Scanning Transmission Electron Microscopy (STEM)

A major advantage of STEM (compared with the conventional transmission electron microscope (CTEM) resides in its ability to provide several kinds of unique information, ranging from the mass distribution in large supramolecular assemblies to the total ion concentrations in small organelles. There are two reasons for the strength of the STEM as a sensitive and quantitative analytical device. First, the various signals generated from a specimen's interaction with a nanometer-sized electron probe can be collected by highly optimized detectors. This is important for analyses of biological specimens that are invariably susceptible to radiation damage. Second, the sequential acquisition of these signals as the electron probe is scanned pixel-by-pixel across the specimen lends itself readily to digital processing methods.

The crucial feature of the STEM is its high-brightness field-emission source consisting of a very sharp tungsten tip placed in a strong electric field. After being accelerated (in our instrument to 100 keV) the electrons are focused by condenser lenses and the objective lens into a sub-nanometer probe on the specimen. The STEM is best conceptualized as an instrument for measuring the ensuing scattering events; signals from a variety of these events can be recorded simultaneously by specific detectors, each of which provides different information about the specimen. A high-resolution map of the projected structure or composition is obtained by scanning the probe (which can be of atomic dimensions). In the original design of the STEM it was recognized that the signal due to elastic scattering could be collected with extremely high efficiency by a annular detector suitably positioned behind the specimen.

Applications of STEM over the past decade have mainly involved molecular weight measurement, a technique established by Wall and Hainfeld (1) and Engel et al. (2). The "mass mapping" technique is based on the fact that the elastic scattering intensity at any pixel is directly proportional to the projected molecular mass. It is normally performed on rapidly frozen, freeze-dried preparations of purified macromolecules. An effective resolution of approximately 2 nm is achievable as limited by the specimen preparation and radiation damage. Although the STEM cannot provide primary or secondary structure of proteins (a goal still within reach of high-resolution CTEM) it provides valuable information about the quaternary structures of large supramolecular assemblies.

The STEM is also a highly sensitive tool for elemental microanalysis. Near single-atom sensitivity has been demonstrated for the first time in our laboratory (3, 4) using electron energy loss spectroscopy (EELS) to analyze individual macromolecules.

Microanalysis is also of great interest to the cell physiologists, because it can be used to determine ion concentrations (e.g., calcium) in very small structures. The field-emission source offers a crucial advantage here too in terms of spatial resolution and sensitivity. Such measurements are obtained from x-rays generated in the sample by the incident electron probe. Thus, energy-dispersive x-ray spectroscopy (EDXS) provides information about elemental concentrations that is complementary to that obtained from EELS. The technique is sensitive to important biological elements including Na, Mg, P, S, Cl, K and Ca.

References

[1] Wall JS and Hainfeld JF. Ann. Rev. Biophys. Biophys. Chem., 15: 355-76, 1986.

[2] Engel A, Baumeister W and Saxton WO. Proc Natl. Acad. Sci., USA, 79: 4050-4054, 1982.

[3] Leapman RD. Detecting single atoms of calcium and iron in biological structures by electron energy loss spectrum-imaging.  J. Microsc. 210: 5-15, 2003.

[4] Leapman RD. Novel techniques in electron microscopy. Current Opinion Neurobiol, 14:591-98, 2004.