Microtechnique

and
Microscopy
Department of histology
and cell biology
 Objectives
By the end of these lectures the student should know:
1. The meaning of microtechnique.
2. Steps of paraffin section preparation and the
importance of each step.
3. Basic idea of other types of tissue section
preparation.
4. Major differences between tissue section preparation
for light microscope and for electron microscope.
5. The idea of different types of staining.
6. The meaning of histochemistry, immunohistochemistry,
immunocytochemistry.
7. Structure and idea of ordinary light microscope
working.
8. Basic idea of other different types of microscopes.
Histology
is the study of the
microscopic structure of
tissues. So, it is the
microscopic anatomy of the
tissues.
Microtechnique
is the art of preparing
objects for examination under
the microscope (light and
electron microscopes) and of
preserving objects so
prepared
Preparation of histological sections
A) For light microscope (LM):
1. Paraffin technique.
2. Celloidin method.
3. Freezing technique.
B) For electron microscope (EM)
Paraffin technique (routine histological section)
1. Selection and obtaining tissue.
2. Fixation.
3. Washing.
4. Dehydration.
5. Clearing.
6. Infiltration and Embedding.
7. Microtomy and Sectioning.
8. Mounting.
9. Deparaffinization.
10. Hydration.
11. Staining.
12. Dehydration.
13. Clearing.
14. Mounting.
15. Cleaning and Labeling.
 1. Selection and obtaining tissue
- Samples must be obtained from living
tissue (biopsy) or very soon and as rapidly
as possible after death (autopsy).
- Samples are taken using very sharp razors
to prevent distortion of tissue during cutting.
- Samples should be small size to allow the
penetration of fluid through it up to the most
interior part.
2. Fixation
- The tissue is put immediately in a suitable fixative.
- The most commonly used fixative is formalin
(buffered 10%).
- Purpose of fixation:
1. Prevent autolysis as it destroys lysosomal enzymes.
2. Prevent putrefaction by killing bacteria.
3. Coagulate the proteins and harden the tissue
making it easier for cutting into thin sections.
4. Some fixatives have mordating effect (increase the
affinity of tissue for stains).
3. Washing
- After the tissue is fixed for the proper length of time,
excess fixative is washed out.
- Washing also removes substances in the fixative
which might interfere with the subsequent processing.
4. Dehydration
- Removal of water is a necessity to embed the tissue
in a wax or resin material to be ready for sectioning.
- Dehydration is usually done by ascending grades of
ethyl alcohol (50, 70, 90, 100%) to prevent sudden
withdrawal of water which may cause distortion and
damage of the tissue.
5. Clearing
Paraffin is not soluble in alcohol. So, alcohol is
replaced by a substance in which paraffin is soluble,
such as xylene, toluene, or benzene.
6. Infiltration and Embedding
-Wax must be surrounding (embedding) and passing
into the inside of the tissue (infiltration).
- Infiltration is done first soft paraffin.
- Soft paraffin, thus, replaces the clearing agent
and hard paraffin is used in the following step to
conform the block around the tissue.
7. Microtomy and Sectioning
- Sectioning is done using the microtome.
- Microtomes in common use are usually
the rotary types although other types
are used in tissue preparation such as
the sliding microtome usually used in
sectioning of dense tissue (bone and
skin).
- Section thickness usually suitable for
routine histological study is 4-7 um.
8. Mounting
Sections are mounted onto a glass slide and left to dry.
9. Deparaffinization
- Stains are usually in the form of aqueous solutions.
- Sections on the slide is surrounded and infiltrated
with paraffin which immiscible with water.
- Paraffin is removed by xylol

10. Hydration
- Hydration of the tissue is a necessity to enable the
stain reach and react with the tissue components.
- Rehydration of the tissue is achieved by descending
grades of alcohol (100, 90, 70, 50%), distilled water
is then used to replace the alcohol.
11. Staining
- The nucleus is stained blue first by haematoxylin then, the
use of acidic dyes (e.g. eosin) to stain the cytoplasm red.
- Washing with water is done between the two steps.
12. Dehydration
by ascending grades of ethyl alcohol (50, 70, 90, 100%).
13. Clearing
by xylene, it makes the stained tissue sections transparent.
14. Mounting
the section is mounted (covered) with a very thin cover slip
using a sticky mount (Canda balsam or De Pe Xe).
15. Cleaning and Labeling
Advantages of paraffin technique
➢ It takes a short time.
➢ It gives serial sections (ribbon).
➢ It gives very thin sections.
➢ The sections are easily stained.
Disadvantages of paraffin technique
➢ The fixatives and heat used may damage the
tissues.
➢ The fat contents of the cells are dissolved during
preparation.
➢ It is not suitable for histochemistry.
➢ It can not used for large pieces of tissues with large
cavities.
 Celloidin method
- The tissue is embedded in celloidin block .
- The sections are cut with a sliding microtome.
Advantages of Celloidin method:
➢ 1. It preserves the relations of the tissues
because no heating is used.
➢ 2. It can be used for cutting large organs with
plenty of folds and lumina.
Disadvantages of Celloidin method:
➢ Time-consuming.
➢ No serial sections can be obtained.
➢ The sections are very difficult to be stained.
 Freezing technique
- It is used to cut fresh specimen.
- The specimens are frozen, hardened and cut by the cryostat
(is an electrical freezing and cutting apparatus widely used to
prepare frozen sections)
Advantages:
➢ It is used in hospitals to study specimens during surgical
procedures.
➢ Allows stained sections to be prepared rapidly (within a
few minutes).
➢ It is effective in the histochemical study of enzymes since
freezing does not inactivate most enzymes.
➢ It preserves lipids.
Disadvantage:
➢ Producing thick sections.
➢ Does not give serial sections.
➢ The section may fragment into small pieces.
Preparation of tissues for electron microscope
Fixation: very small piece of tissue (not exceed 3mm)
is obtained and fixed immediately in glutaraldehyde
followed by osmium tetroxide.
Clearing: in propylene oxide
Embedding: using epoxy resin as araldite or epon
Resin is resistant to the damaging effects of electron
beam. It is much harder than paraffin so very thin
section can be obtained.
Sectioning: ultrathin sections are prepared (0.005-0.1
m) the epoxy resin block is cut by glass or diamond
knives of the ultramicrotome.
Mounting: sections are mounted in copper grids.
NB: The presence of heavy metal salts as uranyl
acetate and lead citrate enhance the contrast in EM
 Staining methods
Hematoxylin and Eosin (H&E)
The combination of H&E is the most commonly used.
Hematoxylin: is a basic dye, stains the cell nucleus and other
acidic structures blue.
Eosin: is an acidic dye , stains the cytoplasm and other basic
structures pink.
Vital staining
It is the staining of the living cells within the living animal
(i.e. in vivo). It is done by injecting a nontoxic dye into living
animals. e.g. staining of phagocytic cells with trypan blue or
Indian ink.
Supravital staining
It is the staining of living cells outside the body (i.e. in vitro).
e.g. staining of mitochondria with Janus green B.
Neutral stains
It is a mixture of acidic and basic stains.
It used to stain the blood elements in a
blood film, e.g. Leishman’s stain.

Trichrome stains
Three stains are used in combinations to
give 3 colors to different tissue
components. In addition to staining the
nuclei and cytoplasm can differentiate
collagen from smooth muscle as
Masson’s stain: it stains collagen green,
nuclei blue and cytoplasm red.
Metachromatic staining
It is the staining of certain cell
components with a color which
is different from that of the dye
used. The phenomenon of
altering the color is called
metachromasia .It is due to the
interaction between the dye and
the cell component, producing
a different compound with
a different color. e.g. the staining of mucopolysaccharide
granules of mast cells by toluidine blue which changes to
the red or violet colors.
 Histochemistry & Cytochemistry
They are methods for localizing substances in tissue
sections. These methods produce insoluble colored
or electron-dense compounds that enable the
localization of specific substances by means of light
or electron microscopy.
Examples:
➢ Demonstration of nucleic acids.
➢ Demonstration of enzymes.
➢ Demonstration of glycogen.
➢ Demonstration of Lipids.
Immunocytochemistry
Is a technique for identifying cellular or
tissue constituents (antigen) by means of
antigen antibody interactions.
MICROSCOPY
Microscopy
is the science of studying subjects through a microscope
Resolving power
is the smallest distance between two particles at which they
can be seen as separate objects.

➢ The human eye can recognize two objects if they are not
closer than 0.1 mm at a normal viewing distance of 25
cm. Any finer detail than this can be resolved by the eye
only if the object is enlarged by the use of microscopes.
➢ The maximal resolving power of light microscope is 0.2
µm.
➢ The resolving power improves as the wavelength of the
illuminating light decreases.
 TYPES OF MICROSCOPES
A) Depend on ordinary visible light
1. Conventional light microscope.
2. Polarizing microscope.
3. Phase contrast microscope.
4. Dark-field microscope.
B) Depend on other than visible light
1. Fluorescent microscope.
2. X-ray microscope.
3. Laser microscopes.
4. Electron microscope.
Conventional light microscope
It performs its function through 2 systems:
The illumination system
Light, from the illumination source (electrical lamp) is directed and
focused onto the specimen (mounted on a glass slide) through the
aperture in the stage. This light then passes through the specimen and
into the objective lens.
The optical system
Condenser: collects and focuses light,
producing a cone of light that illuminates
the object to be observed.
Objective lenses: enlarge and project the
illuminated image of the object in the
direction of the eyepiece.
Eyepiece lens: further magnifies this image.

NB: The total magnification is obtained by multiplying the magnifying

power of the objective and eyepiece lenses.
Polarizing microscope
When light passes through certain substances e.g. crystals or
body tissues as fibers it divides in a way that produce 2 light
rays from one. This called double refraction or polarization
(birefringence).
In its simplest form, the polarizing
microscope is a conventional
microscope in what a Nicol prism
(filter)is interposed in the light path
below the condenser and is called
polarizer which converts all light
passing through the instrument into
plane polarized light. A similar
second prism (filter) termed the analyzer is placed within the
parallel of the microscope above the objective lens.
➢ When the analyzer oriented so that its
polarizing direction is parallel to that of
polarizer, regular image was seen.
➢ However if the analyzer is rotated until its
axis is perpendicular to that of the polarizer,
no light can pass through the ocular lens and
the field is black.
➢ The field will remain black if an isotropic or
singly refractive object is placed on the stage.
➢ A birefringent object however will appear
light upon a dark background when examined
in this manner.
Phase contrast microscope
The principle is based upon the fact
that the light changes its speed and
direction when passing through
cellular and extra-cellular
Substances with different refractive
indices.
These changes cause the structure
to appear lighter or darker related
to each other by using a phase contrast system (phase plate)
that convert the difference in speed into difference in intensity.
Dark-field microscope
For darkfield illumination,
the cone of light illuminating
the specimen must not enter
the microscope objective lens.
Only light that is scattered by
the specimen is detected by
the objective lens. This is
achieved by use special
dark-field substage condensers called cardioid condensers.
Fluorescence microscopy
When exited by radiation
of short wavelengths
(non visible), some substances
will emit light of a longer
wavelength (visible). This
phenomenon is called
fluorescence. The usual light
source for excitation radiation
is ultraviolet (UV).
The fluorescent images
produced can be recorded on
either black and white or color
film.
X-ray microscope
X-rays have a shorter wave length than visible or ultraviolet
light so, it has a higher resolving power (Resolution is not
particularly high and with the instruments available is still far
from theoretical limits).
The specimen placed upon a photpgraphic emulsion and
exposed to soft X-irradiation. The small X-ray picture
obtained is subsequently magnified optically.
Laser microscopes
- Confocal microscope
- Multiphoton (Femto-second)
microscope
Electron Microscopes
Transmission (TEM) and Scanning (SEM)
➢ In TEM the specimens are examined by passing the
electron beam through them, revealing more information of
the internal structure of specimens. The resolution is very
high, reaching up to about 0.2 nm and it can magnify up to
500.000 times.
➢ In the SEM the specimen is scanned with a focused beam
of electrons which produce "secondary" electrons as the
beam hits the specimen. These are detected and converted
into an image on a television screen. The resolution power
of SEM is 10 nm.