Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Abstract

scanning transmission electron microscopes can both measure the mass of single protein complexes and take amazingly clear images thereof, offering a wide range of applications in structural biology. The principle of mass measurement is presented and discussed and the scope of scanning transmission electron microscopy is illustrated by selected examples. Keywords:Mass determination · scanning transmission electron microscopy · sTEM unstained proteins to be clearly visualised under low dose conditions. Each pixel of the digital image is a quantitative scattering experiment from which the mass of the irradiated volume can be calculated [9]. Thus, the mass of a single protein complex can be determined by integrating over all pixel values that lie within its boundary. The method is applicable to macromolecules of widely varying mass and form (reviewed in [10]). When combined with SDS-gel electrophoresis and, where available, sequence information, STEM mass measurements serve to define protein stoichiometry. Further, as electron energy loss spectroscopy [11][12] or energy dispersive X-ray spectroscopy [13] can be performed while imaging, the STEM also offers the possibility of determining the distribution of specific elements in protein complexes. Indeed the feasibility of mapping single atoms bound to protein molecules has recently been demonstrated [14].The high contrast delivered by the dark-field detector system [15] can also be exploited in negative or positive stain microscopy [16]. Here the STEM yields clear single-shot images that are free from phase contrast fringes. These images frequently provide information only otherwise visible after averaging several hundred projections recorded with a transmission electron microscope (TEM). They thus make it possible to document the presence of distinct protein conformations, information that would be lost by averaging techniques. Indeed, STEM images of both negatively stained [17] and unstained [18] protein complexes have been used to reconstruct their 3D structure.The few dedicated STEMs employed in biology today are invaluable. In particular, the coupling of image and mass gives them the power to answer questions not resolvable by ultracentrifugation or mass spectrometry. Here we discuss the use of the STEM both as an imaging tool and to measure mass, and examine the uncertainties to be expected in STEM mass data sets. The Importance and Versatility of STEM Mass MeasurementsWorldwide, only four laboratories routinely use dedicated STEMs to measure the mass of biological samples. Their facilities are open for external collaborations and the many papers in the literature containing STEM mass measurements document the importance of this rare technique to the scientific community. Although the precision of STEM cannot match that of the mass spectrometer, its capability to measure an enormously wide mass range, its independence from shape assumptions and the presence of an image make the STEM an invaluable tool.The methodology of mass spectrometry has develop...

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

“…As noted above and discussed in Müller and Engel (2006), the distinct advantage of mass determination by EM is the possibility to link mass to shape. The statistical analysis subroutine of MAS-DET facilitates this procedure, readily generating galleries displaying ROIs with mass values in user-defined ranges, and also in the case of filaments, the individual average mass profiles.…”

“…As detailed in Müller and Engel (2006) the background contribution, which is closely related to quality of the support film (generally a very thin carbon layer), is of paramount importance to the precision of a mass measurement. Manual optimisation procedures, required in IMPSYS to minimise background contributions (Engel and Reichelt, 1988), are automated in MASDET reducing the processing time from hours to seconds.…”

Aggregation and fibril morphology of the Arctic mutation of Alzheimer’s Aβ peptide by CD, TEM, STEM and in situ AFM

“…The NIH IMAGE program (see ref. Rasband) was used to measure MPL of the fibrils according to standard procedures described in the literature (Thomas et al, 1994; Müller and Engel, 2006; Sousa and Leapman, 2007). …”

Electron microscopy of whole cells in liquid with nanometer resolution

“…This flow cell is placed in the vacuum of a STEM, using a fluid specimen holder. The annular dark field (ADF) detector in the STEM is sensitive to scattered electrons, which are generated in proportion to the atomic number (Z) of the atoms in the specimen (7,8), so-called Z contrast, where the contrast varies with ϷZ 2 . It is thus possible to image specific high-Z atoms, such as gold, inside a thick (several micrometer) layer of low-Z material, such as water, protein, or the embedding medium of a thin section (9).…”