Electron MicroscopyJohn J Bozzola, Southern Illinois University, Carbondale, Illinois, USA

Electron microscopy is a technology for examining the extremely fine detail

or ultrastructure of biological specimens for use in research and medical

situations.

Introduction

Electron microscopy is a technology for examining theextremely fine detail or ultrastructure of biological speci-mens. This methodology opened a totally new vista to thehuman eyewhenbiological ultrastructurewas revealed as adynamic and architecturally complex arrangement ofmacromolecules under the direction of genetic unitstermed genes. Complex subcomponents of the cell, termedorganelles, were clearly visualized and biochemical activ-ities associated with each structure. Pathogenic micro-organisms, the viruses, were finally captured on film andthe simple, yet beautiful, structures of these infectioussubunits provided the first step to organizing them intomajor taxonomic categories, or virus families. Thestructural organization of DNA into the chromosomewas elucidated using electron microscopy and the electronmicroscope provided insight into many disease processes.Electron microscopy is routinely used as a tool in suchdiverse areas as anatomy, anthropology, biochemistry, cellbiology, forensic medicine, microbiology, immunology,pathology, physiology, plant biology, toxicology andzoology.

The two basic types of electron microscopy arescanning electron microscopy, for viewing surfacesof bulk specimens, and transmission electronmicroscopy, for scrutinizing internal as well as externalfeatures of extremely thin specimens. The scanningelectron microscope is so named because a fineprobe of electrons is scanned across the surface of aspecimen to generate an image with a three-dimensionalappearance. In the transmission electron microscope, theelectrons are transmitted through the specimen to reveal atwo-dimensional image of the interior of cells. Althoughthese instruments operate in completely different ways,both use accelerated electrons and electromagnetic lensesto generate images. Both types of electron microscoperequire high vacuum so that electrons, having little mass,can travel down the column of the microscope withoutbeing scattered by air molecules. Images are recordedeither on photographic films or captured digitally usingcomputer interfaces.

In order to appreciate the types of informationobtainable with these instruments, it is importantto understand their basic design and operationalprinciples.

The Transmission Electron Microscope

The conventional transmission electron microscope(TEM, Figure 1) generates a beam of electrons in theelectron gun, usually by heating a tungsten filament andapplying a high voltage of negative potential in therange of2 75 000V. The heat and negative high voltagecause ejection of the valence electrons of tungsten near thetip of the V-shaped filament. A highly negative, cup-likedevice, termed a shield, forces the electrons into a cloudnear the filament tip. An aperture in the shield permitssome of the electrons to exit towards an anode plate. Theelectrons, which are travelling at about half the speed oflight, then enter the magnetic fields of the first and secondcondenser lenses, which focus the electrons onto thespecimen.The lenses in electron microscopes are windings of

copper wire, or solenoids, which generate a magnetic fieldwhen an electrical current is run through the wire. Whenelectrons enter the magnetic fields of the condenser lenses,they come to focus at certain distances, or focal lengths,from the lens. By adjusting the current running through thecondenser lens coil, the focal length is adjusted so that theelectrons illuminate the specimen in the area being studiedin the TEM.After electrons strike cellular organelles in the specimen,

they are deflected to various degrees, depending upon themass of the cellular component. Areas of greatmass deflectthe electrons to such an extent that they are effectivelyremoved from the optical axis of the microscope. Suchareas, where electrons have been subtracted, appear darkwhen ultimately projected onto the viewing screen. Areasthat have less mass, and so scatter the electrons to lesserdegrees, appear brighter on the viewing screen. As a resultof the electron scattering by the specimen, a highly detailedimage forms in the objective lens. This image is then furthermagnified up to onemillion times by the remaining three tofour lenses of the TEM. Even though each lens maymagnify only 100 times or less, the finalmagnification is theproduct of all of the lenses and can quickly reach highmagnifications.

Article Contents

Introductory article

. Introduction

. The Transmission Electron Microscope

. The Scanning Electron Microscope

. Outline of Methods

. Applications

. Future Developments

. Summary

1ENCYCLOPEDIA OF LIFE SCIENCES © 2002, John Wiley & Sons, Ltd. www.els.net

The electromagnetic imaging lenses are constructedin the same way as the illuminating or condenserlenses. Imaging lenses are used to focus and magnifythe image formed as a result of the interaction of theelectrons with the specimen. Ultimately, the electronsare projected onto a viewing screen coated with aphosphorescent material that glows when struck byelectrons. Greater numbers of electrons (from areas ofthe specimen having less density) generate brighter areason the screen. Since photographic emulsions are sensitiveto electrons as well as to photons, the image is recorded bylifting up the viewing screen and allowing the electrons tostrike a photographic film placed underneath. Afterdevelopment, such images are termed electron micro-graphs.

Besides high magnification, which refers to an increasein the size of an object, TEMs produce images havinghigh resolution. High-resolution images have tremendousdetail and can withstand the high magnification thatmakes the details visible. A modern TEM is shown inFigure 2.

The Scanning Electron Microscope

The electron gun and the lenses in the scanning electronmicroscope (SEM, Figure 1) are nearly identical to theTEM. However, instead of forming an image, the SEMlenses focus the beam of electrons to a very tiny spot on thespecimen. Each of the three condenser lenses in the SEMmakes the spot of electrons progressively smaller. Whenthe beam of electrons strikes the specimen, it causes theejection of low-energy, secondary electrons from thesurface of the specimen. Secondary electrons conveyinformation about the surface of the specimen and areused to generate the image.In contrast to the TEM, which illuminates the specimen

area under examination with a single, large spot, the SEMscans a tiny spot of electrons across the specimen. This ismuch like the beam of electrons that is rastered across theinside of a television tube. At each point where the electronbeam of the SEM strikes the specimen, various numbers ofsecondary electronswill be generated, depending primarilyupon topography, angle of entry of the beam into the

TEM

Shield

Filament

Anode

Condenserlenses (2)

Specimen (red)deep insideobjective lens

Projectionlenses (3)

Image planeFluorescent

screen

Specimen

Photo-multiplier

Signal-processingelectronics

Secondaryelectroncollector

Lightguide

Secondary electrons

SEM

Condenserlens 1

Condenserlens 2

Scangenerator

Condenserlens 3

Display monitor

Figure 1 Diagram showing the basic components making up the transmission (TEM) scanning electron microscope (SEM).

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specimen and thickness of raised portions of the specimen.The secondary (image-forming) electrons are stronglyattracted to a secondary electrondetector that is placed at apositive high voltage of around 12 000V. When thesecondary electrons strike a phosphorescent coating onthe detector, this generates a burst of light that travelsdown a light guide and into a photomultiplier, wherephotons are converted to photoelectrons and the weak

signal amplified. Areas of the specimen that generate largenumbers of secondary electrons appear very bright whenviewed on the display screen of the SEM, whereas areaswith fewelectrons are dark.The various shades of grey seenin an image give the impression of depth, much like a blackand white photograph conveys three-dimensionality.Two scanning events take place simultaneously, but in

different locations in the SEM. The electron beam is

Figure 2 This modern TEM has capabilities for scanning transmission electron microscopy (STEM) and X-ray microanalysis.

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scanned across the specimen at the same time that a secondelectron beam is being scanned across a display monitor orviewing screen. For each point on the specimen, there is acorresponding point on the viewing screen, the brightnessof which depends upon the number of secondary electronsgenerated by the specimen. The size of the area scanned onthe viewing screen is fixed, based on the size of the screen(15 cm�20 cm, for example). However, the size of the areascanned on the specimen is completely variable and underthe control of the operator. If one scans a line across thespecimen that is 1 cm in length and thendisplays this line ona 20 cm monitor, the resulting line is magnified 20� . A20 mm line scanned on a specimen when displayed to 20 cmwould represent a magnification of 10 000� .

Magnification in the SEM is expressed as:As the length of the scan decreases on the specimen, it is

necessary to make the electron spot on the specimenproportionally smaller.Otherwise, the imagewill appear tobe out of focus. In the SEM, therefore, focusing consists ofusing the condenser lenses to adjust the spot size on thespecimen to the appropriate size for the magnificationbeing used. For high-magnification work involving verysmall scan lengths, it is necessary to decrease the size of theelectron spot to extremely small spots of 1–2 nm. (For

comparison, the poliovirus is about 30 nm in diameter.) Amodern SEM is shown in Figure 3.

Outline of Methods

Specimen preparation for scanning electronmicroscopy

Biological specimens for SEM examination can be quitelarge compared with the size of specimens used for TEM.In some instances, entire organisms (insects, small plants,and animals up to several inches) may be suitable if theyhave been prepared properly. Hardy organisms withexoskeletons or rigid cell walls may need only air-drying.Themajority of biological specimens, however,will requirechemical fixation in order to stop biological processes andto stabilize the cellular architecture.Although the details will vary considerably, depending

upon the organism, a commonly followed method forspecimen preparation starts with a careful cleaning ofspecimen surfaces using a fine brush, gentle puffs of air ormild streams of aqueous buffer. Following this, the fine

Figure 3 A scanning electronmicroscope (SEM) with capabilities for examining a hydrated specimen in a partial vacuum. This instrument also has X-rayanalytical capabilities. A portion of the X-ray detector is shown in the top left (arrow) corner of this figure.

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structure of the specimen is preserved using glutaraldehydeand osmium tetroxide. After dehydration in ethanol, andcritical-point drying with liquid carbon dioxide, thespecimen is mounted on a stub and coated with conductivemetal.

It is necessary to apply a 40–50 nm thin coating of aninert metal, such as gold, in order to dissipate the high-voltage charge from the accelerated electrons that strikethe specimen. The metal coating, normally applied by ahigh-voltage, sputter-coating process, serves as an excel-lent source of secondary electrons. Figure 4 is a scanningelectron micrograph of several Salmonella bacterial cells

attached to a rayon fibre. The sample was prepared asdescribed in the previous paragraphs.

Specimen preparation for transmissionelectron microscopy

Methods for preparing specimens for TEM examinationare normally more complicated and the specimens moresensitive to processing details than are SEMsamples. Sincewe wish to examine the interior of cells, and since theelectron beamof theTEM is unable to penetrate specimens

Figure 4 Salmonella bacteria adhering to the surface of a rayon fibre. Note the three-dimensional aspects of the image.

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much thicker than 60–80 nm, it is necessary to cutextremely thin slices of most biological tissues. As is donefor light microscopy, the specimen must be chemicallypreserved and embedded in a supportingmatrix in order toprevent collapse of the tissues as the knife sections thetissue. Biological ultrastructure can rapidly deterioratewhen the organism is being prepared, so it is essential to usesmall pieces of tissue (less than 1mm thick) that can bepenetrated quickly by the chemical fixatives.

A typical method for preparing biological tissues wouldemploy glutaraldehyde and osmium fixatives. After thespecimens have been dehydrated in ethanol, they areinfiltratedwith a liquid, epoxy plastic that is then hardenedin an oven. At this point, the water throughout thepreserved cells has been replaced completely with hardplastic and it is now possible to cut extremely thin slices.This is accomplished using an ultramicrotome thatadvances the specimen over a glass or diamond knife andcuts ultrathin sections from the plastic-embedded speci-men. The sections are retrieved onto a specimen carrier, orgrid, stained for contrast using heavymetals such as uranylacetate and lead citrate, and placed into the TEM forviewing.

Figure 5 shows several bacterial cells that were preparedand sectioned as described. A typical plant cell is shown inFigure 6. Two mammalian cells, infected with a tumourvirus that has been released into the space between the cells,are shown in Figure 7. Figure 8 is a close-up of five of thetumour viruses.

Some specimens, such as viruses and isolated subcellularorganelles (ribosomes andmitochondria, for example), arethin and do not require sectioning. In this case, thespecimens are mixed with an aqueous solution of a heavymetal stain (uranyl acetate, potassium phosphotungstate)for contrast and placed directly onto the specimen grid forviewing in the TEM.This procedure is extremely rapid andpermits one to examine a specimenwithinminutes.A singleherpesvirus particle that was negatively stained withpotassium phosphotungstate is shown in Figure 9. Anexplanatory diagram for this virus is presented in Figure 10.

As the accelerating voltage of the TEM is increasedbeyond the 75 000–125 000V available in conventionalinstruments, it is possible to examine much thickerspecimens. Intermediate and high-voltage electron micro-scopes are instruments that operate in the range of200 000–500 000 and 500 000 to one million volts. High-voltage instruments have four advantages over loweraccelerating voltage microscopes: increased resolution,greater penetrating ability in thicker sections, diminishedspecimen damage and considerably greater depth of

Figure 5 A chain of bacterial cells as viewed in the TEM. These bacteria,Streptococcus mutans, are responsible for causing tooth decay. Comparethe two-dimensional appearance of this image to that shown in theprevious figure. These cells have been prepared andultrathin sections of 70nm have been cut through the cells. These bacteria are 0.5 mm in width.

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information. Sections can be examined that are 10–30times thicker than those needed for the conventional TEM.

With the right equipment, it is also possible to examinerapidly frozen specimens that have not been subjected tomost of the chemicals used in preparing specimens in theconventional manner. In one method, cryoultramicrot-omy, rapidly frozen specimens are sectioned at2 80to2 1008C and the sections examined in the TEM. Underideal conditions, sections can be examined in about onehour.

Another instrument, the scanning transmission electronmicroscope, or STEM, is similar to a TEM except that ithas the capability to generate extremely small electron

beams that can be rastered over a thin specimen. Thistechnology is particular useful when one wishes to obtainX-ray analytical data from extremely small spots in thespecimen.

Applications

Both types of electron microscopes have found wide use inthe basic biological sciences as well as in the medical field.Strictly descriptive studies provide crucial data for newlydescribed organisms and comparative investigations are

Figure 6 A typical plant cell as seen in the TEM. Ultrathin sections reveal the interior of the cell in great detail. Note the nucleus (N), nucleolus (Nu),vacuoles (V) and cell wall (CW) surrounding the cell. Courtesy of Microscopy Society of America.

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valuable to pinpoint differences between organisms(evolutionary studies, drug treatment regimes, pathologi-cal changes, cancerous transformation, physiologicalprocesses such as ageing and cell death).

TheTEMhasbecomeamainstay in themedical field as itcan provide definitive evidence for a diagnosis. For

example, locating certain subcellular components canreadily identify certain types of tumours that are difficultto identify using conventional light microscopy. Forcertain types of viral infections, often the quickestdiagnosis may be by electron microscopy. When newlyemergent or previously undiscovered infectious agents are

Figure 7 Sectioned mammalian cells as viewed in the TEM. These cells are infected with a tumour virus, with some virus particles visible in the spacebetween the two cells (arrow). N, nucleus; Nu, nucleolus.

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involved (hepatitis B, HIV, Ebola), electron microscopy isoften the only possible way to identify the pathogen.Besides the conventional approaches to viewing speci-

mens in both types of electron microscopes, there existancillary procedures that may provide additional informa-tion.X-raymicroanalysis, for example, is a technology thatenables one to identify the types of elements present in aspecimen. Useful in SEMs, TEMs and STEMs, theprocedure is possible since the electron beam elicitsthe production of X-rays upon interacting with theatoms in the specimen. Since each element has acharacteristic spectrum of X-rays, one can identify theelements present and sometimes the percentage composi-tion. Thismay be accomplished in a very localizedmanner,for example over a cellular inclusion, or onemay generate adistribution map of the elements present in the specimenbeing viewed.A related procedure, electron energy loss spectroscopy,

EELS, is useful in the TEM for localizing the lighteratomic numbered elements most likely to be present inbiological tissues. EELS has the added advantage of beinga more sensitive procedure for detecting trace levels ofelements present in cells. EELS may also be used toincrease contrast in specimens and improve resolution inthicker sections. Both EELS and X-ray analysis technol-ogies, however, require expensive accessories to theelectron microscope that are nearly half the cost of themicroscope itself.Cytochemistry, immunocytochemistry, in situ hybridi-

zation and autoradiography are specialized TEM techni-ques that allow one to localize molecules such as enzymes,viral proteins, specific sequences of DNAorRNA, cellularbuilding blocks, hormones or practically any molecule ofinterest within the cell. Such studies permit one to trace theroute of targeted molecules in the cell and may revealpathways for the production of macromolecules in thedynamic interior of the cell. Once the molecules have beenlocated in normal cells, this serves as a database forcomparative studies in pathological conditions (geneticdisorders, disease development, tumorigenesis), drugtreatment studies (antimicrobials, anticancer agents, otherpharmaceuticals) and unlimited, basic science, researchinvestigations.

Figure9 Aherpesvirusparticle fromapatient suffering fromshingles. Thisvirus is responsible for causing both chickenpox and shingles. The centralpart of the virus particle, the capsid, is approximately 100 nm in size.

Figure 8 High-magnification viewof five tumour viruses fromcells shownin Figure 7.Note the double-layered nature of themembrane surroundingthe top virus particle. This membrane is derived from the host cell and isapproximately 8 nm in thickness.

Capsid

Envelope

Nucleic acidin core

Nucleocapsid

Figure 10 Diagram showing the various parts of the virus particle inFigure 9.

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Future Developments

It has always been the goal of cell biologists to be able toexamine cells in the hydrated, native state. One of the bestways of doing this involves cryomicroscopy, in which theliving specimen is rapidly frozen and examined while beingmaintained in the frozen state. Such capabilities exist forboth SEM and TEM studies. When one combinescryomicroscopy with high-voltage TEMs and moderncomputers, cryoelectron tomography is possible. Thistechnology can be used to view ultrastructural details inthree dimensions. For example, chromosomes, mitochon-dria, microtubule arrays, sites of viral replication, etc.,which are unfixed, unstained and embedded in vitreous ice,can now be examined in three dimensions.

The environmental SEM, or ESEM, is a speciallydesigned SEM that uses a newly designed, gaseousdischarge detector for capturing imaging electrons. Withthe ESEM it is possible to examine hydrated, unfixed,nonfrozen, uncoated biological specimens with only slightvacuums present in the specimen chamber. This shouldpermit the study of dynamic processes in hydrated, livingcells.

Thanks to computers and digital imaging, electronmicroscopes can be directly interfaced with the Internet sothat students and researchers are able to view images veryshortly after they have been viewed by the microscopist.Ultimately, with faster connections, it will be possible toview the images in real time and even to operate themicroscope remotely, thousands of miles away. Thiscapability would be particularly important for medicaldiagnostic procedures in locations where electron micro-scopes are unavailable.

Summary

Electronmicroscopes enable investigators to obtain highlydetailed images of cells for use in research and medical

situations. The transmission electron microscope (TEM)has imaging and analytical capabilities over one thousandtimes better than light microscopes. TEMs are useful tostudy the interior of thinly sliced cells and subcellularentities such as viruses and may obtain images even downto the molecular level. Scanning electron microscopes(SEM), by contrast, are used to study the three-dimen-sional features of intact specimens (whole insects, cells,portions of organs, etc.). Modern instruments are capableof imaging frozen, hydrated specimens where one mayapply various analytical procedures (elemental analysis,distribution of molecular species, for example). High-resolution, three-dimensional images of fully hydrated,frozen specimens can be obtained in high-voltage TEMs aswell as in the environmental SEM.Modern computers andthe Internet have increased the availability of electronmicroscopes to the wider teaching, research and medicalcommunities. Further information on electronmicroscopymay be obtained by contacting the Microscopy Society ofAmerica website at [http://www.msa.microscopy.com].

Further Reading

Bailey GW, JeromeWG,McKernan S,Mansfield JF and Price RL (eds)

(1999)Microscopy andMicroanalysis, Proceedings of the Microscopy

Society of America and Microbeam Analysis Society. New York:

Springer-Verlag.

Bozzola JJ and Russell LD (1998) Electron Microscopy: Principles and

Techniques for Biologists, 2nd edn. Boston: Jones & Bartlett Publish-

ers.

DykstraMJ (1992) Biological ElectronMicroscopy: Theory, Techniques,

and Troubleshooting. New York: Plenum Press.

Flegler SL, Heckman JW and Klomparens KL (1993) Scanning and

Transmission Electron Microscopy: An Introduction. New York: WH

Freeman and Co.

Glauert AM and Lewis PR (1998) Biological Specimen Preparation For

Transmission Electron Microscopy, 2nd edn, vol. 17 of Practical

Methods in Electron Microscopy, Glauert, AM (ed.). Princeton, NJ:

Princeton University Press.

Hayat MA (1989) Principles and Techniques of Electron Microscopy:

Biological Applications. Boca Raton, FL: CRC Press, Inc.

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