Microbiology and Immunology

Electron microscopic examination of microorganisms WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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The microscope built by Ruska and Knoll is similar in

principle to a compound microscope. A beam of electrons is

directed at a specimen sliced thin enough to allow the beam to

pass through. As they travel through, the electrons are

deflected according to the atomic structure of the specimen.

The beam is then focused by the magnetic coil onto a photo-

graphic plate; when developed, the image on the plate shows

the specimen at very high magnification.

Scientists worldwide immediately embraced Ruska’s

invention as a major breakthrough in microscopy, and they

directed their own efforts toward improving upon its precision

and flexibility. A Canadian-American physicist, James Hillier,

constructed a microscope from Ruska’s design that was nearly

20 times more powerful. In 1939, modifications made by

Vladimir Kosma Zworykin enabled the electron microscope to

be used for studying viruses and protein molecules.

Eventually, electron microscopy was greatly improved, with

microscopes able to magnify an image 2,000,000 times. One

particularly interesting outcome of such research was the

invention of holography and the hologram by Hungarian-born

engineer Dennis Gabor in 1947. Gabor’s work with this three-

dimensional photography found numerous applications upon

development of the laser in 1960.

There are now two distinct types of electron micro-

scopes: the transmission variety (such as Ruska’s), and the

scanning variety. The Transmission Electron Microscope

(TEM), developed in the 1930’s, operates on the same physi-

cal principles as the light microscope but provides enhanced

resolution due to the shorter wavelengths of electron beams.

TEM offers resolutions to approximately 0.2 nanometers as

opposed to 200 nanometers for the best light microscopes. The

TEM has been used in all areas of biological and biomedical

investigations because of its ability to view the finest cell

structures. Scanning electron microscopes (SEM), instead of

being focused by the scanner to peer through the specimen, are

used to observe electrons that are scattered from the surface of

the specimen as the beam contacts it. The beam is moved

along the surface, scanning for any irregularities. The scan-

ning electron microscope yields an extremely detailed three-

dimensional image of a specimen but can only be used at low

resolution; used in tandem, the scanning and transmission

electron microscopes are powerful research tools.

Today, electron microscopes can be found in most hos-

pital and medical research laboratories.

The advances made by Ruska, Knoll, and Hillier have

contributed directly to the development of the field ion micro-

scope (invented by Erwin Wilhelm Muller) and the scanning

tunneling microscope (invented by Heinrich Rohrer and Gerd

Binnig), now considered the most powerful optical tools in the

world. For his work, Ruska shared the 1986 Nobel Prize for

physics with Binnig and Rohrer.

See alsoBiotechnology; Laboratory techniques in immunol-

ogy; Laboratory techniques in microbiology; Microscope and

microscopy; Molecular biology and molecular genetics

ELECTRON MICROSCOPIC EXAMINATION

OF MICROORGANISMSElectron microscopic examination of microorganisms

Depending upon the microscopeused and the preparation

technique, an entire intact organism, or thin slices through the

interior of the sample can be examined by electron

microscopy. The electron beam can pass through very thin

sections of a sample (transmission electron microscopy) or

bounced off of the surface of an intact sample (scanning elec-

tron microscopy). Samples must be prepared prior to insertion

into the microscope because the microscope operates in a vac-

uum. Biological material is comprised mainly of water and so

would not be preserved, making meaningful interpretation of

the resulting images impossible. For transmission electron

microscopy, where very thin samples are required, the sample

must also be embedded in a resin that can be sliced.

For scanning electron microscopy, a sample is coated

with a metal (typically, gold) from which the incoming elec-

trons will bounce. The deflected electrons are detected and

converted to a visual image. This simple-sounding procedure

requires much experience to execute properly.

Samples for transmission electron microscopy are

processed differently. The sample can be treated, or fixed, with

one or more chemicals to maintain the structure of the speci-

men. Chemicals such as glutaraldehyde or formaldehyde act to

cross-link the various constituents. Osmium tetroxide and

uranyl acetate can be added to increase the contrast under the

electron beam. Depending on the embedding resin to be used,

the water might then need to be removed from the chemically

fixed specimen. In this case, the water is gradually replaced

with ethanol or acetone and then the dehydrating fluid is grad-

ually replaced with the resin, which has a consistency much

like that of honey. The resin is then hardened, producing a

block containing the sample. Other resins, such as Lowicryl,

mix easily with water. In this case, the hydrated sample is

exposed to gradually increasing concentrations of the resins,

to replace the water with resin. The resin is then hardened.

Sections a few millionths of a meter in thickness are

often examined by electron microscopy. The sections are

sliced off from a prepared specimen in a device called a micro-

tome, where the sample is passed by the sharp edge of a glass

or diamond knife and the slice is floated off onto the surface

of a volume of water positioned behind the knife-edge. The

slice is gathered onto a special supporting grid. Often the sec-

tion is exposed to solutions of uranyl acetate and lead citrate

to further increase contrast. Then, the grid can be inserted into

the microscope for examination.

Samples can also be rapidly frozen instead of being

chemically fixed. This cryopreservation is so rapid that the

internal water does not form structurally disruptive crystals.

Frozen thin sections are then obtained using a special knife in

a procedure called cryosectioning. These are inserted into the

microscope using a special holder that maintains the very cold

temperature.

Thin sections (both chemically fixed and frozen) and

whole samples can also be exposed to antibodies in order to

reveal the location of the target antigenwithin the thin section.

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