In scanning electron microscopy, the electron beam does not pass through the specimen but is scanned across its surface.
In many ways, the images obtained from SEM more closely resemble those seen on a television screen than on the TEM monitor. They are three-dimensional in appearance and portray the surface structure of an examined sample. For the examination of most tissues, the sample is fixed, dehydrated by critical point drying, coated with an evaporated gold–carbon film, mounted on an aluminum stub, and placed in the specimen chamber of the SEM. For mineralized tissues, it is possible to remove all the soft tissues with bleach and then examine the structural features of the mineral.
Scanning is accomplished by the same type of raster that scans the electron beam across the face of a television tube. Electrons reflected from the surface (backscattered electrons) and electrons forced out of the surface (secondary electrons) are collected by one or more detectors and reprocessed to form a high-resolution three-dimensional image of a sample surface. In earlier models of microscopes, images were captured on high-resolution cathode ray tube (CRT) or photographic plate; modern instruments, however, capture digital images using sensitive detectors and CCD for display on a high-resolution computer monitor.
Other detectors can be used to measure X-rays emitted from the surface, cathodoluminescence of molecules in the tissue below the surface, and Auger electrons emitted at the surface.
The scanning-transmission electron microscope (STEM) combines features of the TEM and SEM to allow electron-probe X-ray microanalysis.
The SEM configuration can be used to produce a transmission image by inserting a grid holder at the specimen level, collecting the transmitted electrons with a detector, and reconstructing the image on a CRT. This latter configuration of an SEM or scanning-transmission electron microscope (STEM) facilitates the use of the instrument for electron-probe X-ray microanalysis.
Detectors can be fitted to the microscope to collect the X-rays emitted as the beam bombards the section; with appropriate analyzers, a map can be constructed that shows the distribution in the sections of elements with an atomic number above 12 and a concentration sufficient to produce enough X-rays to analyze. Semiquantitative data can also be derived for elements in sufficient concentration. Thus, both the TEM and the SEM can be converted into sophisticated analytical tools in addition to being used as “optical” instruments.
The atomic force microscope has emerged as one of the most powerful tools for studying the surface topography at molecular and atomic resolution.
One newer microscope that has proved most useful for biologic studies is the atomic force microscope (AFM). It is a nonoptical microscope that works in the same way as a fingertip, which touches and feels the skin of our face when we cannot see it. The sensation from the fingertip is processed by our brain, which is able to deduce surface topography of the face while touching it.
In the AFM, an ultrasharp, pointed probe, approaching the size of a single atom at the tip, scans the specimen following parallel lines along the x-axis, repeating the scan at small intervals along the y-axis. The sharp tip is mounted at the end of a highly flexible cantilever so that the tip deflects the cantilever as it encounters the “atomic force” on the surface of the specimen (Fig. 1.13). The upper surface of the cantilever is reflective, and a laser beam is directed off the cantilever to a diode. This arrangement acts as an “optical lever” because extremely small deflections of the cantilever are greatly magnified on the diode. The AFM can work with the tip of the cantilever touching the sample (contact mode), or the tip can tap across the surface (tapping mode) much like the cane of a blind person (Fig. 1.13 insets).