Serial block-face scanning electron microscopy
Serial block-face scanning electron microscopy (SBEM, SBSEM or SBFSEM) is a method to generate high resolution three-dimensional images from small samples, often biological samples such as brain tissue. A serial block-face scanning electron microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by similar methods to transmission electron microscopy, typically by fixing the sample with aldehyde, staining with heavy metals such as osmium and uranium 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 less than 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 SBFSEM was been invented in 2004 by Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc.
One of the first applications of SBFSEM was to analyze the connectivity of axons in the brain. The resolution is sufficient to trace even the thinnest axons and to identify synapses. SBFSEM generates extremely large data sets, and development of algorithms for automatic segmentation of the very large datasets generated is a challenge. The EyeWire project harnesses human computation in a game to trace neurons through images of a volume of retina obtained using SBEM.
Many different samples can be prepared for SBFSEM 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.