Observing Microorganisms

The Microbial
Through a Microscope
World and
You

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Source:Tortora
Tortora1313 Edition
Edition
Microscopy: The Instruments

1. Light microscope

2. Electron microscope
Microscopy:
Types
Light Microscopy

Bright field
Dark field
Phase contrast
Differential interference contrast
Polarized light
Fluorescence
Confocal
Two-Photon
Scanning Acoustic Electron Microscopy
Transmission Electron
Microscopy Scanning Electron
Microscopy

Scanned-Probe Microscopy
Scanning Tunneling Microscopy
Electron Microscopy
Objects smaller than about 0.2 μm, such as viruses or the internal structures of
cells, must be examined with an electron microscope.

In electron microscopy, a beam of electrons is used instead of light. Like light,

free electrons travel in waves.

The resolving power of the electron microscope is far greater than that of the
other microscopes described here so far.

The better resolution of electron microscopes is due to the shorter

wavelengths of electrons; the wavelengths of electrons are about 100,000
times smaller than the wavelengths of visible light.

Thus, microscopes are used to examine structures too small to be resolved

with light microscopes.
Electron Microscopy
Images produced by electron microscopes are always black and white, but
they may be colored artificially to accentuate certain details.

Instead of using glass lenses, an electron microscope uses electromagnetic

lenses to focus a beam of electrons onto a specimen.

There are two types: the transmission electron microscope (TEM) and the
scanning electron microscope (SEM).
Transmission Electron Microscopy
A finely focused beam of electrons from an electron gun passes through a
specially prepared, ultrathin section of the specimen (Figure 3.11a).

The beam is focused on a small area of the specimen by an electromagnetic

condenser lens that performs roughly the same function as the condenser of a
light microscope—directing the beam of electrons in a straight line to
illuminate the specimen.

Instead of being placed on a glass slide, as in light microscopes, the specimen

is usually placed on a copper mesh grid.
Transmission Electron Microscopy
The beam of electrons passes through the specimen and then through an
electromagnetic objective lens, which magnifies the image.

Finally, the electrons are focused by an electromagnetic projector lens (rather

than by an ocular lens as in a light microscope) onto a viewing screen and
saved as a digital image.

The final image, called a transmission electron micrograph, appears as many

light and dark areas, depending on the number of electrons absorbed by
different areas of the specimen.

The transmission electron microscope can resolve objects as close together as

10 pm, and objects are generally magnified 10,000 to 10,000,000X.

Because most microscopic specimens are so thin, the contrast between their
ultrastructures and the background is weak.
Transmission Electron Microscopy

TEM: Electrons pass through the specimen and are scattered. Magnetic lenses focus the image
onto a fluorescent screen. This colorized transmission electron micrograph (TEM) shows a thin
slice of Paramecium. In this type of microscopy, the internal structures present in the slice can be
seen.
Transmission Electron Microscopy
Contrast can be greatly enhanced by using a “dye” that absorbs electrons and
produces a darker image in the stained region.

Salts of various heavy metals, such as lead, osmium, tungsten, and uranium,
are commonly used as stains.

These metals can be fixed onto the specimen (positive staining) or used to
increase the electron opacity of the surrounding field (negative staining).

Negative staining is useful for the study of the very smallest specimens, such
as virus particles, bacterial flagella, and protein molecules.
Transmission Electron Microscopy
In addition to positive and negative staining, a microbe can be viewed by a
technique called shadow casting.

In this procedure, a heavy metal such as platinum or gold is sprayed at an

angle of about 45° so that it strikes the microbe from only one side.

The metal piles up on one side of the specimen, and the uncoated area on the
opposite side of the specimen leaves a clear area behind it as a shadow.

This gives a three-dimensional effect to the specimen and provides a general

idea of the size and shape of the specimen
Transmission Electron Microscopy
Scanning Electron Microscopy

SEM: Primary electrons sweep across the specimen and knock electrons from its surface. These
secondary electrons are picked up by a collector, amplified, and transmitted onto a viewing
screen. (Right) In this colorized scanning electron micrograph (SEM), the surface structures of
Paramecium can be seen.
Scanned-Probe Microscopy

Since the early 1980s, several new types of microscopes, called scanned-probe
microscopes, have been developed.

They use various kinds of probes to examine the surface of a specimen using electric
current, which does not modify the specimen or expose it to damaging, high-energy
radiation.

Such microscopes can be used to map atomic and molecular shapes, to characterize
magnetic and chemical properties, and to determine temperature variations inside
cells.

Among the new scanned-probe microscopes are the scanning tunneling microscope
and the atomic force microscope.
Scanning tunneling microscope (STM)

A sharp tip is mounted on a scanning device known as an xyz scanner, which allows
three-dimensional positioning in the x, y, and z directions with subatomic precision.

The tunneling tip is typically a wire that has been sharpened by chemical etching or
mechanical grinding.

Tungsten (W), Platinum-iridium alloy (PtIr), or pure iridium (Ir) are often chosen as
the tip material.

A bias (fixed DC voltage or current) voltage Vt is applied to the sample, and when
the distance between tip and sample is in the range of several angstroms, a tunneling
current It flows between the tip and sample. This current is used as the feedback
signal in a z-feedback loop.
Scanning tunneling microscope (STM) or
atomic force microscope (AFM)
Atomic force microscope (AFM)

In atomic force microscopy (AFM), a metal-and-diamond probe is gently

forced down onto a specimen. As the probe moves along the surface of the
specimen, its movements are recorded, and a three-dimensional image is
produced (Figure 3.12b).

As with STM, AFM does not require special specimen preparation.

AFM is used to image both biological substances (in nearly atomic detail) and
molecular processes (such as the assembly of fibrin, a component of a blood
clot).
Figure 3.12 Scanned-probe microscopy. (a) Scanning tunneling microscopy (STM) image of a
double-stranded DNA molecule. (b) Atomic force microscopy (AFM) image of perfringolysin O
toxin from Clostridium perfringens. This protein makes holes in human plasma membranes.