The various types of light microscopy include bright-field, dark-field, fluorescence, and
phase contrast microscopy. Each method has specific applications and advantages, but the
most commonly used is brightfield microscopy.
Light Microscopes: Bright-field microscopy produces an image made from light that is
transmitted through a specimen. The specimen restricts light transmission and appears
"shadowy" against a bright background. Contrast between the specimen and background can
be improved with the application of stains to the specimen.
Image formation begins with light coming from a light source. 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 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 magnification produced by each lens is marked on the lens.
Total magnification of the specimen can be calculated by the following formula: Total
Magnification = Magnification of the Objective Lens X Magnification of the Ocular Lens
Clarity of an image is called resolution. The limit of resolution is an actual measurement of
how far apart two points must be in order for the microscope to view them as being separate.
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.
In dark field microscopy, 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.
In this type of microscopy, a dark back ground is produced against which objects are
brilliantly illuminated. For this purpose the light microscope is equipped with a special kind
of condenser that transmits a hollow core of light from the source of illumination. Thus, if the
aperture of condenser is allowed to open completely, and a dark field stop inserted below the
condenser, the light rays reach the objects form a hollow core.
Any object within this beam of light will reflect some light into the objective and will be
visible. This method of illuminating an object where the object appears self-illuminous
against a dark field, called dark-field illustration.
The condensers used are Abbe condenser, paraboloid condenser and cardoid condenser. Dark
field microscopy is particularly valuable for the examination of unstained microorganisms
suspended in fluid wet mount and hanging drop operations.
Phase contrast microscopy 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
cancellation 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.
According to this principle, light waves have variable character for frequency and amplitude.
Human eyes cannot perceive a phenomenon when two light rays have similar amplitude and
frequency but different phases.
The phase contrast microscope is an ordinary bright field microscope with two additional
plates, namely annular diaphragm and phase shifting plate, which enables the usage forming
rings to be phase shifted with respect to others. Annular diaphragm allows only a ray of light
to pass through the condenser and then to object.
The phase shifting plate is placed at the rear focal plane of objective lens. This disc has a ring
of optical dielectric material on which the ring of light from annular diaphragm is focused.
The ring has the property of retarding or advancing the phase of light of a quarter of a
wavelength traversing it.
From each translucent or transparent particle in the object, consider a single ray of incident
light. From this two rays result, one, the direct (undiffracted) ray comes through the annular
diaphragm passes through the objective and focused on phase- shifting ring which either
retards or advances the ray one quarter wavelength with respect to second ray.
The second ray is also derived from the incident ray, but modified by being scattered or
diffracted in passing around margin of the object.
This ray does not pass through the phase-shifting ring but traverse the other area of
transparent disc. Its wavelength is neither retarded nor advanced. Thus, there is one quarter
wavelength out of phase. The contrast between the two is called phase-contrast. It is valuable
device in wet mounts and hanging drops. It increases visualization of cellular structure and
traverse the other area of transparent disc.
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 over the light
microscope.
In electron microscope the source of illumination is electron beam. The construction and principle of
electron microscope are easily related to those of light microscope. The range of wave length of visible
light used in light microscope is 4000 Å – 7800 Å, while with an electron microscope employing 60-
80 KV electron, the wave length is only 0.05 Å.
In the instrument, the electron gun generates electron beam. These electrons are concentrated by
other components of electron gun producing a fast moving narrow beam of electron. Electrons are
focused by electromagnetic lenses. Electromagnetic lens consists of wire encased in soft iron casing.
When electric current is passed through the coil, it generates an electromagnetic field through which
electrons are focused.
Transmission Electron Microscopy:
The electron source is commonly a tungsten filament of 30-150 KV potential. The electron beam
passes through the centre of ring like magnetic condenser and becomes converged on the specimen.
After being transmitted through the specimen (hence transmission electron microscope (TEM), the
magnetic objective focuses the electron into a first (real) image of the object which is enlarged (2000
times). The magnetic projector lens then magnifies a portion of the first image producing
magnification upto 240,000 or more.
The final enlarged image can be reviewed by striking a fluorescent screen which makes it visible. The
image can also be thrown upon a photographic plate for permanent record. Portions of the photo-
graphs may be enlarged four to six times giving the picture in the range of two million times as large
as the object.
0 = Magnifies 2 million times → 2 mm diameter
4 million max or 2.5 miles in diameter.
Molecules in the microscope interferes with the movement of electrons. To prevent this, the interior of
the microscope is kept in the state of high vacuum, around 10-4-10-6 mm Hg. It is also necessary to
have specimen ultra thin. The electron beams have a very poor penetrating power, therefore, only
small objects or very thin sections of the specimen can be examined.
Scanning Electron Microscopy (SEM):
Scanning electron microscopes combine the mechanism of electron microscopy and television. SEM
became commercially available in early 1960’s and the researchers were Knoll, Von Ardenne,
Zworytein etc.
In SEM, electrons are not transmitted through the very thin specimen from below but impinge on its
surface from above. The specimen may be opaque and of any manageable thickness and size.
If the specimen is an electron conductor, it needs only to be held on an appropriate support. If it is
non-conductor, it is allowed to dry but if moist, freeze dried in liquid nitrogen is necessary. The
specimen is then coated with metal vapour (gold) in vacuum.
The electrons originate at high energy (20,000 V) from a hot tungsten or lanthanum hexoboride
cathode “gun”. These electrons are sharply focused, adjusted and narrowed by an arrangement of
magnetic fields.
Instead of forming a broad inverted cone of rays, in SEM a needle sharp probe (about 5 – 10 mm in
diameter) is made. This primary beam (probe) acts only as an exciter of image forming secondary
electrons emerging from the surface of the specimen.
The probe scans the specimen like that on a blank TV screen. The probe can impinge on depth and
heights with equal speed and accuracy giving great depth of field and producing images with three
dimensions. Images are elicited from wherever the probe strikes the metal coated areas of the
specimen. Magnification is the ratio of final image to the diameter of area scanned.
Any of the secondary electrons with sufficient energy can emerge from the surface. Those that emerge
not too far from the point of impact of the probe can be used to form an image. The useful secondary
electrons are magnetically deflected to a collector or detector. Here, they produce a signal that
represents at any single moment, only 5-10 mm area or spot of impingement of the probe on the
specimen.
The successive signals from the collector are amplified and transmitted to a cathode ray (TV) tube.
The scanning beam and TV tube beam are synchronized.
The image scan by the eye on TV screen is thus the sequence of signals representing in araster pattern,
the successive areas traversed by the primary probe beam. Exposure may range from a few second to
one-half hour or more. The TV image may be photographed, video taped or processed in motion on a
computer.
The transmission electron microscope (TEM) 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 theimage. The
TEM is useful for studying a cell's interior, its ultrastructure.
A scanning electron microscope (SEM) 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.