INTRODUCTION :-
A microscope may be defined as an optical instrument consisting of a lens or combination of lenses for
making enlarged or magnified images of minute objects. The science dealing with all aspects of a
microscope is called microscopy. Microscopes may be classified as follows:
1. Depending on number of lenses:
Microscope – Simple Microscope (Single lens)
Compound Microscope (Two lenses)
Microscope
2. Depending on number of eyepiece:-
Microscope -Monocular Microscope (Single eyepiece)
Binocular Microscope (Two eyepieces)
3. Depending on source:
(i)Light or optical microscope: Uses optical lenses and visible light.
(ii) Electron microscope: Uses electromagnetic lenses and an electron beam.
The light microscope can magnify images upto about 1000 times while an electron microscope can
produce magnifications upto a million fold.
Contents
Microscope
(1)Light or Optical Microscopes
Bright-field Microscopes
Dark-field Microscopes
Fluorescence Microscopes
Phase-contrast Microscopes
(2)Electron Microscopes
Transmission Electron Microscopes
Scanning Electron Microscope
COMPOUND MICROSCOPE
The compound microscope essentially consists of three major systems:
1. Support system: It comprises a base, stage and body tube. (Diagram)
2. Illumination system: It comprises a light source or mirror, iris diaphragm and a condenser. The
light source may be a plain or concave mirror or electrically illuminated by a tungsten filament
lamp or a halogen lamp. A mirror and electric light source are generally interchangeable.
3. Magnification system: It consists of two sets of lenses. The lens system nearest to the specimen
is called the objective. The second lens system is called the eyepiece or ocular. The image seen
by the eye has a magnification equal to the product of the magnification of the two lens
systems.
Important Lenses of Compound Microscope:
(A) Oculars:-
The functions of the ocular or eyepiece are as follows:
1. It magnifies the real image of the object as formed by the objective.
2. It corrects some of the
defects of the objective.
3.It is used for observation of images.
Different types of eyepieces are used depending upon the kind of objective located on The microscope.
The most commonly used oculars are Huygenian, Ramsden and Compensating.
(a) Huygenian oculars are constructed with the plain surface of two lenses facing upwards and the
diaphragm is situated between them at the focus of the upper lens.
The lower or field lens
collects the rays from as wide a field of view of image as possible and focusses them at or near
the plane of the diaphragm. Huygenian eye-pieces are also called as ‘negative oculars’ because
the focus occurs Within the eye-piece.
(b) Ramsden or positive oculars are constructed with the convex surface of both lenses Facing
inwards. In this type of ocular, the diaphragm is placed externally below the Lower lens. Hence,
they are used for micrometry and give more accurate results than Huygenian oculars.
(C)Compensating oculars or positive oculars usually contain a triplet system as the lower lens
component. Aberrations are carefully corrected by the makers and they Are designed specifically for use
with particular objectives.
(B)Objectives:
The objectives are the most important lenses of a microscope because its properties Make the final
image. The functions of objective lenses are as follows:
1. Magnify the real image of object.
2. To unite the light at a point of the image.
3. To gather the light rays coming from any point of the object.
There are three major types of objectives mainly used in microscopy such as achromatic, fluorite and
apochromatic. The achromatic objectives are the simplest in construction and Least expensive.
These objectives are used for all the microscopical work of clinical microbiology and most research
purposes. Correction of colour and spherical aberrations is quite easy in the low power objectives as
compared to high power objectives.
(rays going away from the right path) are largely eliminated by the
use of fluorite and apochromatic objectives. Apochromatic objectives represent the highest degree of
optical perfection. These objectives are very costly hence; they are only used for critical research work
and photomicrography. Apochromatic objectives are always used with ‘compensating’ eyepieces and a
properly centered condenser.
Oil-immersion objectives are most frequently used in microbiology because of their greater
magnification and resolution. Most microbial cells are observed with achromatic or apochromatic
objectives designed for use with oil immersion. These objectives increase the angle of the cone of rays
from the object that enters the objective. With a ‘dry’ objective, air is present between the object on the
surface of the glass slide and the objective lens.
The refractive index of air (n = 1) is lower than that of glass (n = 1.55) and as light rays pass from the
glass slide into the air, they are bent or refracted (Fig. 5.6, EBFG) so that they do not pass into the
objective lens. This would cause a loss of light which would reduce the numerical aperture and diminish
the resolving power of the objective lens.
Loss of refracted light can be compensated by using cedar
wood oil (n = 1.5), which has the same refractive index as that of glass, between the slide and the
objective lens (Fig. 5.2, ABCD). In this way, decreased light refraction occurs and more light rays enter
directly into the objective lens producing a vivid image with high resolution.
(C)Condensers:
In microbiology, two methods are commonly used for illuminating the object under the microscope.
1. Illumination by transmitted light:
A condenser may be defined as a series of lenses for
illuminating transmitted light, an object to be studied on the stage of the microscope. It
is located under the stage of the microscope between the mirror and the object. It is
also called as substage condenser.
A condenser is necessary for the examination of an
object with an oil immersion objective to obtain adequate illumination. A condenser is
also preferable when working with high power dry objectives.
A good condenser sends light through the object under an angle sufficiently large to fill the aperture of the back lens of the objective. Therefore, it must be properly positioned during the microscopy.
Generally, condensers are also incorporated with an iris diaphragm and a filter holder.
An iris diaphragm is used to control light intensity. The Abbe condenser, variable focus
condenser and achromatic condensers are commonly used for bright field illumination.
The Abbe condenser (DIAGRAM) utilizes only two lenses. It is extensively used for
general microscopy because of its simplicity and good light-gathering ability. It is not
corrected for spherical aberration (fails to bring the whole microscopic field into
simultaneous focus) and chromatic aberration (produces coloured fringes around
objects in the field).
The variable-focus condenser (DIAGRAM) is a two lens system in which the upper lens
element is fixed and the lower element is flexible. It is possible to fill the field of low
power objectives without the necessity of removing the top element.
This condenser is
basically similar to the Abbe condenser when the lower lens is raised to its top position.
The achromatic condenser (DIAGRAM) is corrected for both chromatic and spherical
aberrations. Hence, it is mainly used for research microscopy and for colour photomicrography where the highest degree of perfection in the image is desired.
2.Dark field illumination or Dark field microscopy:
The microscope which forms a Bright
image against a dark background is called dark field microscopy. Many transparent and
semi-transparent objects are not easily visible in a bright field. Visibility is dependent
upon contrast between the object and its background and can be improved by using a
dark Background.
The cone of light normally illuminating an object does not enter the objective, but light
scattered or reflected by the specimen is seen by the objective. If a dark-field stop of
suitable size is selected, all the direct rays from the condenser can be made to pass
outside the objective. Any object within this beam of light will reflect some light into the
objective and be visible.
There are three requisites for adapting an ordinary microscope for dark background
illumination.
(i) A dark background condenser, which focusses only oblique rays of light on the
specimen,
(ii) A suitable high-intensity lamp, and
(iii) A funnel stop which reduces the numerical aperture of the objective to less than
1.0. The Abbe condenser, paraboloid condenser and cardioid condenser are
commonly used for dark field illumination.
The Abbe condenser (diagram) is more commonly employed than the other condensers because it is
suitable for objects that do not require the highest magnification to make them visible. It may be
employed either by inserting a dark-field stop below the condenser or substituting the top part of the
condenser by a dark-field element.
The paraboloid condenser is designed to be used with oil-immersion objectives and an intense source of
light. The specimen or object must be mounted in a liquid or in cement and protected with a cover slip.
The numerical aperture of the objective must not be greater than that of the condenser.
The cardioid condenser (diagram) is best employed with a strong arc lamp. Ordinary glass slides and
cover slips are not used because of high concentration of light. It is better to employ fused quartz object
slides and fused quartz cover slips. The cardioid condenser is especially designed for the examination of
colloidal solutions and suspensions.
Dark-field microscopy is very useful for the examination of unstained micro-organisms Suspended in
fluid i.e. by wet mount techniques and hanging drop preparations. Dark-field Examination is useful in
the detection of Treponema palladium in the early diagnosis of Syphilis.
PHASE-CONTRAST MICROSCOPY
The phase contrast principle was discovered by Fritz Zernike for which he was awarded the Nobel Prize
in Physics in 1953.
The basic construction of phase contrast microscope is like a bright field microscope except a special
type condenser and a phase plate. The condenser has a special diaphragm consisting of an annual stop.
The annular stop allows only a hollow cone of lights rays to pass through the condenser. The phase plate
is special optical disc located in the rear focal plane of the objective. It has a special ring coated with a
material that can either advance or retard the direct rays.
The rays that pass through the object in a straight line are called direct (undiffracted) rays and are unaltered in amplitude and phase. The rays that are bend and slowed down due to differences intensity of medium are called diffracted rays.
The phase of light rays is altered when they pass through a specimen to be observed under a light
microscope. This change of phase is a manifestation of the depth and density of the Cell and its internal
parts. Since there is very little difference or contrast in the refractive indices or density of the specimen,
its internal structures and the medium, it is not made visible by bright-field microscopy.
In phase
contrast microscopy, the small phase differences are intensified and translated into differences in light
intensity with the help of special optical devices. A diagram illustrating an optical system and light
transmission through a phase contrast microscope is shown in DIAGRAM.
An annular aperture In the diaphragm placed in the focal plane of the sub-stage condenser, controls the
illumination of the object. The aperture is imaged by the condenser and the objective at the real focal
plane of the objective. In this plane a phase shifting disk or phase plate is placed. Undiffracted light rays
are transmitted through the object and pass through the altering ring on the phase plate. At this point
they acquire a one quarter wavelength advance over the diffracted light rays by the object.
While
diffracted (indirect) rays pass through the transparent region of the phase plate and are unchanged by
the missing phase ring they are already retarded by one quarter (1/4) wavelength due to the object.
Finally, all rays including undiffracted rays and diffracted rays are brought together by an eyepiece lens.
Apparent brightness or darkness in an image is proportional to the square of the amplitude of light
waves. The image will be four times brighter or darker as seen in the bright field microscope. Hence, it is
possible to visualise microorganisms without staining.
Phase contrast microscopy provides a second method for observing unstained living microorganisms
with good contrast and high resolution. It does not show very small objects like in dark field microscopy
but it is more useful for the study of the structure and structural changes in larger microorganisms and
tissue cells. Phase contrast microscopy is also useful in examination of growth and cell division in
bacteria, flagellar movements, spore and capsule formation and cytopathic effect of viruses in tissue
culture.
ELECTRON MICROSCOPY
An electron microscope (EM) is an instrument, which utilises short wavelength of electrons as a source
of illumination for observing objects at a greater magnification. The major significance of an electron
microscope is that it has the highest resolution and magnification. German engineers Max Knoll and
Ernest Ruska in 1931 developed the first electron microscope.
The electron microscope works on the principle similar to that of a light microscope. An electromagnetic
field and a beam of electrons act in a way similar to the action of a glass lens and a beam of light. The
better resolution of electron microscopes is due to the shorter wavelengths of electrons. The
wavelengths of electrons are about 100,000 times smaller than the wavelengths of visible light. Electron
microscopes are used to examine structures too small to be resolved with light microscopes. It is
possible to resolve objects as small as 10 A° by electron microscopy.
Electron microscope is classified into two types:
1. Transmission electron microscope (TEM).
2. Scanning electron microscope (SEM).
Transmission Electron Microscope:
In a transmission electron microscope (Fig. 5.6), a beam of electrons is projected from an electron gun
and is passed through a series of electromagnetic lenses (Fig. 5.7). The electron beam is produced by an
electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electrons
accelerated by the high voltage energy are forced through a collimating aperture which renders the rays
in parallel lines and fashions them into a beam. The beam is focused on a small area of the specimen by
an electromagnetic condenser lens. The condenser lens is a magnetic coil which corrects the aberrations
(bending) in the beam. Electrons get scattered, transmitted through the object and pass through the
objective lens which magnifies the image of the object (diagram).
The strength of the magnetic lens depends on the amount of current that is allowed to flow through it.
The electron beam that has been partially transmitted through the very thin specimen carries
information about the structure of the specimen. The entire electron microscope must be in a vacuum
or otherwise the electrons get scattered due to collisions with air molecules and fail to get focused. The
specimen must be absolutely dry. Specimen supporting grid is usually a collodion film or Fonnvar or
polymerized plastic plus a screen grid of copper. The electron beam passes through object is scattered
depending on the varying refractive index of the specimen. The electron beam from specimen passes
through the second set of magnetic coils (objective lens), which focus the electrons and form an
intermediate image. A third set of magnetic lenses (projection lens), further magnifies the image and
projects it on the fluorescent screen or photographic film. The electron image is converted into visible
form by projecting on a fluorescence screen.
An electron beam has low penetration power through solid matter. Hence, very thin sections of
specimen can be examined under an electron microscope. The degree of scattering of electrons by the
specimen is related to the number and mass of the atoms that lie in the electron path. Since most of the
constituent elements in biological matter are of low mass and the contrast of these materials is weak.
The contrast of such materials can be enhanced by staining with salts of heavy metals such as uranium
or tungsten. These metals may be fixed on the specimen (positive staining) or used to increase the
opacity of the surrounding area (negative staining).
Scanning Electron Microscope:
The scanning electron microscope (SEM) was built by Van Ardene in 1938. SEM (Fig. 5.9) is primarily
used for visualizing the surface architecture of the specimen rather than the internal details. A SEM
provides striking three-dimensional views of specimens. The construction plan and working principle of
a SEM is different from that of TEM.
In SEM, an accelerated beam of electrons is produced from the electron gun and is focused on the
specimen by the condenser lens. The magnetic lenses of a SEM are responsible to produce an extremely
thin beam of electrons called the primary electron beam. These electrons pass through electromagnetic
lenses and directed over the surface of the specimen (diaGRAM).
The primary electron beam knocks
electrons out of the surface of the specimen. This causes the release of secondary electrons from the
specimen surface. The intensity of these secondary electrons depends on the shape and the chemical
composition of the irradiated object. The secondary electrons are collected by a detector which
generates an electron signal. The signals are then scanned in the manner of a television system to
produce an image on a Cathode Ray Tube (CRT).
During this process, some of primary electrons are also reflected and transmitted to the collector but
their number is less than the secondary electrons. As a result, the image signal is developed more by the
secondary electrons than the primary electrons.
The secondary electrons deflected out of the specimen
will be a replica of the refractive index of the surface and thus produce an image on the CRT screen
revealing all the topographical details. Image contrast is mainly dependent on surface topography which
determines the number of secondary electrons reaching the detector.
SEM also has a resolution equalto that of TEM. A resolution from 1 to 10 nm is possible with a corresponding magnification from 10,000to 100,000. This microscope is especially useful in studying the surface structures of intact cells andviruses. In the pharmaceutical field, SEM is very useful in studies associated with the surfacecharacteristics of drug particles and morphological studies of antibiotic producing microorganisms and their spores.
Limitations of Electron Microscopy:
1. The specimen being examined is in a chamber that is under a very high vacuum. Thus, cells
cannot be examined in a living state.
2. Drying process may change some morphological characteristics.
3. Thin sections are required to observe internal structures of the cell because of low penetration
power of the electron beam.
4. Numerical aperture of an electron microscope lens is very small.
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