Tag Archives: electron diffraction

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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LET’S JOIN IN CELEBRATING THE INTERNATIONAL YEAR OF CRYSTALLOGRAPHY!!

 

Schematic of ordering in 1, 2, and 3 dimensions.  Top view looks downward onto the flat object; side view is at 90 degrees and looks onto the edge.  1-D ordering is a row of 5 associated subunits.  2-D ordering has 4 associated rows of 5 subunits. 3-D ordering has a layer of 12 subunits on top of the 20 subunits in the 2-D array.   (http:dr-monsrs.net)
Schematic of ordering in 1, 2, and 3 dimensions. Top view looks downward onto the flat object; side view is at 90 degrees and looks onto the edge. 1-D ordering is a row of 5 associated subunits. 2-D ordering has 4 associated rows of 5 subunits. 3-D ordering has a layer of 12 subunits above the 20 subunits in 2-D array.  (http://dr-monsrs.net)

2014 is the 100th year since the discovery that a beam of x-rays directed onto a single crystal comes out as multiple beams forming discrete spots.  That monumental finding has been called one the most important scientific research discoveries of all time.  Everyone in the entire public now is invited to celebrate this special 2014, designated by the United Nations as the International Year of Crystallography (IYCr2014)!  Below, I give the briefest possible non-mathematical introduction to the basic essentials of crystals and crystallography, along with some recommended internet websites where much further information is available.

What are crystals? 

We all encounter crystals every day, but most know little about them.  Crystals consist of matter whose smallest subunits (i.e., molecules or macromolecules) are very highly ordered in all 3 dimensions (3-D), as depicted in the opening figure above.  Some typical examples of the numerous crystals we commonly see are table salt (sodium chloride), diamonds in rings and jewelry (carbon), ice (water), and small granules of sugar (glucose).  If you have a magnifier, take a look now at what is in your salt shaker and you will observe many tiny crystals!

The 3-D ordering of the components making up crystals is so perfect that each and every subunit has exactly the same orientation and positioning as do all its neighbors.  This means that the atoms making up each subunit also have identical positioning to those in other subunits.  When a beam of x-rays is directed onto a single crystal in a suitable manner, each atom in the highly ordered complex scatters the incoming radiation identically into discrete spots.  The array of different spots produced from one crystal is the sum of the rays scattered by all its component atoms; because the innumerable atoms are so highly ordered, the many scattered rays join to form discrete spots.

What is diffraction? 

Diffraction is a fundamental property of atoms whereby incoming x-rays, electrons, or neutrons are scattered at characteristic angles and intensities.  The numerous scattered rays form an ordered array of diffraction spots and rings known as a diffraction pattern.  A single crystal produces one set of periodic diffraction spots, 2 crystals produce 2 sets of spots (at different rotations), and multiple crystals in polycrystalline materials will produce diffraction rings (i.e., many sets of the same spots at numerous different rotations).  The periodic order in diffraction patterns directly corresponds to the ordered position of the different atoms inside crystals; this means that diffraction patterns show atomic structure of the material making up each crystal.  The diffraction pattern is totally distinctive for each crystalline material, since the locations (atomic spacings) and brightness (kinds and numbers of atoms) of spots or rings are unique for each kind of crystalline matter.

What is crystallography? 

Crystallography is a research methodology for studying crystals.  Diffraction patterns are used in crystallography to tell us about the arrangement of the component atoms inside many different materials.  Since the beginning of x-ray crystallography one century ago, many thousands of materials have been crystallized and examined by crystallography; these include catalysts, enzymes, metals, minerals and biominerals, newly synthesized chemical compounds, proteins, salts, viruses, etc.   Because the atoms inside crystals are so highly ordered in an identical manner, diffraction patterns can be recorded, measured, and then processed by computation to determine structure down to the level of individual atoms.  This atomic structure determination of crystalline matter is the magic of crystallography!

Crystallography is a global activity for both science and industry, and almost all countries have scientists working as crystallographers.  X-ray diffraction is the most frequently used approach for crystallography, and now is quite automated through the use of computers to carry out the extensive numerical calculations needed to define an unknown structure to a high level of resolution.  Crystallography can be performed with laboratory x-ray sources or with very powerful and very fast x-rays produced by synchrotrons.   The hugely expensive synchrotron facilities are rather few in number, but have well-organized programs permitting their use by many visiting scientists.  Diffraction of electrons or neutrons also provides valuable special knowledge about structure at the atomic level.  When all is said and done, crystallography simply is a special way of looking at structure.

Not all materials are crystalline

Not all substances are naturally crystalline or can be induced to form crystals.  If the atoms in some substance are not ordered at all (i.e., they are randomly distributed), then this material is said to be in the amorphous state.  Examples of amorphous materials we see frequently include liquid water, many plastics, and air.  Inducing the formation of very highly ordered crystals is an essential requirement for structure determination by x-ray crystallography, since amorphous materials do not produce any diffraction spots or rings.

How does crystallography matter to you and me? 

Why do research scientists spend so much time and effort to use the magic of crystallography for determining the atomic structure of many kinds of physical, chemical, and biological materials?  The answer is that this knowledge about structure always provides information about functional capabilities and mechanisms for activities.  As one example, consider what can be derived from new knowledge about the high resolution structure of a virus; this will often increase  understanding about its biogenesis, mechanism for infecting host cells, immunoreactivity, and differences from other viruses.  Knowledge about functional capabilities  always is immensely valuable for both science and industry; for example: functioning of some inorganic catalyst or enzyme (e.g., mechanisms for activity and activation), interactions with other ions and molecules (e.g., changed functioning upon binding), formation of functional complexes (e.g., complex multi-protein assemblies), arrangement to form some more complex object (e.g., associations of 2-D polymers), changes producing specific toxic effects, prerequisites for binding to various ligands. sequential steps in genesis, characteristics of new materials (e.g., nano-materials made in university or industrial labs), etc.

Where can more information be found about crystals, crystallography, and IYCr2014? 

In the year-long world-wide celebration of crystallography and crystallographers during IYCr2014, many very fascinating programs for non-scientists now are being featured on the internet.  A large directory of instructive videos about crystals and crystallography for IYCr2014 is available at:  http://iycr2014.org/learn/watch .  Dr.M gives a rating of ‘outstanding’ to “Diving into the Heart of the Molecules of Life”, which shows how modern protein crystallography is done (http://www.youtube.com/watch?v=GfOyZch6llo ); regardless of which branch of science you prefer, Dr.M encourages everyone to see this video.

The International Union of Crystallography, which coordinates the international congresses on crystallography, has a special area on its website explaining the current celebration of IYCr2014 (see  http://www.iycr2014.org/about/video , and,  http://www.iycr2014.org/about  ).  Other large areas provide a listing of many internet resources and web tutorials dealing with crystals and crystallography; these include educational materials for students and teachers, and, recipes and instructions for growing your own crystals ( http://www.iycr2014.org/learn/educational-materials ).  The American Crystallographic Association has annual meetings that always include a special presentation aimed to instruct ordinary people about crystals and crystallography ( http://www.amercrystalassn.org/ ); this and many other national or regional crystallography societies also feature special IYCr2014 programs on their websites.

Visitors to Dr.M’s website are urged to take a look at any of these internet resources.  You don’t have to be a scientist to love crystals!  The IYCr2014 is for everyone, and that includes you!

 

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