, sequential imaging of the block face after each cut, which could be combined to create detailed three-dimensional reconstructions (3).
What does a SBF-SEM system include?
Diamond knife: The diamond knife is a pivotal (no pun intended) component of the ultramicrotome integrated within the SBF-SEM system. Given that the slices can be as thin as tens of nanometers, the cutting edge is incredibly sharp with a radius of curvature down to 1 - 2 nm. This ensures that the cut surface is clean with minimal deformation or damages. The diamond surface is usually treated to facilitate the formation of debris in a continuous manner.
Precision Z-stage: The precision Z-stage is the mechanical platform on which the specimen block is mounted. This stage is critical for the accurate positioning of the sample relative to the diamond knife cutting plane. The stage must have a high stiffness to withstand any downward force exerted during the cutting process. The stage motion often incorporate a feedback loop to achieve repeatable precision stepping and correction for drifting.
Backscattered electron detector: The backscattered electron detector (BSED), typically positioned under the pole piece or integrated within the column for an in-lens configuration, is designed to capture electrons that are scattered back from the sample. In SBF-SEM imaging, there is usually very limited information in images obtained from secondary electrons due to the smoothness of the diamond knife-cut surface. However, the BSED can produce compositional contrast between different biological structures, as heavy metal stains bind selectively to various cellular components. A high-sensitivity BSED detector used in SBF-SEM often features a very thin passivation layer to improve its collection efficiency, which, in turn, may shorten the lifespan of the detector diodes.
How to prepare samples for SBF-SEM?
Preparing a sample for SBF-SEM involves several key steps to ensure the biological specimen retains its native state as closely as possible, while also being suitable for producing contrast when imaging with an electron beam. The first step is fixation which stablise the sample's structure. Perfusion fixation is often used for SBF-SEM samples as it occurs rapidly and throughout. This is done using a combination of solutions like cacodylate, formaldehyde and glutaraldehyde. This is followed by staining with heavy metals to enhance contrast under the electron microscope. The sample is immersed in heavy metal salts solutions such as osmium tetroxide or osmium ferrocyanide, uranyl acetate, lead aspartate along with thiocarbohydrazide. The staining procedure is often performed multiple times to enhance contrasting of cellure or extracellular constituents. The final steps involve dehydrating the sample, usually using ethanol or acetone, and embedding it in a resin, which is harden either by heat or UV. The choice of resin depends on factors like fluorescence preservation and stability under imaging conditions.
In summary, sample preparation for vEM is a meticulous process tailored to the specific requirements of the sample and the imaging modality, aiming to maintain the biological integrity while ensuring compatibility with the imaging environment.
1. Leighton, S. B. (1981). SEM images of block faces, cut by a miniature microtome within the SEM — A technical note. Scanning, 4(1), 85-89.
2. Leighton, S. B., & Kuzirian, A. M. (1981). U.S. Patent No. 4,253,837. Washington, DC: U.S. Patent and Trademark Office.
3. Denk, W., & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLOS Biology, 2(11), e329.