MICROSCOPY

Definition

Microscope is an instrument with magnifying lenses that is used for the observation of objects
not visible to the naked eye. The set of lens system in the instrument amplify and convert

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information (light or electrons) to form an image that is discernible by the eye. It utilizes light
(energy). The use of such an instrument is termed microscopy.

Classification of magnifying systems

a. Simple magnifying system – uses a single lens unit to form an enlarged image of an
object for viewing or projecting e.g. reading glasses, pocket magnifiers, slide projectors.
b. Compound magnifying system – here magnification takes place in two stages (2 lens
system units) and is the one used in the compound light microscope. The observer looks
at the first or 1o image (real image) with a lens that produces an enlarged 2 o image called
the virtual image, the image the eye perceives.

Properties of light
a. Travels in a straight line to infinity
b. Described by amplitude, which is the strength of the energy or brightness of the light, and
diminishes in the medium. The wavelength (distance between the apex of one wave and
the next measured in nm (1 x 10-9m) is referred to as lambda. The frequency is the
number of waves per second.
c. The speed (frequency per second) is slowed down (retardation) by the medium through
which the light passes (proportional to density of medium).
d. Refraction – deviation of light as it passes at any other angle (apart from 90 o) of a glass.
In a curved glass, retardation + refraction.

Image formation
Parallel rays of light entering a simple lens are brought together by refraction to a single point,
the principal focus or focal point where a clear image of the object will be formed. Focal length
is the distance between optical center of the lens and the focal point.
Types of Microscopy
Based on the source of energy, there are two main kinds of microscopes, one that utilises the
light energy (light microscope) and the other utilising electron beam (electron microscope).
Within the first group, we distinguish the following subtypes, some of which are specialized.

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a. Light microscope (the ordinary compound microscope)
b. Phase contrast microscope
c. Dark field microscopy
d. Interference light microscope
e. Polarizing microscope
f. Fluorescence microscope
g. Ultraviolet microscope
h. Stereo/dissecting microscope

In the second category i.e. Electron Microscopy, there are principally two types:
a. Transmission Electron Microscope (TEM)
b. Scanning Electron Microscope (SEM)

The light microscope

Synonyms: Compound microscope or Bright-field microscope. This is the most widely used
microscope. It is used to study normal and pathological structure of tissues (Histology and
Histopathology), the structure of the very small organisms (Microbiology and Parasitology), and
thus for routine diagnostic procedures as well as in research.

The Components of a light Microscope

1. Light source – The sources of light energy to the microscope, can be sunlight, oil lamp or
electric lamp.
2. Aperture diaphragm. This is the exit point of light. In some of the microscopes the
aperture diaphragm is fixed whereas in others it has the facility to regulate its diameter,
hence, the light beam. Others have voltage control to vary the strength or brightness of
light.
3. Condensers (substage condenser).
This is the first major optical component. In here, light is directed into it, either directly
or by a mirror or prism in the base of the microscope. The condenser focuses the beam of
light (illumination) on the specimen (object).

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 Construction
a. Some microscopes have vertically adjustable condensers in order to allow for
varying heights or thickness of slide.
b. Some condensers are provided with adjustment screws for centering the light
path.
c. Some condensers are provided with condenser diaphragm (field iris diaphragm) to
control the diameter of the light beam.
d. Others have a swing – out top lens. The lens is turned into the light path when the
higher power objectives are in use. It focuses better the light into the field
particularly when using the objective whose front lens have smaller diameter.

4. Object stage
A rigid platform with an aperture through which the light may pass to illuminate the object. The
function of the stage is to support the glass slide bearing the specimen and therefore the latter is
oriented perpendicular to the optical path.
Modifications
a. With mechanical stage attached or built in to allow controlled movement in two
directions.
b. Movable stages with vernier scales to enable operator return the specimen to exact
location at a later occasion.

5. Adjustment knob
These are of two kinds, and whose use is to move the stage up and down. One (always
the inner) knob is for coarse focusing. This is also known as the stage position
adjustment knob. The other knob (always the outer and in many cases smaller) is the
focus adjustment knob, and it is used for fine focusing after obtaining a coarse focus with
the coarse focus adjustment knob.

6. Objectives
These are the most important components of the microscope. Within each objective, are
lens elements (5-15 in number) depending on quality and type. The objective collect the

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 maximum amount of light possible from the object, unite it and form a high quality,
magnified, real image, some distance above. The magnifying power ranges from 1:1 to
100:1, and the objectives are marked x 4 or 4; x 10 or 10; x 25; x 40 or 40 and x 100 or
100. Coloured rings (most cases red for x 100 i.e. oil immersion) are found in most
objectives. Other markings on the objectives are:-
a. Numerical aperture (NA) which is the ability of the objective to resolve details.
The resolving power of the objective is the ability to resolve details that can be
measured.
NA = n x sinµ, where n = refractive index of the medium between the
cover glass over the objective and the front less of the objectives. It can
be air, water or immersion oil.

µ= angle between optical axis and the outermost ray of light.

The resolution is the smallest distance between two dots or lines at which they can
be seen as separate entities and it depends on the NA and the wavelength of the
light used.

b. Mechanical tube length (M.t.l.) It is the distance between the objective front and
the top of the draw tube. Most manufacturers use a M.t.l. of 160mm, (universal)
others are 170, 200 and 210. Objectives are designed for specific tube length.

c. An objective for use, without a cover-glass is in addition indicated by 160/-

(optical mechanical tube length followed by a minus sign).

Glass cover thickness – e.g. 160/0.17 requires cover glass of 0.17mm thickness.
Slides are normally 1mm thick.

Type of objectives
1. Achromatic – for routine purposes, corrected for two colours, blue + red, and
producing a 2o spectrum of yellow/green.

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2. Apochromatic – often incorporates fluorite glass and has more lens components –
higher resolution.

7. Revolving nose piece

The objectives (up to 6) are fitted onto a revolving nose piece. This enables a rapid
change from one objective to another.

8. Body tube: Above the nose piece are the following.

i. Observation tube can be one (monocular) or
two (Binocular). In some binocular microscopes there is an adjustment for the
inter-pupillary distance between the observation tubes.
ii. Combined photo – Binocular (trinocular). Here a system of prisms is added to
distribute the light to several targets e.g. to the eye piece, the photographic film
plate, and the exposure control unit.
iii. In some others, there are attachments to extensions such discussion tube, video
monitors, etc.

9. Eye piece
The final stage of the optical path of microscope is the ocular piece. In here, the real
image formed by the objective within the body tube is magnified and presented to the eye
as virtual image.

Modification
i. With measuring graticules (an eye piece calibrated with stage micrometer slide) for
measuring microscopic structures size.
ii. Focus adjustment

Magnification values
The total magnification is a product of the magnification of:
a) eye piece
b) objective

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 c) additional lens, eg. Tube lens factor of 1.25
NB: Total magnification should not exceed combined magnifications of objective and eye
piece x NA of the objective e.g. with 100/1.30 maximum magnification is 1300.

10. Object
The object that is looked at, is normally a stained specimen, that has been sectioned (by a
microtome) in such a thickness that allows light to go through. The staining aids the
formation of the image by absorbing part of the light (some of the wave lengths), and
producing an image of amplitude differences and color. Normally, the specimen or
object (thickness of 1 to 7 microns) is mounted on a glass slide that is usually 76mm x
25mm and 1mm (length x breadth x thickness), and covered with a coverglass (square,
circle or rectange). The thickness of coverglass is either 0.17 or 0.18mm.
NB: Unstained sections or living cells, have refractive indices close to that of the
medium in which they are suspended and thus they are difficult to be seen by
bright field technique. When examining such objects, dark background or
darkfield microscopy is employed. The method prevents direct light from
entering the front of the objectives, the only light gathered being that reflected by
structures within the specimen. It is employed in studying or observing
spirochetes, parasites, cell suspension and in flow cell technique. The method is
achieved through:
a. Use of modified or special condensers to form a hollow cone of direct light that
will pass through the specimen but outside the objective.
b. Closing down the iris diaphragm of the condenser.

Specialized light microscopy

1. Phase contrast light microscope
It is used to view unstained living biological specimens. In here, the microscope uses
modified objectives and condensers (normally marked Ph.) and the latter carries a series
of annular diaphragms made up of opaque glass. The objectives are modified by adding a
phase plate at the back focal plane of the objectives, which then provides interference
into the light and provide the phase difference. In addition, the specimen retards some

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 light rays with respect to those which pass through the surrounding medium providing
image contrast.
The microscope is used in the examination of living cells and tissues, and unstained
plastic thick sections.

2. Interference light microscope

This modification of the phase constrast microscope allows for quantitative analyses of
tissue mass. It is thus used as phase contrast microscope but here the retarded waves are
entirely separated from the direct or reference rays thus allowing improved image
contrast, color graduation and quantitative measurements.

3. Differential interference light microscope

This is special for assessing surface properties of cells and other biological objects.

4. Polarizing light miscroscope

This is a simple modification of the light microscope in which polarized light is passed
through the specimen and another polarizer is used as a rotator to detect molecular
orientation within a tissue sample.
Such a microscope uses 2 polarisers – one beneath the condenser and the other (analyzer)
between objective and eye piece. They employ use of birefringent substances or crystals
capable or splitting light into two light paths each with its own vibration direction and
velocity. Crystalline substances and well ordered fibrous structures alter the plane of
entering polarized light and the altered light plane is noted with the detector lens.
Rotation of polarized light due to molecular orientation of tissue components is referred
to as birefringence. Such a microscope is used mainly in diagnostics, in the detection of
pigments such as amyloid deposits bones.

5. Fluorescence light microscope (FM)

This type of microscopy is based on the detection of molecules that fluoresce; that is,
emit light of wavelengths in the visible range. Some substances when illuminated by
light of a certain wavelength will emit light of a longer wavelength. In fluorescence light

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 microscopy the light source is a halogen or mercury lamp burned. Ultraviolet light (=
360nm) is produced by introducing filters in the pathway of the light from the lamp
burner and is shine to a tissue with fluorescing material that is reflected and visualized.
Fluorescence microscopy is thus used to:-
a. To detect natural occurring fluorescent (autofluorescent) molecules, for instance
Vitamin A.
b. To detect introduced fluorescence, such as antigens or antibodies that have been
coupled with fluorescence (in immunocytochemical/histochemical staining
producers) and used diagnosis and research.
c. To trace specific fluorescent molecules that may be injected into an animal or
directly into cells. Such methods (Tracer techniques) are used in studying
intercellular (gap) junctions, tracing pathways of nerve fibres, markers of
mineralized tissues, etc.

6. Ultraviolet light microscope

It uses ultraviolet light source and depends on the absorption of ultraviolet light by
molecules in the specimen. A series of filters is thus employed to filter unwanted light
wavelengths. This kind of microscopy is employed in the detection of nucleic acids,
especially the purine and pyrimidine bases of the nucleotides.

7. Stereo microscope
It also sometimes referred to as dissection microscope. This is a simplified compound
microscope without condenser and objectives (where the latter is present normally is
without magnifying power (i.e. x1). The object is placed immediately on top of the light
source. Its magnifying power is thus low, and it is used to study unstained organism or
structures e.g. worms and motility of spermatozoa.

Electron Microscopy
Three kinds of electron microscopes are used to obtain extrafine morphological data i.e.
the transmission electron microscopes (TEM), the Scanning Electron Microscope (SEM)
and the Laser Confocal Scanning Electron Microscope.

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1. Transmission Electron Microscope (TEM)
The TEM, utilizes a beam of electrons instead of light in producing an image. It consists
of:-
a. Cathode:
A heated tungsten filament which is the electron source
b. An Anode
With a very high current of 60,000 to 100,000 Volts which accelerates the
electrons through the column.
c. A system of electromagnets that serves as the condenser lens, objective lens, and
projection lens. The magnets create electromagnetic fields which blend and
thereby focus the beam of electrons.
d. Specimen holder
A very thin plastic or resin section of a tissue on a copper grid through which
electrons pass. The section thickness is in the range of 8 to 100 nanometers (0.1
micrometer). The sections are said to be ultrathin.
e. Viewing screen
Where the image of the section can be viewed. There is a provision for the image
to fall on a photographic film, which are normally placed under the viewing
plane.

Image formation in TEM is based on the fact that some electrons do not pass
through the specimen to fall on a photographic film or the viewing screen, rather,
they are absorbed, retarded or deflected by substances of high mass normally
within the specimen or added to the specimen during fixation and staining such as
osmium tetroxide in fixatives. In contrast to the LM, tissues are embedded in
plastics, cut by diamond knife on special microtome (ultramicrotome) and are
placed on copper-mesh grids and then stained by stains containing heavy metals
eg. Urany1 acetate and lead citrate.

2. Scanning electron Microscope

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 Unlike the TEM, in SEM, the electrons do not pass through the specimen as part
of the image-making process. Instead a beam of electrons is made to scan the
sample surface. Electrons that are deflected from the surface are collected by a
detector and processed so that are displayed as a three dimensional image on a
viewing screen or a pathographic film.

3. Laser Confocal Scanning electron Microscopy

Unlike the SEM, the electrons pass through the specimen as part of the image-
making process. Several images are produced as the beam of electrons scan
through the specimen. These are reconstructed into a three-dimension image and
view on a screen or on a photographic film.

Proper use of the light Microscope

Before using a microscope, ensure the following:-

a. Objectives are firmly screwed home
b. Optical parts are clean and dust free (lens cleaning tissue).
c. There is source of light (working).

Setting up the microscope

a. Switch on the microscope and place a suitable prepared specimen on the stage (to
establish plane of object).
b. Select lowest power, and swing into position the condenser if the magnification is
10x and above, (not 4x).
c. In microscope with diaphragm aperture regulation devices, open the aperture
diaphragm in the condenser and then close the field diaphragm aperture in the
foot of microscope. An image of the field diaphragm is seen in the field of view.
Adjust the height of the condenser until the image of the diaphragm is sharply
focused, and then center the diaphragm image with the centering screws in the
condenser.

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 d. Open the aperture diaphragm until it just disappears from view.
e. Adjust the condenser aperture diaphram to obtain the best resolution and
constrast. In the microscopes without diaphragm apertures: after switching on the
microscope and with just enough light and with the lowest magnification
objective in portion, with the coarse focusing knobs, raise the stage towards the
objectives, as you watch from the side until it is almost in contact.
f. While looking in the eye piece, carefully focus up with the coarse adjustment until
the specimen come into focus first. Touch up the proper focus by adjusting the
fine focus.
g. Ensure illumination is adequate
h. Adjust the binocular tubes/trinocular head.
NB:
1. Never look at the object as you raise the stage as you risk; (a) breaking the slide
and (b) destroying the objective.
2. When changing slides always lower the stage before removing the slide.
3. When changing the objective, lower the stage first, and then swing in the
appropriate objective and ensure that it is well centered.
4. Use oil immersion only for an immersion objective (mostly the 100x), and after a
session with immersion objectives, clean with lens cleaning tissue (± xylene or
petroleum spirit). Never alcohol or acetone as they may seep into the mount and
dissolve the mount.

You can obtain optimal performance of the optic instrument by ensuring that:
i. illumination and observation beam paths are centered and correctly adjusted
ii. The settings and alignment of the optic pathway are properly set.

Recognition of minute (fine) details in the specimen, and good display of colour for the visual
image and for photomicrography are achieved by adopting what is known as Kohler
illumination. This can be obtained through the following steps:

1. Focus the specimen

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2. Close the field diaphragm
3. Focus the field diaphragm image by moving the condenser up and down until the
outline of its field diaphragm appears sharp. (Both the specimen and leaves of the
diaphragm are in sharp focus). When correct position obtained;
4. Remove the eye piece and observe the exit pupil of the objective. (You will
visualize an illuminated circular field, the radius of which is directly proportional
to numerical aperture of the objective. This setting results in the best compromise
between resolution and contrast.
5. Replace the eyepiece and observe the field of view.
6. Open the field diaphragm until it is just outside the field of view.
7. Open and close (adjust) the condenser or field iris diaphragm to obtain the best
resolution and constrast.

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HISTOLOGICAL TECHNIQUES

Definition: Various methods/techniques employed in the microscopic study of cells, tissues

and organs.

Approach: based on aim/goal

a. Living cells
(i) Short term investigation of living cells eg. Leucocytes in a drop of blood (supravital).
The cells may be stained or unstained
Slide, cover slip, petroleum jelly (seal), warm stage.

(i) In tissue culture (vital preparations) – isolate single cells or cloves.

NB: requires – nutritive media
- control of atmospheric gases
- control of sterility.

Observation - bright field microscope

- phase microscope
- interference microscope
- polarizing
- fluorescence.

Structures observed: nucleus + motility

Cytoplasm
Mitochondria
Golgi apparatus
Ceatuoles
Pleasure membrane in pinocytosi/phagocytosis.

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b. Fixed cells
Through fixation, given cellular structure or activity is preserved
or stabilized for subsequent viewing with the microscope.

Methods for fixation

1. Chemicals – these induce chemical changes in the cells e.g. precipitation of protein. The
process is achieved by either immersion of the tissue in a solution of the fixative or the
chemical solution is perfused into the animal body/organ. The chemicals include heavy
metals eg. Osmium tetroxide (react with the carboxy/group) and aldehydes eg.
Formaldehyde and glutaradehyde which react with the amino acids.

2. Physical means – eg. Heat denaturation, air drying or freezing. The latter is very useful
in the study of enzyme activity (enzyme/histochemistry) and in the study of membrane
architecture (freeze fracture technique). An instrument used for sectioning frozen
sections is termed cryostat

After the tissue is fixed, the tissues must then be sectioned into sufficiently thin slices so that
details can be revealed by microscopy (normally 5 um for light microscopy (LM) and 8-100 nm
for transmission electron microscopy (TEM).

The slicing is done with an instrument known as microtone (for thick sections) and
ultramicrotome (for semithin 0.1 – 2.5 um) and thin (8-100nm) sections. The instrument consist
of a chuck holding the tissue, a knife and an advance mechanism.

However, after fixation, tissues are brittle and impossible to cut in thin slices. It must therefore,
be infiltrated with a stiff flexible material that can be cut eg. Waxes. For light microscopy,
paraffin wax or celloidin are used, and recently resins eg. Historesin have been introduced. For
TEM, epoxy resins e.g. epon or araldite are used. The fixed tissue must therefore be dehydrated
either manually or by a machine termed histokenettle

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The tissues an passed through a series of increasingly concentrated aqueous solutions of ethyl
alcohol, acetone or other dehydrating agents that are missible with both water and fat e.g. 50, 70,
80, 95% and then absolute alcohol.

Either directly or indirectly, through an intermediate organic solvent like toluene or xylene, the
tissue is placed in the embedding agent in a liquid phase. The embedding agent replaces the
solvent and thoroughly anfillrates the tissues.

After the tissue is sectioned, it is mounted on a glass slide and stained (LM) or on copper grids
and stained it heavy metals e.g. Pb. Acetate (TEM). Unstained tissues may be observed by phase
contrast or interference microscope.

SPECIMEN PRESERVATION
Aim: Stop decay (bacterial attack or antolysis) of the specimen and be kept to as close to their
living state as possible.

Methods depend on future use of the specimen

1. Temperatures – temperatures that enzymes and bacteria are inactive.

–20oC freezer
-70o liquid nitrogen.

2. Fixation – state of rendering the tissues nonsuitable for bacteria or enymatic decay by the
use of physical or chemical agents.
(a) physical – heat
(b) chemical – use of fixative – aldehydes – formaldehyde, glutaraldehyde
- oxidizing agent – KMn04, Kdichromate
- protein dematuring agent such as acetic acid, alcohol.
- Other – picric acid, Hgcloride.

3. Plastination – infiltrate and replace all tissue fluids with polymers/resins e.g. perspex

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- retain shape + size.

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