BSC 310 Lecture Notes - Lecture 6: Numerical Aperture, Antibody, Scanning Electron Microscope
Introduction to Microscopes
Since microorganisms are invisible to the unaided eye, the essential tool in microbiology
is the microscope. One of the first to use a microscope to observe microorganisms
was Robert Hooke, the English biologist who observed algae and fungi in the 1660s. In
the 1670s, Anton van Leeuwenhoek, a Dutch merchant, constructed a number of
simple microscopes and observed details of numerous forms of protozoa, fungi, and
bacteria. During the 1700s, microscopes were used to further elaborate on the microbial
world, and by the late 1800s, the sophisticated light microscopes had been developed.
The electron microscope was developed in the 1940s, thus making the viruses and the
smallest bacteria (for example, rickettsiae and chlamydiae) visible.
Microscopes permit extremely small objects to be seen, objects measured in the metric
system in micrometers and nanometers. A micrometer (μm) is equivalent to a millionth of a
meter, while a nanometer (nm) is a billionth of a meter. Bacteria, fungi, protozoa, and
unicellular algae are normally measured in micrometers, while viruses are commonly
measured in nanometers. A typical bacterium such as Escherichia colimeasures about two
micrometers in length and about one micrometer in width.
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.
find more resources at oneclass.com
find more resources at oneclass.com
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.
An alternative microscope is the dark field microscope‐, which is used to observe live
spirochetes, such as those that cause syphilis. This microscope contains a special
condenser that scatters light and causes it to reflect off the specimen at an angle. A light
object is seen on a dark background.
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
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Since microorganisms are invisible to the unaided eye, the essential tool in microbiology is the microscope. One of the first to use a microscope to observe microorganisms was robert hooke, the english biologist who observed algae and fungi in the 1660s. In the 1670s, anton van leeuwenhoek, a dutch merchant, constructed a number of simple microscopes and observed details of numerous forms of protozoa, fungi, and bacteria. During the 1700s, microscopes were used to further elaborate on the microbial world, and by the late 1800s, the sophisticated light microscopes had been developed. The electron microscope was developed in the 1940s, thus making the viruses and the smallest bacteria (for example, rickettsiae and chlamydiae) visible. Microscopes permit extremely small objects to be seen, objects measured in the metric system in micrometers and nanometers. A micrometer ( m) is equivalent to a millionth of a meter, while a nanometer (nm) is a billionth of a meter.
