Types of Microscopy

The earliest microscopes used visible light to create images and were little more than magnifying glasses. Today, more sophis-
ticated compound light microscopes (Figure 3-1) are routinely used in microbiology laboratories. The various types of light
microscopy include bright-field, dark-field, fluorescence, and phase contrast microscopy (Figure 3-2). Each method has
specific applications and advantages, but the most commonly used in introductory classes and clinical laboratories is bright-
field microscopy. In many research applications, electron microscopy is used because of its ability to produce higher quality
images of greater magnification.

Light Microscopes
Bright-field microscopy produces an image made from light
that is transmitted through a specimen (Figure 3-2A). The
specimen restricts light transmission and appears "shad-
owy" against a bright background (where light enters the
microscope unimpeded). Since most biological specimens
are transparent, contrast between the specimen and back-
ground can be improved with the application of stains to
the specimen (see Sections 4 and 5). The price of improved
contrast is that the staining process usually kills cells. This
is especially true of bacterial staining protocols.
Image formation begins with light coming from an
internal or an external light source (Figure 3-3). It passes
-•
through the condenser lens, which concentrates the light
and makes illumination of the specimen more uniform.
Refraction (bending) of light as it passes through the
objective lens from the specimen produces a magnified real
• I ;,image. This image is magnified again as it passes through
the ocular lens to produce a virtual image that appears
below or within the microscope. The amount of magnifica-
tion produced by each lens is marked on the lens (Figure
3-4A and B). Total magnification of the specimen can be Figure 3·1. A Binocular Compound Microscope. A quality micro-
calculated by the following formula: scope is an essential tool for microbiologists. Most are assembled
with exchangeable component parts and can be customized to suit
Total Magnification = Magnification of the Objective Lens the particular needs of the user.
X Magnification of the Ocular Lens Photograph courtesy of Olympus America Inc.

23
24 A PHOTOGRAPHIC ATLAS FOR THE M,CROB,OLOGY LABORATORY

Figure 3-2. Types of Light Microscopy.

fA) Bright-field micrograph of a stained
specimen of the fungus Penicillium.
otice that in this whole mount very little
of the specimen is in focus due to its
thickness. To observe it properly, the user
needs to continually adjust the fine focus
as the field is scanned. (8) Dark-field
micrograph of several Paramecium.
Notice the contrast between internal
components even without stain. (C) Phase
contrast micrograph of Amoeba. Again,
notice the contrast produced without
stain. (0) Fluorescence micrograph of
Mycobacterium kansasii.
Photograph C courtesy of Gary D. Wisehart,
San Diego City College.

The practical limit to magni-

fication with a light microscope is
around 1300X. Although higher
magnifications are ppssible, it
becomes increasingly difficult
Condenser to maintain image clarity as the

/U~>< ".,.
Objective Lens

. .""",
~

.
Ocular Lens

~
Observer's
magnification increases. Clarity
of an image is called resolution.
The limit of resolution Iorre-
solving power) is an actual meas-
Specimen urement of how far apart two
Eye
t
Real Image
points must be in order for the
microscope to view them as
Virtual Image (formed by objective lens) being separate. Notice that reso-
(formed by ocular lens)
lution improves as resolving
~ power is made smaller.
The best limit of resolution
achieved by a light microscope
Figure 3-3. Image Production in a Compound light Microscope. light from the source is focused is about 0.2 rm. (That is, at its
on the specimen by the condenser lens. It then enters the objective lens, where it is magnified to absolute best, a light microscope
produce a real image. The real image is magnified again by the ocular lens to produce a virtual cannot distinguish between two
image that is seen by the eye. (After Chan, et a/., 1986) points closer together than 0.2
um.) For a specific microscope,
-
SECTION THREE: MICROSCOPY 25

Figure~3-4. Markings of Magnification and Numerical Aperture on Microscope Components. (A) Three plan apochromatic 'objective lenses
on the nosepiece of a light microscope. Plan means the lens produces a flat field of view. Apochromatic lenses are made in such a way that
chromatic aberration is reduced to a minimum. From left to right, the lenses magnify lOX, 20X, and 40X, and have numerical apertures of
0.40, 0.70, and 0.85. The 20X lens has other markings on it. The mechanical tube length is the distance from the nosepiece to the ocular
and is usually ~etween 160 to 210 mm. However, this 20X lens has been corrected so the light rays are made parallel, effectively creating
an infinitely long mechanical tube length (00). This allows insertion of accessories into the light path without decreasing image quality. The
thickness of cover glass to be used is also given (0.17 ± 0.01 mm). (8) A lOX ocular lens. (C) A condenser (removed from the microscope)
with a numerical aperture of 1.25. The lever in the upper right is used to open and close the iris diaphragm and adjust the amount of light
entering the specimen.

the actual limit of resolution can be calculated with the
following formula:
D=
N.A,condenser + N.A·Objective
where D is the minimum distance at which two points can
be resolved, A is the wavelength of light used, and N.A. is the
numerical aperture of the condenser lens and objective lens.
Numerical aperture is a measure of a lens's ability to
"capture" light coming from the specimen and use it to
make the image. As with magnification, it is marked on
the lens (Figures 3-4A and C). Using immersion oil between
the specimen and the objective lens increases its numerical
aperture and in turn, makes its limit of resolution smaller.
(If necessary, oil may also be placed between the condenser
lens and the slide.) The result is better resolution.
The light microscope may be modified to improve its
ability to produce images with contrast without staining,
which often distorts or kills the specimen. In dark field
microscopy (Figure 3-2B), a special condenser is used so
that only light reflected off the specimen enters the objective.
The appearance is of a brightly lit specimen against a dark
'-'. background, and often with better resolution than that of
the bright field microscope.
Phase contrast microscopy (Figure 3-2C) uses special
optical components to exploit subtle differences in the
refractive indices of water and cytoplasmic components to
produce contrast. Light waves that are in phase (that is,
their peaks and valleys exactly coincide) reinforce one
another and their total intensity (due to the summed
amplitudes) increases. Light waves that are out of phase by
exactly one-half wavelength cancel each other and result in
no intensity; that is, darkness. Wavelengths that are out of
phase by any amount will produce some degree of cancella-
tion and result in brightness less than maximum, but more
than darkness. Thus, contrast is provided by differences
in light intensity that result from differences in refractive
indices in parts of the specimen that put light waves more
or less out of phase. As a result, the specimen appears as
various levels of darks against a bright background.
Fluorescence microscopy (Figure 3-2D) uses a fluorescent
dye that emits fluorescence when illuminated with ultraviolet
light. In some cases, specimens possess naturally fluorescing
chemicals and no dye is needed.
The Electron Microscope
The electron microscope uses an electron beam to create an
image, with electromagnets acting as lenses. The limit of
resolution is improved by a factor of 1000 (theoretically
down to 0.1 nm, but more realistically down to 2 nm) over
the light microscope.
The transmission electron microscope (TEM) (Figure
3-5) produces a two-dimensional image of an ultrathin
section by capturing electrons that have passed through the
specimen. The degree of interaction between the electrons
and the heavy metal stain affects the kinetic energy of the
electrons, which are collected by a fluorescent plate. The
light of varying intensity produced is directly proportional
to the electron's kinetic energy and is used to produce the
26 A PHOTOGRAPHIC ATlAS FOR
image. The TEM is useful for studying a cell's interior, its
ultrastructure. A sample transmission electron micrograph
is shown in Figure 3-6.
Figure 3-5. Transmission Electron Microscope. The transmission
electron microscope produces an image using electrons that pass
through the specimen. The image is then viewed on the monitor.
This particular model magnifies from 8X up to 630,000X.
Photogroph courtesy of Carl Zeiss NTS GmbH
Figure 3-6. Transmission Electron Micrograph. The TEM
produces images of sectioned specimens. Since light is not
used, the image is not in color. These cells were magnified
12,500X. Photogroph courtesy of Corl Zeiss NTS GmbH
THE MICROBIOlOGY LABORATORY
A scanning electron microscope (SEM) (Figure 3-
used to make a three-dimensional image of the specimen'
surface. In this technique, a beam of electrons is passed over
the stained surface of the specimen. Some electrons are
reflected (backscatter electrons), whereas other electrons
(secondary electrons) are emitted from the metallic stain.
These electrons are captured' and used to produce the three- •
dimensional image. A sample scanning electron micrograph ' "-
is shown in Figure 3-8.
Figure 3-7. Scanning Electron Microscope. This scanning elecfr -
microscope has the ability to magnify from 12X to 900,000X "
resolving power of as little as 1.0 nm.
Photogroph courtesy of Carl Zeiss NTS G- -
i
Figure 3-8. Scanning Electron Micrograph. Like the TEM, the
image produced by the SEM has no color, but it is three-dirnensi
This micrograph is of E. coli. Photograph courtesy of Carl Zeiss S &:;;--