Few research instruments are as widely used in science as are microscopes. I will present a very brief description of microscopy and the many different types of microscopes by this series of articles. These are not in-depth discussions, but rather are designed to provide an understandable background about microscopy for teachers, technicians, students, parents, and other beginning users. Since I want to keep everything concise and suitable for non-experts, I will not give the usual optical equations and mathematics, ray path diagrams, or standard instructions about how to use these microscopes!
The fundamental concepts and general terms for using microscopes and understanding microscopy were covered by “Part 1” , and light microscopy was presented by “Part 2” . Part 3 now presents electron microscopy; all beginners should first study Part 1.
Waves and optics: electrons and photons.
Electron waves/particles have several differences from light waves and photons: (1) electron waves are much smaller, meaning that resolution in electron microscopes is better than in light microscopes; (2) electrons are negatively charged, while photons are neutral, meaning that electron microscopes must utilize electromagnetic lenses rather than the glass lenses used for light microscopes; (3) electrons can be transmitted through only very thin specimens (e.g., 50-100 nanometers in thickness), meaning that the usual 5-10 micrometer thickness of slices used for light microscopy are not usable for electron microscopy because far too few electrons will be transmitted to reach the detector; and, (4) unlike photons, electrons interact very strongly with all atoms and molecules, therefore necessitating keeping their pathway inside electron microscopes at a high vacuum level. Beyond these prominent distinctions, the optics of electrons in electron microscopy have counterparts with the optics of photons in light microscopy; however, a multitude of controls for the vacuum system, high voltage generation, coordinated electronics and monitors, cameras, and associated accessories make electron microscopes much more complex than any light microscope.
General design of electron microscopes.
The chief components in electron microscopes are shown in the highly schematic diagram given above under the title. Many other parts are not depicted (see text for details!). This diagram can be readily compared to that given for compound light microscopes in the previous article (see: “Part 2”). Electron microscopes commonly are divided into 2 fundamental types depending upon how the specimen is irradiated by the beam of electrons (i.e., all at once, or point by point).
Different kinds of electron microscopes: common transmission electron microscopes.
For these instruments. an entire area of a specimen is irradiated by the electron beam all at once. Major components are kept in a high vacuum inside the column. Electrons are generated at high voltage (e.g., 50-1,000kV) from the electron gun (electron source) by emission induced from a hairpin or a pointed filament. An anode in the gun then draws the stream of electrons down the column into the several condenser lenses; these focus the beam onto the specimen. After transmission through a very thin specimen, the beam then passes into the objective lens. This strong lens contains an objective aperture (i.e., a sheet or disk of metal with a precise very small hole centered on the optical axis); this intercepts those transmitted electrons which have been strongly scattered by atoms in the specimen and prevents them from reaching the plane of detection, thereby creating image contrast. A series of several other electromagnetic lenses follows and acts to increase the magnification of the transmitted image; magnifications can range from 100X up to 1,000,000X. The transmitted electrons finally are received by an electron detector in a photographic or digital camera which records the image (i.e., an electron micrograph). In addition to images, electron diffraction patterns from crystalline specimens also can be recorded. Special attachments to transmission electron microscopes extend the capabilities of these instruments for diverse samples (e.g., frozen-hydrated specimens with cryomicroscopy, special specimen chambers for chemical reactions with in-situ microscopy and analysis, etc.).
Different kinds of electron microscopes: scanning electron microscopes.
These electron microscopes are functionally analogous to dissecting light microscopes, in that the natural or sliced surface of specimens is imaged. The beam of electrons is focused to a fine point by condenser lenses, and then is directed onto a specimen with a raster pattern, similarly to the way a television image is formed. Unlike transmission electron microscopes, minute parts of the specimen area to be examined are irradiated consecutively rather than all at once. Scanned imaging uses different electron detectors to capture one of several available signals (e.g., secondary electrons emitted by the specimen surface in response to being hit by the incoming primary electrons, backscattered electrons reflected from the specimen surface, etc.); these electron signals are received by a detector located above the specimen (i.e., the electrons forming an image are not transmitted through the specimen). Magnifications generally range from 10X to 30,000X.
Contrast in scanned images is mainly due to differences in topography and atomic composition of the specimen. These mechanisms produce different numbers of detected electrons, thus providing image contrast. Images from secondary electrons in scanning electron microscopes often have a 3-dimensional character due to shadowing by neighboring parts of the specimen. Image resolution levels usually are influenced by the characteristics of each specimen. Scanning electron microscopes mostly are used to image much finer details in surface structures than are given by a dissecting light microscope; however, resolution is poorer than that produced by transmission electron microscopes.
Different kinds of electron microscopes: scanning-transmission electron microscopes.
A third version of electron microscopes also exists, and is a hybrid of the two described above. Scanning-transmission electron microscopes irradiate the sample in a sequential raster pattern like scanning electron microscopes, but still form images from those electrons that are transmitted through the specimen (i.e., the electron detector is on the far side of the specimen, unlike the case for scanning electron microscopes). This optical arrangement can achieve atomic resolution and is utilized particularly for compositional mapping and for very high resolution imaging.
A number of specialized and experimental electron microscopes also are available for research usage, but will not be covered in this introductory presentation.
Specimen preparation for electron microscopy.
For study by transmission electron microscopy, good preparation of samples is vital in order to achieve high quality, reproducible, and artifact-free results. Samples most frequently are mounted onto a very thin film of carbon or plastic; this support film is held upon a metallic grid (i.e., similar to a window screen, but much thinner and smaller). Rocks and minerals, tissues, organs, and industrial products all must be prepared by slicing, thinning, or polishing into a thin enough state to permit the electron beam to penetrate through the specimen. In biology, specimens are chemically (i.e., buffered cross-linkers) or physically (i.e., very rapid freezing) fixed, then are dehydrated and embedded, and finally are sliced into ultrathin sections using an ultramicrotome (i.e., a special finely controlled cutting machine); these slices commonly are stained by heavy metal solutions in order to increase the image contrast. Electron microscope immunocytochemistry with specific antibodies is used to locate various protein components in ultrathin sections. Rapid freezing is used to prepare macromolecules and cells for electron cryomicroscopy; the frozen-hydrated unstained specimens are kept at liquid nitrogen or liquid helium temperature inside the electron microscope, thereby maintaining their native structure.
For scanning electron microscopy, non-conductive specimens must be treated by coating them with a conductor so they become conductive. Sample preparation aims to produce specimens that are (1) dry (i.e., simply putting a moist specimen into the high vacuum of an electron microscope will cause its collapse and other structural changes), (2) conductive (i.e., non-conducting samples give bad images due to their becoming charged under the beam), (3) producing a high level of signal (i.e., coating with a thin layer of metal produces increased numbers of secondary electrons, thus giving a brighter image), (4) compatible with higher resolution imaging, and, (5) free from artifacts.
What are electron microscopes actually used for?
The several different kinds of electron microscopes are used very extensively for imaging, diffraction, and analysis in all 3 branches of science, and also in industry. For research, they are utilized to examine normal, abnormal, and experimental structure, along with the amount and distribution of compositional elements. Other major uses include atomic level imaging, spectroscopy, and experimental electron optics. For crystallography in bioscience and materials science, electron diffraction patterns are essential for structural characterization; electron crystallography is an important special branch of applied electron optics. Enormous efforts have been devoted to producing better specimen preparation, since that has such a clear importance for determining exactly what can be imaged, detected, and meaningfully studied.
Correlative microscopy uses electron microscopes to obtain higher resolution details for specimens that first were imaged at moderate resolution and magnifications (e.g., by light microscopy). Their enormous range of magnifications can permit correlative microscopy to be conducted by a single transmission or scanning-transmission instrument. As one real example, defects and inclusions in semiconductor devices are first characterized by scanning electron microscopy and then analysis of their elemental distribution is mapped with a scanning-transmission electron microscope.
For those of us using electron microscopes in our daily work, they also provide quite a lot of fun! Electron microscopists are analogous to airline pilots looking down at a landscape!
The chief advantages and the chief problems of electron microscopes.
All electron microscopes stand out for their ability to image structure at higher resolution levels than can be achieved by light microscopy. Atomic-level structure now can be directly imaged; this capability is usable for many kinds of specimens, and excels for nanomaterials and materials science.
Electron microscopes are quite costly and purchase often can be justified only when made for a group of multiple users. Routine and special specimen preparations frequently are expensive, hazardous (due to exposure to toxic chemicals and nanoparticles), and give good results only with much training and experience of the technician or microscopist. The biggest problems for electron microscopy of biosamples, polymers, and wet materials are that: (1) they must be either frozen or dried, both of which easily can cause undesired changes in their native structure, and, (2) the same illuminating electrons that enable imaging also cause radiation damage to the specimen, thereby changing their native structure. Good images of artifacts are commonplace.
Recent developments in electron microscope instrumentation.
Modern electron microscopes have become increasingly sophisticated and specialized in their capabilities. The recent commercial production of correctors for electron optical lens aberrations now permits the measured level of resolution to be equal to the calculated theoretical resolution limit; this permits better atomic imaging and better compositional analysis to be achieved. New experimental approaches for the electron source, camera, and optical design are progressing nicely; new instrumentation accessories and new software are being developed every year.
Electron microscopy in science education.
Electron microscopy is very widely used in science education at secondary schools and colleges, but all that is almost completely hidden from students by their teachers! The source and nature of the many images from electron microscopy shown in classrooms are only rarely indicated! Examples of this silent treatment include cells and tissues, organelles and macromolecules, bacteria and viruses, solid state devices, polymers, fibers, minerals, metals and alloys, nanomaterials, etc.
Courses on electron microscopy mostly are found only in larger universities and specialized educational institutions. Recently, some manufacturers and certain institutions are offering opportunities for students and classes to use scanning or transmission electron microscopes having computerized control systems, either via the internet or by visiting a working facility.
The different kinds of electron microscopes have a high practical importance for enabling diagnosis of kidney diseases by examination of renal biopsies, reliable detection of causes for manufacturing defects and malfunctions in semi-conductors, advancement of understanding of normal and pathological cell substructure, detection and identification of disease microbes, development of nanomaterials and nanomachines, etc., etc. Technology developments for electron microscopes and for advanced specimen preparation are progressing vigorously in the modern world.
Let us now take a look at some images from electron microscopes!
Examples of images produced from all 3 kinds of electron microscopes are easily available on the internet. The following are recommended to you by Dr.M.
(1) A GOOD PLACE TO START: The semi-popular monthly journal, Microscopy Today, will give a good taste about what is going on currently (see: http://microscopy-today.com/jsp/common/home.jsf ). Most manufacturers of electron microscopes and related accessories have full-page advertisements in each issue. Articles about microscopy in education are a regular feature of this publication.
(2) A GALAXY OF IMAGES: For galleries with a multitude of images and diagrams, look up each of the 3 kinds of instruments (“transmission electron microscope”, “scanning electron microscope”, and “scanning-transmission electron microscope”) in the image section of your favorite internet browser. When you find something of personal interest among the many hundreds of panels shown, click on its thumbnail and you will be taken to the explanatory details directly provided by its source.
(3) ELECTRON MICROSCOPY OF NANOPARTICLES: Electron microscopy excels with specimens from nanoscience! Go to the website of the Nanoparticle Information Library at http://www.nanoparticlelibrary.net/results.asp and enter a search for “electron microscopy”; you will receive electron micrographs for 24 quite different nanoparticles, along with a brief report for each.
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