Microscopy & Types of Microscopes

Various types of microscopes are available for use in the microbiology laboratory.
The microscopes have varied applications and modifications that contribute to their
usefulness.

The light microscope. The common light microscope used in the laboratory is

called a compound microscope because it contains two types of lenses that
function to magnify an object. The lens closest to the eye is called the ocular, while
the lens closest to the object is called the objective. Most microscopes have on their
base an apparatus called a condenser, which condenses light rays to a strong
beam. A diaphragm located on the condenser controls the amount of light coming
through it. Both coarse and fine adjustments are found on the light microscope
(Figure ).

To magnify an object, light is projected through an opening in the stage, where it hits
the object and then enters the objective. An image is created, and this image
becomes an object for the ocular lens, which remagnifies the image. Thus, the total
magnification possible with the microscope is the magnification achieved by the
objective multiplied by the magnification achieved by the ocular lens.

A compound light microscope often contains four objective lenses: the scanning

lens (4X), the low‐power lens (10X), the high‐power lens (40 X), and the oil‐
immersion lens (100 X). With an ocular lens that magnifies 10 times, the total
magnifications possible will be 40 X with the scanning lens, 100 X with the low‐power
lens, 400 X with the high‐power lens, and 1000 X with the oil‐immersion lens. Most
microscopes are parfocal. This term means that the microscope remains in focus
when one switches from one objective to the next objective.

The ability to see clearly two items as separate objects under the microscope is
called the resolution of the microscope. The resolution is determined in part by the
wavelength of the light used for observing. Visible light has a wavelength of about
550 nm, while ultraviolet light has a wavelength of about 400 nm or less. The
resolution of a microscope increases as the wavelength decreases, so ultraviolet
light allows one to detect objects not seen with visible light. The resolving power of
a lens refers to the size of the smallest object that can be seen with that lens. The
resolving power is based on the wavelength of the light used and the numerical
aperture of the lens. The numerical aperture (NA) refers to the widest cone of light
that can enter the lens; the NA is engraved on the side of the objective lens.

If the user is to see objects clearly, sufficient light must enter the objective lens. With
modern microscopes, entry to the objective is not a problem for scanning, low‐power,
and high‐power lenses. However, the oil‐immersion lens is exceedingly narrow, and
most light misses it. Therefore, the object is seen poorly and without resolution. To
increase the resolution with the oil‐immersion lens, a drop of immersion oil is
placed between the lens and the glass slide (Figure ). Immersion oil has the same
light‐bending ability (index of refraction) as the glass slide, so it keeps light in a
straight line as it passes through the glass slide to the oil and on to the glass of the
objective, the oil‐immersion lens. With the increased amount of light entering the
objective, the resolution of the object increases, and one can observe objects as
small as bacteria. Resolution is important in other types of microscopy as well.

Other light microscopes. In addition to the familiar compound microscope,

microbiologists use other types of microscopes for specific purposes. These
microscopes permit viewing of objects not otherwise seen with the light microscope.

A second alternative microscope is the phase‐contrast microscope. This

microscope also contains special condensers that throw light “out of phase” and
cause it to pass through the object at different speeds. Live, unstained organisms
are seen clearly with this microscope, and internal cell parts such as mitochondria,
lysosomes, and the Golgi body can be seen with this instrument.

The fluorescent microscope uses ultraviolet light as its light source. When

ultraviolet light hits an object, it excites the electrons of the object, and they give off
light in various shades of color. Since ultraviolet light is used, the resolution of the
object increases. A laboratory technique called the fluorescent‐antibody technique
employs fluorescent dyes and antibodies to help identify unknown bacteria.

Electron microscopy. The energy source used in the electron microscope is a

beam of electrons. Since the beam has an exceptionally short wavelength, it strikes
most objects in its path and increases the resolution of the microscope significantly.
Viruses and some large molecules can be seen with this instrument. The electrons
travel in a vacuum to avoid contact with deflecting air molecules, and magnets focus
the beam on the object to be viewed. An image is created on a monitor and viewed
by the technologist.

The more traditional form of electron microscope is the transmission electron

microscope (TEM). To use this instrument, one places ultrathin slices of
microorganisms or viruses on a wire grid and then stains them with gold or palladium
before viewing. The densely coated parts of the specimen deflect the electron beam,
and both dark and light areas show up on the image.

The scanning electron microscope (SEM) is the more contemporary form electron

microscope. Although this microscope gives lower magnifications than the TEM, the
SEM permits three‐dimensional views of microorganisms and other objects. Whole
objects are used, and gold or palladium staining is employed.
 Figure 1

Light microscopy. (a) The important parts of a common light microscope. (b) How
immersion oil gathers more light for use in the microscope.

Interference microscopy uses a prism to split light into two slightly

diverging beams that then pass through the specimen. It is thus based on
measuring the differences in refractive index upon recombining the two beams.
Interference occurs when a light beam is retarded or advanced relative to the other.
There are three types of interference microscopy: classical, differential contrast, and 

fluorescence
 contrast. Since its introduction in the late 1960s differential interference contrast
microscopy (DIC) has been popular in biomedical research because it produces high-
resolution images of fine structures by enhancing the contrasted interfaces. The
image produced is of a thin optical section and appears three-dimensional, with a
shadow around it. This creates a contrast across the specimen that is bright on one
side and darker on the other.

Figure: Path of light in differential interference contrast microscopy (DIC):

Two parallel light beams pass through the specimen and combine to produce an
image.

The Interference Microscope

The microscope is a bright field light microscope with the addition of the following
elements: a polarizer between the light source and the condenser, a DIC beam-
splitting prism, a DIC beam-combining prism, and an analyzer. Manipulating the
prism changes the beam separation, which alters the contrast of the image. When
the two beams pass through the same material across the specimen they produce
no interference. When the two beams pass through different material across the
specimen such as on the edges, they produce alteration when combined.

Fluorescence
 differential interference contrast (FLIC) microscopy was developed by combining 
fluorescence
 microscopy with DIC to minimize the effects of photobleaching on fluorochromes
bound to the stained specimen. The same microscope is equipped to
simulataneously image a specimen using DIC and 
fluorescence
 illumination.

Key Points
 Interference microscopy is superior to phase-contrast microscopy in its ability
to eliminate halos and extra light.
 In differential interference contrast microscopy (DIC), the optical path
difference is determined by the product of the refractive index difference
(between the specimen and its surrounding medium) and the thickness
traversed by a light beam between two points on the optical path.
 Images produced by DIC have a distinctive shadow-cast appearance.