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MICROSCOPY FOR RESEARCH, EDUCATION, AND FUN! PART 3: ELECTRON MICROSCOPY.

 

Schematic Diagram showing Major Parts in a Transmission Electron Microscope.   (http://dr-monsrs.net)
Schematic Diagram showing the Major Parts in a Transmission Electron Microscope.    (http://dr-monsrs.net) 

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.

Concluding remarks. 

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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MICROSCOPY FOR RESEARCH, EDUCATION, AND FUN! PART 1: FUNDAMENTALS FOR BEGINNERS.

 

Different kinds of microscopy present a wealth of information.    ( http://dr-monsrs.net )

Microscopy gives a wealth of information!  (http://dr-monsrs.net)

 

Very few research instruments have as widespread a usage in science as do microscopes.  They also are a very useful tool for industries (e.g., failure analysis and monitoring fidelity at a fabrication and production facility), hospitals (e.g., pathology diagnosis, identification of microbial infections, determining hematology status, etc.), minerology, metallurgy, crystallography, etc.  In recent years, microscopes have become more available and more utilized for science education in primary and secondary schools.  For those of us using microscopes for our work, they additionally provide quite a lot of fun!

In this short series of articles, I will present a very brief and readily understandable description of microscopy and the different types of microscopes.  These are not in-depth discussions, but are designed to give an introductory background about microscopy for teachers, technicians, parents, students, and beginning users.  I have tried to make everything concise and good for non-experts.  Although simplified explanations will be given, some recommended resources for deeper coverage also are provided.

The initial article gives an overview of the most fundamental concepts for microscopes and microscopy.  These topics precede actual usage of any microscopes.  The following articles will briefly explain the main kinds of microscopes used in 2015.  A final article outlines utilization of microscopes for education in primary and secondary schools.  

How do microscopes actually work? 

Microscopes permit observation of structure, function, and composition that cannot be seen with the naked eye.  All the common kinds of microscopes are governed by the branch of physical science known as optics; this describes exactly how microscopes use lenses to form images.  A common example of a single lens is the magnifying glass; one need not know anything at all about optics to have fun using one!  Compound lenses have multiple single lenses working together to give higher magnification of specimens.  As magnification is increased, good compound lenses will reveal smaller and smaller details.  Magnifications for typical ordinary uses range from 3 times (3X) to several hundred times (300X) larger than the natural size; for special microscopes, magnifications can go all the way up to a million times their natural size (1,000,000X). 

The size of small details that can be visualized with sufficient magnification is limited by the level of resolution.  Resolution can range from detection of specimen details that cannot quite be seen with the naked eye (i.e., low resolution), up to visualizing individual atoms (i.e., very high resolution).  The resolution level for microscopes is determined by optics, and varies with the kind of lenses and microscope being used.  

The functioning of microscopes is generally analogous to the production of images by our eyes.  That involves light waves bouncing off some object, passing through our pupils and ocular lenses, and then being detected by our retinas.  Most imaging in microscopy uses shining waves onto or through a specimen, then passing them through lenses, and finally registering them on a detector; detectors for microscopy record the waves hitting them via cameras that use either photographic film or digital memory.  For microscopy, lenses first focus waves onto the specimen, and then onto the detector.  Imaging requires contrast (i.e., relative amount of lighter vs. darker components); this is produced in most microscopes when the specimen causes some portion of the waves to not be transmitted to the detector, due to being absorbed or scattered.  

The several compound lens sysytems in microscopes provide enough magnification and sufficient resolution to resolve some small details in specimens.  Recorded images give a permanent record of what was observed, and also can be used to make measurements and counts of the small details.  Basically, resolution determines the information content of images made with any microscope.  In some cases, the smallest details known to be present in a specimen are not able to be imaged because the lenses lack enough resolution even at high magnifications; this is empty magnification.

Information about chemical composition of a specimen also is available from some types of microscopes.  Analytical microscopy detects the amount of some element or compound, and/or their location, within the specimen being examined.  Resolution here corresponds to the ability to accurately measure amounts for several elements or compounds that differ only slightly.  Compositional information is usually displayed as a spectral histogram, with the vertical axis denoting quantity and the horizontal axis showing a scale differentiating the elements or compounds.  The compositional data also can be displayed superimposed upon a regular image of the specimen; this mapping shows exactly where some element or chemical component is located. 

The different kinds of microscopes. 

The most general way of characterizing microscopes is by the type of waves used to view the specimen.  Our own eyes produce images using light waves coming from (e.g., stars, neon signs, etc.) or reflected off different specimens (e.g., birds, leaves, other people, etc.).  Different portions of the electromagnetic spectrum are used by the 2 main kinds of microscopes: (1) light waves, ranging from ultraviolet, through all the visible colors, and on into infrared, are used in light microscopes, and, (2) electron waves, which are very much smaller than light waves, are used in electron microscopes

The wavelengths utilized, and the quality of the lenses present, determine the level of resolution given by each microscope.  Smaller wavelength and higher quality lenses give higher resolution (i.e., the ability to see and image finer details in a specimen).  Bacteria are too small to be observed with the naked eye or with a magnifying glass, but can be seen with a good light microscope; electron microscopes use wavelengths very much smaller than those found in visible light, and so are able to not only easily image bacteria and viruses, but also can show very small details within those objects (i.e., substructure). 

There are several other important special types of microscopes, but they will not be included here since this article presents only an introductory coverage.

How is microscopy important for ordinary people?

Microscopes are used for very many different purposes, including usage for research.  Images from microscopy show enough details to permit detection, identification, and authentification of many different objects and conditions.  The discipline of pathology in clinical medicine uses microscopy extensively for the diagnosis of disease states and the identification of microbes causing infections.  Microscopy provides an ideal tool for making size measurements of small objects and smaller details within them; thus, it is fundamental for analysis of all levels of structure.  Microscopy often is used to evaluate quality (e.g., perfection of small crystals to be used for x-ray diffraction; status of solid-state semi-conducting components).  Developing new high technology directly depends upon microscopy.  Dynamic imaging of specimens that are changing with time reveals the course of changes and positions of constituent parts; this capability is a major feature of microscopy at both low and high magnifications. All these capabilities make microscopy very widely used, meaning that microscopes are very important for everyone!  

The “simplest microscope” of all is fun and can be useful for science education! 

The very simplest microscope often is not recognized as such!  A magnifying glass (e.g., a single plastic or glass lens within a holder, provides a magnification of 2-5X) uses white light waves in the visible spectrum to show us some smaller details that cannot be discerned with the naked eye.  A magnifying glass is a single lens; light and electron microscopes use compound lenses made from several single lenses working together.  Just as you focus images with a magnifying glass by moving either the lens or the specimen along a line towards your eyes, so do light microscopes focus by moving either compound lenses up and down from a specimen, or by moving the specimen relative to stationary lenses. 

Teachers should recognize that magnifying glasses are inexpensive, difficult to break, and easy to use by all students.  The concepts of a lens, magnification, resolution, and focusing become rapidly understood from hands-on usage, and some unexpected small details often are discovered by young students.  Easy specimens for examination with a magnifying glass are table salt or granular sugar, a leaf from a plant, a piece of Kleenex tissue, a cut piece of any fruit, and, skin hairs and scratches on the student’s own forearm.  

Kerry Ruef has developed very successful teaching programs for primary school students which use magnifiers extensively ( http://www.the-private-eye.com ).  I highly recommend to all teachers presenting science in primary schools Kerry Ruef’s very recent article, “The Private Eye ®– (5X) Looking/Thinking by Analogy”, just published in Microscopy Today (May 2015, volume 23, pages 52-57) .  This now is available on the internet as a PDF (see: http://content.yudu.com/web/14lmv/0A3cxwn/MicroscopyTodayV23N3/flash/resources/52.htm?refUrl=http%3A%2F%2Fwww.the-private-eye.com%2Findex.html ).  The topic of “Microscopy in Education” is a subject frequently published in this journal coming from the Microscopy Society of America (see:  https://www.microscopy.org ).  

Concluding remarks. 

Even though we have not yet looked at any actual microscope or images, you now should have a good very basic understanding about microscopy, what are the different types of microscopes, and how is microscopy so very important in the modern world.  In the next article of this series, we will take a closer look at light microscopy. 

 

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