Serial block-face scanning electron microscopy is a method to generate high resolution three-dimensional images from small samples. The technique was developed for brain tissue, but it is widely applicable for any biological samples.
A serial block-face scanning electron microscope consists of an
ultramicrotome mounted inside the vacuum chamber of a
scanning electron microscope
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that ...
. Samples are prepared by methods similar to that in
transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...
(
TEM), typically by fixing the sample with aldehyde, staining with heavy metals such as
osmium
Osmium () is a chemical element; it has Symbol (chemistry), symbol Os and atomic number 76. It is a hard, brittle, bluish-white transition metal in the platinum group that is found as a Abundance of elements in Earth's crust, trace element in a ...
and
uranium
Uranium is a chemical element; it has chemical symbol, symbol U and atomic number 92. It is a silvery-grey metal in the actinide series of the periodic table. A uranium atom has 92 protons and 92 electrons, of which 6 are valence electrons. Ura ...
then embedding in an epoxy resin. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section (typically around 30 nm) from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire many thousands of images in perfect alignment in an automated fashion. Practical serial block-face scanning electron microscopy was invented in 2004 by
Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc., Thermo Fisher Scientific (VolumeScope) and ConnectomX.
Applications
One of the first applications of serial block-face scanning electron microscopy was to analyze the connectivity of
axons
An axon (from Greek ἄξων ''áxōn'', axis) or nerve fiber (or nerve fibre: see spelling differences) is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action pot ...
in the brain. The resolution is sufficient to trace even the thinnest axons and to identify synapses. By now, serial block face imaging contributed to many fields, like developmental biology, plant biology, cancer research, studying neuro-degenerative diseases etc. The technique can generate extremely large data sets, and development of algorithms for automatic
segmentation of the very large data sets generated is still a challenge. However much work is being done on this area currently. The
EyeWire project harnesses
human computation in a game to trace neurons through images of a volume of retina obtained using serial block-face scanning electron microscopy.
Many different samples can be prepared for serial block-face scanning electron microscopy and the ultramicrotome is able to cut many materials, therefore this technique has wider applicability. It is starting to find applications in many other areas ranging from cell and developmental biology to materials science.
Advantages and disadvantages
A disadvantage encountered with the SBEM method is that the thickness of the slice which can be removed with the ultra-microtome is limited (~25 nm), thus the resolution in the depth direction is limited. An advantage of the SBEM technique is that the specimen is stationary what improves the alignment in the stacks of images. Another advantage of the SBEM technique is the ability to acquire large data sets with a high level of detail. Because cutting by the ultra-microtome is extremely fast (comparing to the milling process in FIB-SEM), it can expose a wide area of the material (x and y directions) every sectioning. Additionally, by fast cutting, we can acquire many images in z-direction in a short period of time.
See also
*
Focused ion beam
References
External links
Original Publication in PLOS Biology
Gatan's 3View
Cell Centered Data Base, SBEM datasets
{{Electron microscopy
Electron microscopy