Development and application of STEM for the biological sciences
Highlights
► We review, with a historical perspective, current applications of STEM in the biological sciences. ► The most widely used application of biological STEM is mass determination of proteins. ► Dark-field STEM enables localization of ultrasmall bionanoparticles containing heavy atoms. ► STEM-EELS hyperspectral imaging enables elemental mapping of subcellular compartments. ► Axial bright-field STEM tomography provides 3D ultrastructure in micrometer thick sections.
Introduction
From the outset Albert Crewe and his coworkers recognized the potential of their newly developed scanning transmission electron microscope (STEM) for applications to biological systems. In fact, the earliest paper from Crewe’s Laboratory in 1970 describing the full design of a “scanning microscope with 5 Å resolution” was published in the Journal of Molecular Biology, and includes the use of STEM to study a variety of biological structures including viruses, bacteriophages, catalase crystals, and ferritin molecules [1]. It was quickly realized that the flexible configuration of detectors in the STEM not only enabled a very high collection efficiency for signals generated by interaction of the finely focused electron probe with the specimen but it also provided the means for obtaining quantitative information pixel by pixel. This is particularly clear in the case of the annular dark-field detector (ADF), which yields a signal that is proportional to the mass of the biological structure that is contained within a defined volume illuminated by the probe.
We shall begin our review by describing two types of applications of ADF-STEM. First, ADF imaging can be used to determine the molecular masses of macromolecular assemblies, which has led to the vast majority of STEM applications in biology over the past 30 years [2], [3]. Second, the strong electron elastic scattering produced by high atomic number elements, first exploited by Crewe and coworkers in the early 1970s to image single atoms [4], can be applied more broadly to visualize small heavy atom clusters that are used for labeling specific amino-acid sequences of isolated protein assemblies or for localizing specific proteins in their cellular context [5], [6], [7].
Another technique originally developed for application to biological materials in Crewe’s laboratory was electron energy loss spectroscopy (EELS). The initial work by Isaacson et al. in the early 1970s included acquisition of high resolution spectra of low-loss and core-edge fine structure from DNA bases and other compounds, as well as an assessment of how radiation damage degraded this information [8], [9]. Subsequently, Isaacson and Johnson set out the formalism for determining detection limits for elemental nanoanalysis based on the cross sections for core edge excitations, and reached the prescient conclusion published in the first volume of Ultramicroscopy that single atom detection should be feasible [10]. The next section of this review summarizes the present capabilities and limitations of EELS nanoanalysis of biological specimens, as well as nanoanalysis by energy-dispersive x-ray spectroscopy (EDXS).
The capability today of carrying out high-performance STEM in transmission electron microscope columns has the potential to offer many more researchers the opportunity to apply STEM to biological systems. In this regard, amongst the biology community there has been rapidly growing interest in the use of electron tomography to image cellular ultrastructure in three dimensions. Recently, it has been demonstrated that STEM tomography provides some important advantages over conventional TEM tomography for imaging thicker sections of cells [11], [12], [13], [14]. The final section of our review describes this new technique, which has considerable potential for imaging micrometer-sized volumes of complex cellular architecture at a spatial resolution of approximately 5 nm.
Thus the aim of the present paper is to demonstrate how the STEM has evolved into a flexible tool with capabilities for (1) determining the masses of individual protein molecules, (2) localizing specific sites on protein assemblies labeled with ultrasmall heavy-metal nanoparticles, (3) analyzing the elemental composition of macromolecular assemblies as well as subcellular organelles by EELS and EDXS, and (4) elucidating the 3D structure of large volumes of complex eukaryotic cells. Fig. 1 shows in schematic form the principal modes of operation of the STEM microscope in connection with the types of applications discussed in this review. All these methods are complementary to TEM techniques, which can now be performed on the same instrument.
Section snippets
Mass measurement of macromolecular assemblies
STEM imaging in the ADF mode provides a unique means with which to determine the molecular mass of isolated protein assemblies [2], [15], [16], [17], [18], [19]. Importantly, because the mass of the protein is computed from an image, molecular mass can be directly correlated with molecular shape. Thus, it becomes feasible not only to measure the total mass of a complex and to determine its oligomeric state, i.e., tertiary and quaternary structure, assuming that the subunit mass is known, but
Visualization of ultrasmall heavy-metal clusters
Development of the STEM by Albert Crewe and colleagues was motivated by the prospect of visualizing single atoms of high atomic number elements supported on a lower atomic number matrix. This was indeed proved possible in 1970, in a milestone paper in Science [4]. Since then, the capability of ADF-STEM to image single heavy atoms has found innumerable applications in materials science (e.g., [43], [44]). In biology however, the electron dose required to visualize single atoms exceeds by orders
Biological nanoanalysis
In STEM the capability of collecting multiple signals originating from the interaction of the incident electron probe with atoms in the specimen enables point-by-point compositional analysis [61], [62]. Electrons that are scattered inelastically by the specimen carry quantitative information about the numbers of atoms of specific elements. This information is acquired by means of a magnetic prism spectrometer that disperses the electrons across a detector consisting of a scintillator coupled to
Three-dimensional imaging of micrometer-thick sections
Electron tomography (ET) in the TEM is a well-established technique for generating detailed 3D views of cellular ultrastructure at nanoscale (3–10 nm) spatial resolution [82], [83], [84]. For 3D ultrastructural studies of large eukaryotic cells and tissues, most work is carried out on samples prepared by rapid freezing/freeze substitution, heavy metal staining and sectioning in an ultramicrotome. The characteristic sample thickness for a 300 kV microscope falls in the range of 100 to 400 nm, with
Conclusion
Forty years after the development of the STEM by Albert Crewe and colleagues, the technique has matured to the point where it can be applied almost routinely to biological structures, and the new generation of (S)TEMs has made the technique more generally available. Mass mapping based on imaging with the annular dark-field signal, as originally developed by Wall [17] and by Engel [15], has been STEM’s most widespread application. This technique enables the analysis of large and heterogeneous
Acknowledgments
We would like to acknowledge Dr. Guofeng Zhang and Dr. Martin Hohmann-Marriott for providing the specimens of C. reinhardtii and pancreatic beta cells; Mr. Daniel Cox for assistance in tomographic reconstruction and 3D modeling; Dr. Christopher Ackerson for valuable discussions on the synthesis of the Au144 clusters; and Ms. April Adams for help in synthesizing the Au144 clusters. This work was supported by the Intramural Research Programs of the National Institute of Biomedical Imaging and
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2022, Journal of Structural BiologyCitation Excerpt :Meanwhile, we also compared iDPC-STEM and ABF-STEM techniques on the biological sections with the minimal staining. Previous studies have suggested ABF-STEM can yield better images with better contrast in comparison with the conventional TEM (Hohmann-Marriott et al., 2009; Sousa and Leapman, 2012; Wolf et al., 2014). Our present study also confirmed this observation.
Exploring valence states of abnormal mineral deposits in biological tissues using correlative microscopy and spectroscopy techniques: A case study on ferritin and iron deposits from Alzheimer's disease patients
2021, UltramicroscopyCitation Excerpt :Likewise such experiments have contributed to notable advances, particularly for early stage cancer detection and treatment [15,16]. Some particularly well-known examples of phys-EM techniques in the biological field concern the use of holographic analysis of magnetic fields in magnetotactic bacteria [17] and various STEM techniques (EELS, EDS and tomography) and energy filtered TEM (EFTEM) in pathological tissues [18,19]. As background for the present research, it is known that inorganic trace elements, such as calcium, magnesium, chromium, iron and copper, are essential to human health [20].
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