Development and application of STEM for the biological sciences

Abstract

The design of the scanning transmission electron microscope (STEM), as conceived originally by Crewe and coworkers, enables the highly efficient and flexible collection of different elastic and inelastic signals resulting from the interaction of a focused probe of incident electrons with a specimen. In the present paper we provide a brief review for how the STEM today can be applied towards a range of different problems in the biological sciences, emphasizing four main areas of application. (1) For three decades, the most widely used STEM technique has been the mass determination of proteins and other macromolecular assemblies. Such measurements can be performed at low electron dose by collecting the high-angle dark-field signal using an annular detector. STEM mass mapping has proven valuable for characterizing large protein assemblies such as filamentous proteins with a well-defined mass per length. (2) The annular dark-field signal can also be used to image ultrasmall, functionalized nanoparticles of heavy atoms for labeling specific amino-acid sequences in protein assemblies. (3) By acquiring electron energy loss spectra (EELS) at each pixel in a hyperspectral image, it is possible to map the distributions of specific bound elements like phosphorus, calcium and iron in isolated macromolecular assemblies or in compartments within sectioned cells. Near single atom sensitivity is feasible provided that the specimen can tolerate a very high incident electron dose. (4) Electron tomography is a new application of STEM that enables three-dimensional reconstruction of micrometer-thick sections of cells. In this technique a probe of small convergence angle gives a large depth of field throughout the thickness of the specimen while maintaining a probe diameter of <2 nm; and the use of an on-axis bright-field detector reduces the effects of beam broadening and thus improves the spatial resolution compared to that attainable by STEM dark-field tomography.

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.