Types of Imaging, Part 1: Electron Microscopy

This article is the first in a series of articles that are intended to provide a brief overview of the types of microscopy currently used in the biological sciences. The high-resolution electron microscope is a powerful tool for analyzing molecular structure, interactions, and processes. This article will discuss the types, characteristics, and practical application of electron microscopy. Electrons differ from X-rays and neutrons by their ability to form images and small probes. An electron microscope uses electrostatic and electromagnetic lenses to control an electron beam and focus it to form an image. There is a wide range of different methods in electron microscopy that use the various signals arising from the interaction of the electron beam with the sample to obtain information regarding structure, morphology, and composition. The first transmission electron microscopy (TEM) was built in 1931. TEMs use electrons as the illumination source and their much shorter wavelength than visible light enables a resolution that is at least 1,000 times higher than with a light microscope. A TEM basically consists of a user console, an electron gun, electron lenses that are magnetic coils, primary lenses to form and magnify the image, and apertures that limit the size of the electron beam that passes through it. A TEM produces an image of the entire object, including the surface and the internal structures (Fig. 1A,B). In TEM, a beam of electrons is transmitted through an ultrathin specimen and they interact with the specimen as they pass through its entire thickness of the sample. Objects are able to be observed to the order of a few angstroms (10−10 m). Small details in the cell or different materials down to atomic levels can be examined. The ability for high magnifications has meant that the TEM is a valuable tool in medical, biological, and materials applications. (A) Electron micrograph from a longitudinal section of striated muscle. (B) The same section as “A” at a higher magnification. Black bars indicate 1 μm. A, A band; I, I band; M, mitochondrion; and Z, Z line. The image was provided by Cynthia Jensen, PhD, the University of Auckland. The major advantages of TEM include the following. (1) TEM provides the most powerful magnification, potentially over one million times or more. (2) TEM has a wide range of applications, including providing information on element and compound structure as well as surface features, shape, size, and structure. (3) Images are of high quality and detailed. (4) Lastly, TEMs' are easy to operate with proper training. Disadvantages of the TEM technique include the following. (1) Many materials require extensive sample preparation to produce a sufficiently thin sample to be electron transparent, making TEM analysis a lengthy process. (2) The structure of the sample can also be changed during the preparation process. (3) Additionally, the field of view is relatively small, and, therefore, the region analyzed may not be representative of the whole sample. (4) Lastly, the sample may be damaged by the electron beam. High-resolution TEM (HRTEM), also known as phase contrast TEM, is used to investigate crystal structure. It uses a self-supporting thin sample (usually tens of nm thick) illuminated by a highly collimated kilovolt electron beam (Kirkland et al.,2007; Kirkland,2010). Phase contrast is the basic mechanism behind image formation in HRTEM. Formation of an HRTEM image involves three steps: (1) electron scattering in the specimen; (2) formation of a diffraction pattern in the back focal plane of the objective lens; and (3) formation of an image in the image plane. The atomic structure of a specimen can often be directly investigated by HRTEM. It can provide real-space images of the atomic configuration at localized structural irregularities and defects in materials. Instrumentation for HRTEM is continuing to be developed and substantial advances have been made in the recent development of monochromators, aberration correctors, and energy filters. The scanning TEM (STEM) is a powerful and versatile instrument capable of atomic resolution imaging and nanoscale analysis (Nellist,2007). For imaging of biological samples, STEM makes use of dark-field microscopy, and is more efficient than conventional TEM, enabling high contrast imaging of biological samples, without the need for staining. STEM is similar to the scanning electron microscope (SEM). STEM is distinguished from conventional TEM in that the electron beam is focused into a narrow spot, which is scanned over the sample in a raster pattern. Scattered electrons are detected and their intensity is plotted as a function of probe position to create an image. STEM has better spatial resolution, is capable of additional analytical measurements, and requires more sample preparation than SEM. Most dedicated STEM instruments have an electron gun that may be configured at the bottom or the top of the column. The low-voltage electron microscope (LVEM) is the smallest commercial TEM in the world and it comprises all the standard imaging modes that can be found in conventional TEMs as well as further applications. The LVEM can work in transmission (TEM) or diffraction (selected area electron diffraction) modes as well as in scanning modes (STEM and SEM with BSE—backscattered electrons) with nanometer spatial resolution. An LVEM operates at accelerating voltages of a few kiloelectronvolts or less. Advantages to imaging under LVEM include the ability to produce high quality images for samples that would be otherwise impossible to visualize under conventional electron microscopy techniques (e.g., specimens that do not exhibit sufficient contrast in conventional TEM). Additionally, a considerable decrease in electron energy results in a better contrast. This improved contrast permits a significant reduction, or elimination, of the heavy metal negative staining step for TEM imaging. Limitations are that low-voltage microscopes obtain resolutions of 2 nm–3 nm and this thickness has to be less than that required for TEM or STEM. Fluorescence-integrated TEM images show TEM images with a fluorescent perspective. High-pressure freezing techniques can be used for correlative light and electron microscopy on the same sample. Laser scanning confocal microscopy (LSCM) is used for its ability to collect fluorescent, as well as transmitted and back scattered light images at the same time. The light LSCM and EM images collected may be mirror images of each other. The LSCM image from a single focal plane can be opened in Photoshop and pasted over a TEM image (Sims and Hardin,2007). Fluorescent information from a whole mount or from thin sections can be displayed as a color overlay on TEM images. The first electron diffraction (ED) experiment was carried out in 1927 (Davisson and Germer,1927). ED is an important tool for the study of both crystal structure and molecular structure. Analyses are usually performed using a TEM or a SEM. Electrons are accelerated by an electrostatic potential to achieve the desired energy and determine their wavelength before they interact with the sample of interest. Each atom in the structure being studied scatters the incident wave in all directions, with an intensity that is determined by the structure of the individual atoms. The angles at which coherent scattering takes place contain information regarding the geometrical arrangement of the atoms in the lattice. Therefore, the scattering angles of the diffracted beams are of interest to a solid state physicist who wants to determine the structure of a cresol, while the intensity of the beams are important to a biologist who wants to determine the structure of a protein molecule. The major advantages of ED are the extremely short wavelength (approximately equal to 2 pm), the strong atomic scattering, and the ability to examine tiny volumes of matter. Recent developments have improved the quantitative analysis of ED intensities and resulted in new types of highly accurate ED techniques for structure refinement and structure factor measurement. There are three types of ED: (1) low energy ED (LEED); (2) transmission high energy ED (THEED); and (3) reflection high energy ED (RHEED). LEED and RHEED are used to characterize surfaces. THEED is usually associated with TEM and molecular scattering. In 1981, a new means of sample preservation for electron microscopy investigations was discovered, using the cryotechnique. Cryo-electron microscopy, or electron cryo-microscopy, was designed to overcome radiation damage of biological specimens (Plitzko and Baumeister,2007). Cryopreparation is one of the most important developments in biological electron microscopy. Instead of chemical fixation and dehydration, which elute molecular constituents and cause shrinkage artifact, respectively, the biological sample is embedded within water or its original buffer solution by rapid freezing at very low temperatures. This technique was revolutionary because, for the first time, biological samples were able to be investigated ultrastructurally in their native state (Fig. 2). However, single two-dimensional (2D) images are insufficient for complete structural characterization, and, therefore, three-dimensional (3D) images are required. Cryo-electron tomography (CET) was developed whereby a 3D reconstruction of a sample is created from tilted 2D images (Frank,1992). Cryo-scanning electron micrograph of the pleural space in situ in sheep. Liquid Freon, cooled with liquid nitrogen, was sprayed onto the chest wall to freeze the lung juxtaposed to the chest wall. The pleural space is located between the opposed arrowheads. The pleural space has a uniform width of ∼18 μm. The visceral pleura (VP) of the lung and parietal pleura (PP) of the chest wall are composed of connective tissue. Alveoli (A) are open. Refer to Albertine et al., J Appl Physiol 70:123–134, 1991. The micrograph was contributed by Kurt H. Albertine, Ph.D., University of Utah. The major advantage of cryo-electron microscopy is that it uses low doses of radiation, and therefore, the electron beam causes less damage to the sample. Additionally, there is no staining to distort the sample and the sample is always in solution and does not come into contact with an adhering surface (i.e., the true shape of the hydrated molecule in solution has not been distorted). The major advantage of CET is that it can be used to obtain structural details of complex cellular organizations at subnanometer resolution. Three techniques are used for 3D characterization: electron crystallography, single-particle electron microscopy, and electron tomography. Electron crystallography is used to determine the arrangement of atoms in solids. Single-particle electron microscopy involves cryo-electron microscopy of individual noncrystallized macromolecular assemblies. Unlike the other two methods, electron tomography does not involve implicit or explicit averaging of the specimen, and therefore, it is a suitable method for imaging of pleiomorphic objects. It is used to image entire cells or nonsymmetric viruses. It also has the potential to determine the structure as well as molecular interactions of the various macromolecules inside a cell. Electron tomography is the only 3D imaging technique that can image cells or organelles in a close-to-native state at molecular resolution. Low-energy electron microscopy (LEEM) is an imaging method that uses elastically BSE with energies below approximately 100 eV, frequently less than 10 eV (Bauer,2007). In contrast to TEM, which generally involves electrons in the 100 keV range where backscattering is negligible, the backscattering cross-sections for low-energy electrons are large enough to provide surface imaging. LEEM is used to image atomically clean surfaces, atom-surface interactions, and thin (crystalline) films. High-energy electrons (15 keV–20 keV) are emitted from an electron gun, focused using a set of condenser lenses, and sent through a magnetic beam deflector. The “fast” electrons travel through an objective lens and begin decelerating to low energies (1 eV–100 eV) near the sample surface because the sample is held at a potential near that of the gun. The low-energy electrons are termed “surface-sensitive” and the near-surface sampling depth can be varied by tuning the energy of the incident electrons. The major advantage of LEEM is that it offers both imaging and diffraction information regarding surfaces. Other advantages of LEEM include the routine availability of vacuums below 10,210 Torr for cleanliness in surface-specific work, and the exceptional surface specificity of the low energy beam, as well as ready access to the active surface at temperatures as high as 1,700 K. A LEEM can become a tandem machine fitted with an in situ beam of energetic ions (Ondrejcek et al.,2009). This has enabled advances in the science of surface behavior. Practical applications include quantitative measurements of fundamental properties such as surface diffusion coefficients and chemical potentials, which can be difficult to determine. The first electron beam scanner able to produce an image of the surface of a bulk sample with emitted secondary electrons was developed in 1935. Submicroscopic resolution with an SEM was achieved a few years later using the transmission mode (STEM, see above). In the SEM, a tiny electron beam is focused onto the sample (Reichelt,2007). While the beam is being scanned across a selected sample area, generated signals are being recorded, and an image is formed pixel by pixel. The signals contain information on the sample's surface topography, composition, and other properties such as electrical conductivity. The SEM can produce various types of signals, including secondary electrons, back-scattered electrons, light (cathodoluminescence), characteristic X-rays, specimen current, and transmitted electrons. In contrast to TEM methods, which require very thin samples, SEM can examine large samples. Valuable information regarding morphology, surface topology, and composition, can be obtained. SEM microscopes achieving resolutions below 1 nm are currently available. SEM is a well-established method for the characterization of surfaces in ultrahigh vacuum, high vacuum, and low vacuum in many different fields. It is used to investigate materials, such as metals, alloys, ceramics and glasses, surface sciences, semiconductor research, polymer and food research, geology, mineralogy, archaeology, and biology. The major advantage of the SEM is the tremendous depth of focus (Fig. 3). The brilliant image contrast and the relatively simple sample preparation for imaging of surfaces are other advantages. SEM micrograph showing a nylon fiber that was snapped while still warm. The fiber was imaged using a Leica S440 SEM in secondary electron mode at 15 kV. It is an example of the large depth of field, topographic information and perceived 3D quality characteristic of 2nd electron SEM images. The image was provided by Dane Gerneke, the University of Auckland. SEM instrumentation was improved by the development of the field emission SEM (FESEM), which became commercially available in the 1980s. A field emission gun is used instead of a thermionic gun for electron beam generation. High-resolution imaging by FESEM became possible with further improvements in electron optics and electron detectors as well as in specimen preparation. Development of the environmental SEM (ESEM) enabled use of hydrated specimens, omitting the dehydration procedure. The accumulation of electric charge on the surfaces of non-metallic specimens can be avoided by using ESEM because the specimen is placed in an internal chamber at higher pressure. In ESEM, investigations of specimens using secondary or BSE for imaging can be performed in a low vacuum, in contrast to SEM and FESEM, which are mostly restricted to a high vacuum. This is useful for samples that consist of materials or those that contain dirt or fluids (e.g., natural specimens containing water or oil). The technique of secondary ion mass spectrometry (SIMS) is where material desorbed from a surface by energetic particle bombardment is analyzed by mass spectrometry (Lockyer,2007). Originally developed in the 1950s to analyze metals and oxides, SIMS has evolved into a powerful and versatile analytical tool to investigate surfaces. A primary ion beam is directed at a material and is used to “sputter,” or eject, positively and negatively charged secondary ions from the surface (Castner,2003). Development of the instrumentation and better understanding of the underlying physics have resulted in the development of various operational modes. In the static mode (where <1% of the surface is struck by incoming primary ions), the secondary ions come from the outermost nanometer of the sample, which provides the desired surface sensitivity. A wide range of atomic and molecular secondary ions can be produced, which depends on the type and energy of the incident primary ions and the nature of the material. These ions can then be analyzed using mass spectrometry. Applications in the life sciences are varied, including molecular biology and medicine. A major advantage of static SIMS is that it does not require derivitization/labeling of compounds to be detected, unlike many other techniques for biological imaging. Other advantages of the technique include its high sensitivity and specificity, especially for small molecules such as drugs and metabolites, and its high lateral resolution. The capability of SIMS for surface imaging has been achieved by several different approaches. One such approach is the use of “time-of flight” (TOF) mass analyzers. TOF mass spectrometry is a method of separating ions of different masses based on the time required for the ions to traverse a fixed distance. TOF-SIMS has improved the efficiency of ion detection with its very high transmission (>50%) and parallel detection of all masses. TOF-SIMS, as well as other surface analytical techniques, such as X-ray photoelectron spectroscopy, is used to examine the detailed surface chemical composition, orientation, and spatial arrangement of tailored biomaterials. Another area of development is determination of new primary ion sources to produce a higher yield of secondary ions in SIMS experiments. Liquid-metal-ion guns have become popular for imaging SIMS applications, with gallium as the most widely used element, but indium and gold are also currently used. The development of polyatomic or “cluster ion” beams has resulted in an increased yield of high-molecular-weight fragments with low sample damage. Recent progress in instrumental developments, notably cluster ion beams, promise further advances in this field of microscopy. An energy filter can be used as an accessory with TEM, which enables the establishment of a method for elemental microanalysis, and this is called electron energy loss spectroscopy (EELS) (Egerton,1982). 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A of is the in the preparation of very thin electron (LEEM) (LVEM) The Dane Gerneke, Cynthia and Kurt Albertine for providing the images.