Microscope and microscopy WORLD OF MICROBIOLOGY AND IMMUNOLOGY388•
MMicroscope and microscopyICROSCOPE AND MICROSCOPY
Microscopy is the science of producing and observing images
of objects that cannot be seen by the unaided eye. A micro-
scope is an instrument that produces the image. The primary
function of a microscope is to resolve, that is distinguish, two
closely spaced objects as separate. The secondary function of
a microscope is to magnify. Microscopy has developed into an
exciting field with numerous applications in biology, geology,
chemistry, physics, and technology.
Since the time of the Romans, it was realized that cer-
tain shapes of glass had properties that could magnify objects.
By the year 1300, these early crude lenses were being used as
corrective eyeglasses. It wasn’t until the late 1500s, however,
that the first compound microscopes were developed.
Robert Hooke(1635–1703) was the first to publish
results on the microscopy of plants and animals. Using a sim-
ple two-lens compound microscope, he was able to discern the
cells in a thin section of cork. The most famous microbiologist
was Antoni van Leeuwenhoek (1632–1723) who, using just a
single lens microscope, was able to describe organisms and
tissues, such as bacteriaand red blood cells, which were pre-
viously not known to exist. In his lifetime, Leeuwenhoek built
over 400 microscopes, each one specifically designed for one
specimen only. The highest resolution he was able to achieve
was about 2 micrometers.
By the mid-nineteenth century, significant improve-
ments had been made in the light microscope design, mainly
due to refinements in lens grinding techniques. However, most
of these lens refinements were the result of trial and error
rather than inspired through principles of physics. Ernst Abbé
(1840–1905) was the first to apply physical principles to lens
design. Combining glasses with different refracting powers
into a single lens, he was able to reduce image distortion sig-
nificantly. Despite these improvements, the ultimate resolu-
tion of the light microscope was still limited by the
wavelength of light. To resolve finer detail, something with a
smaller wavelength than light would have to be used.
In the mid-1920s, Louis de Broglie (1892–1966) sug-
gested that electrons, as well as other particles, should exhibit
wave like properties similar to light. Experiments on electron
beams a few years later confirmed de Broglie’s hypothesis.
Electrons behave like waves. Of importance to microscopy
was the fact that the wavelength of electrons is typically much
smaller than the wavelength of light. Therefore, the limitation
imposed on the light microscope of 0.4 micrometers could be
significantly reduced by using a beam of electrons to illumi-
nate the specimen. This fact was exploited in the 1930s in the
development of the electron microscope.
There are two types of electron microscope, the trans-
mission electron microscope (TEM) and the scanning electron
microscope (SEM). The TEM transmits electrons through an
extremely thin sample. The electrons scatter as they collide
with the atoms in the sample and form an image on a photo-
graphic film below the sample. This process is similar to a
medical x ray, where x rays (very short wavelength light) are
transmitted through the body and form an image on photo-
graphic film behind the body. By contrast, the SEM reflects anarrow beam of electrons off the surface of a sample and
detects the reflected electrons. To image a certain area of the
sample, the electron beam is scanned in a back and forth
motion parallel to the sample surface, similar to the process of
mowing a square section of lawn. The chief differences
between the two microscopes are that the TEM gives a two-
dimensional picture of the interior of the sample while the
SEM gives a three-dimensional picture of the surface of the
sample. Images produced by SEM are familiar to the public, as
in television commercials showing pollen grains or dust mites.
For the light microscope, light can be focused and bent
using the refractive properties of glass lenses. To bend and
focus beams of electrons, however, it is necessary to use mag-
netic fields. The magnetic lens, which focuses the electrons,
works through the physical principle that a charged particle,
such as an electron that has a negative charge, will experience
a force when it is moving in a magnetic field. By positioning
magnets properly along the electron beam, it is possible to
bend the electrons in such a way as to produce a magnified
image on a photographic film or a fluorescent screen. This
same principle is used in a television set to focus electrons
onto the television screen to give the appropriate images.
Electron microscopes are complex and expensive. To
use them effectively requires extensive training. They are
rarely found outside the research laboratory. Sample prepara-
tion can be extremely time consuming. For the TEM, the sam-
ple must be ground extremely thin, less than 0.1 micrometer,
so that the electrons will make it through the sample. For the
SEM, the sample is usually coated with a thin layer of gold to
increase its ability to reflect electrons. Therefore, in electron
microscopy, the specimen can’t be living. Today, the best
TEMs can produce images of the atoms in the interior of a
sample. This is a factor of a 1,000 better than the best light
microscopes. The SEM, on the other hand, can typically dis-
tinguish objects about 100 atoms in size.
In the early 1980s, a new technique in microscopy was
developed which did not involve beams of electrons or light to
produce an image. Instead, a small metal tip is scanned very
close to the surface of a sample and a tiny electric current is
measured as the tip passes over the atoms on the surface. The
microscope that works in this manner is the scanning tunnel-
ing microscope (STM). When a metal tip is brought close to
the sample surface, the electrons that surround the atoms on
the surface can actually “tunnel through” the air gap and pro-
duce a current through the tip. This physical phenomenon is
called tunneling and is one of the amazing results of quantum
physics. If such phenomenon could occur with large objects, it
would be possible for a baseball to tunnel through a brick wall
with no damage to either. The current of electrons that tunnel
through the air gap is very much dependent on the width of the
gap and therefore the current will rise and fall in succession
with the atoms on the surface. This current is then amplified
and fed into a computer to produce a three dimensional image
of the atoms on the surface.
Without the need for complicated magnetic lenses and
electron beams, the STM is far less complex than the electron
microscope. The tiny tunneling current can be simply ampli-
fied through electronic circuitry similar to circuitry that iswomi_M 5/7/03 7:52 AM Page 388