Lecture 1

INTRODUCTION TO HISTOLOGY
& MICROTECHNIQUES
 Histology

 Histology is the microscopic study of the

various normal tissues of the body; how
these tissues appear, how they interact with
each other and how they are arranged to
constitute an organ

 Features of tissues cant be seen by the eye.

Therefore, their study required the use of a
magnifying tool– the Microscope
 Cells are the smallest units of life, and are named
according to their function. Cells are arranged in
organized groups called tissues that carry out specific
functions.
 Tissues are organized together to form organs, which
carry out more complex functions.
 Organs work together in groups to form systems, which
in turn carry out higher order functions. Systems make up
the individual organism.
* Tissues: Group of similar cells which have:
- Common embryonic origin.
- Common function.
• Histology: The microscopic study of structure of normal
tissue
• Pathology: The study of diseased tissue and conditions
and processes of a disease
- Tissue-anything surgically removed from the body of an
organism must be processed through the histological
procedure.
 Origin of Tissues
 Primary germ layers within the embryo
- Endoderm
- Mesoderm
- Ectoderm
 Tissue derivation:
- Epithelium from all three germ layers
- Connective tissue and muscles from mesoderm
- Nerve tissue from ectoderm
Tissues are made of two interacting components:
1- Cells
2- Extracellular matrix
- It consists of many kinds of molecules, most of which are
highly organized and form complex structures, such as
collagen fibrils and basement membranes.
- Extracellular matrix produced by the cell
 Component of Tissues

Cells
+
Extracellular Matrix
(Fibers, organic and inorganic molecules and
water
The main functions of extracellular matrix
1- Furnish mechanical support for the cells
2- Transport nutrients to the cells
3- Carry away catabolites and secretory products.
- The small size of cells and matrix components
makes histology dependent on the use of
microscopes.
Preparation of tissues for study
 The most common procedure used in the study of tissues
is the preparation of histological sections or tissue slices
that can be studied with the aid of the light microscopy.

 Under the light microscope, tissues are examined via a

light beam that is transmitted through the tissue.
 Because tissues and organs are usually too thick for
light to pass through them, they must be sectioned to
obtain thin, translucent sections and then attached to
glass slides before they can be examined.
 Ideal histological technique should
result in minimum deviation of the
tissue from the living state, but still
permit maximum resolution of the
components.
 Section – a thin slice of tissue laid flat
on glass slide
Routine production of Histological Slides:

• Paraffin histological microtechinque:

• It involves 15 basic steps in the
preparation of histological slide. All
procedure can be carried out manually
although several procedures can be now be
automated.
1. Selecting and obtaining tissue
A.Sacrifice an animal and dissect out small
pieces of tissue as soon as possible after the
death of the animal to be placed in a
fixative
B.Biopsy and autopsy specimens of animal
(human) tissue
1.Biopsy specimens
2.Autopsy specimens
2. Fixation
Post-mortem changes: Degenerative stages of the
cells and tissues of the body rendering them subject
to significant alteration when used for microscopic
study.If a permanent section is desired, tissues must
be fixed or as soon as possible after removal from
the animal's or human body for the following
reasons:
- To preserve the tissue as close as possible to the living state and
to preserve the structure and molecular composition
- To prevent degeneration and avoid tissue digestion by enzymes
present within the cells (autolysis) or by bacteria
How fixatives preserve tissues?
 Denature proteins by coagulation
 Crosslink macromolecules by forming additive
compounds.
 Disable intrinsic biomolecules (especially
proteolytic enzymes).
 Protect from extrinsic damage (microorgnisms).
 Increase cellular mechanical strength and stability.
 Some common chemical reagents used in
combination to form fixatives:
- Acetic acid - Acetone
- Ethyl alchol - Formaldehyde (formalin)
- Picric acid - Mercuric chloride
- Potassium dichromate - Trichloroacetic acid

- The most famous fixative is Formalin (an

aqueous solution of formaldehyde)
3. Washing
- To prevent over
fixation

4. Dehydration
- Graded series of ethanol
solutions
5. Clearing
 Removal of alcohol
 Paraffin is not soluble in alcohol
 The alcohol is replaced by a substance in
which paraffin is soluble, such as xylene,
toluene or benzene. These substances are
called clearing agents
 Clearing agents also increases hardening of
the tissue and makes the tissue translucent.
Dehydration & clearing
- The water is first extracted from the fragments to be
embedded by bathing them successively in a graded
series of mixtures of ethanol and water, usually from
70% to 100% ethanol (dehydration).
- The ethanol is then replaced with a solvent miscible
with both alcohol and the embedding medium. As the
tissues are infiltrated with this solvent, they generally
become transparent (clearing).
6. Infiltration
 In order for tissue to be sectioned into thin sections,
it should be supported by permeating it with a
medium that will give it a proper consistency for
sectioning.
 Paraffin wax is commonly used as an infiltration
medium
 After clearing, the tissue is placed in several
changes of melted paraffin for a specific period of
time
 Infiltration occurs under heat
7. Embedding
 The paraffin infiltrated tissue is placed
into a mold into which liquid paraffin
is poured
 Then paraffin block containing the
tissue is then removed from the mold
 Result is a block with proper
consistency for sectioning`
 Embedding in solid medium will facilitate
sectioning.
 Embedding materials include paraffin and plastic
resins. Paraffin is used routinely for light
microscopy; resins are used for both light and
electron microscopy.
Preparing Paraffin Blocks
Preparing Paraffin Blocks
Removing Paraffin Blocks from Molds
8. Microtomy or sectioning
The solid paraffin block containing the
tissue is sliced into very thin sections on
an instrument called the microtome
Sectioning
 The thick tissue do not allow light to pass through them.
Therefore they must be cut into thin slices. The hard
blocks containing the tissues are then placed in an
instrument called a microtome. The sections are floated
on water and then transferred to glass slides.
Rotary Microtome with Mounted Tissue Block
Serial Sectioning and Capturing of Tissue Ribbons
9. Attaching or Mounting sections on
slides and spreading the Sections

 Warming Plate Method

 Flotation Method
Mounting Tissues:
Single or multiple sections
 Microscope Slides on Slide Warmer –
Stretching and Adhesion of Tissue Ribbons
10. Staining
Theoretical Aspects for Staining:
 To increase the contrast among the tissue
components
 Hematoxylin and eosin (H & E)
 Triple Stain (Masson’s, Mallory’s, Pollak’s
 Alcian blue
 Periodic acid-Schiff (PAS)
Why you need to do Staining?

 To be studied microscopically sections must typically be

stained or dyed because most tissues are colorless.

 The dyes stain tissue components more or less

selectively.

 Most of these dyes behave like acidic or basic

compounds and have a tendency to form electrostatic
(salt) linkages with ionizable radicals of the tissues.
The main principal of staining

 Acid + Base ----------------- Salt and water

 Components of cells with a net negative charge react with

basic dyes (which are usually blue). These components are
called Basophilic. Example: DNA, RNA,
Glycosaminoglycan and others.

 Components of cells with a net positive charge react with

acidic dyes (which are usually red). These are called
Acidophilic Examples: proteins (as in collagen fibers and
mitochondria) and others
 Acid dyes (eg, orange G, eosin, acid fuchsin) stain the
acidophilic components of tissues such as
mitochondria, secretory granules, and collagen.

Of all dyes, the simple combination of hematoxylin and

eosin (H&E) is used most commonly. Hematoxylin stains
DNA of the cell nucleus and other acidic structures (such as
RNA-rich portions of the cytoplasm and the matrix of
cartilage) blue. In contrast, eosin stains other cytoplasmic
components and collagen pink.
Examples of basic dyes are toluidine blue and methylene
blue. Hematoxylin behaves like a basic dye, that is, it
stains the basophilic tissue components. The main tissue
components that ionize and react with basic dyes do so
because of acids in their composition (nucleic acids,
glycosaminoglycans, and acid glycoproteins).
Etching Microscope Slides with Diamond Pen
11. Dehydration and Clearing of Stained
Tissue sections
12. Mounting
Permount mounting medium is put on the
section and a cover slip is mounted over the
section.
13, 14, 15 Cleaning, Labelling and
Cataloguing the Slide in the Slide Box
Register
Microscopy

The specimen on the microscopic slide is a thin section of

the fixed tissue or organ. The section is stained by one or
more dyes. Without staining the section would be nearly
invisible with the microscope.

Components of the specimen generally stain selectively

and, on this basis, various regions of the specimen may be
differentiated from each other.
1. Bright-Field Microscopy

With the bright-field microscope, widely used by students

of histology, stained preparations are examined by means of
ordinary light that passes through the specimen.
The microscope is composed of:

1- Mechanical components

2- The optical components consist of three systems of

lenses.

- The condenser collects and focuses light

- The objective lenses enlarge and project the illuminated

image of the object in the direction of the eyepiece.
The total magnification is obtained by multiplying the
magnifying power of the objective and ocular lenses.

The critical factor in obtaining a crisp, detailed image

with a light microscope is its resolving power, defined as
the smallest distance between two particles at which they
can be seen as separate objects
The Light Microscope

1) Bright-Field Microscope

This image showing various

parts of a Bright-field light
microscope.
2. Fluorescence Microscopy

 In fluorescence microscopy, tissue sections are usually

irradiated with ultraviolet (UV) light and the emission is
in the visible portion of the spectrum.

 Fluorescent compounds with affinity for specific cell

macromolecules may be used as fluorescent stains.
• Example of fluorescent
substances:

1) Diaminophenylindole
(DAPI) binds to DNA
 Blue
2) Phalloidin binds to
actin filaments 
Red
3) Tetracyclin bind to
newly formed bone
 Green
3. The Electron Microscope

 Uses a beam of electrons instead of light photons.

 It gives a much higher resolution than the light

microscope (resolving power around 3nm).

 It’s either a Transmission Electron Microscope

(TEM) or a Scanning Electron Microscope (SEM).
1) TEM
 The beam of electrons
interact differently with
the different parts of the
section.

 Some are deflected,

some pass through and
some are reflected.

 Electrons passing
through are detected to
produce an image.
Fig.4: Schematic drawing of TEM.
Fig.5: A TEM image of the cell membrane.
2) SEM
 The specimen is first
coated with a metal that
reflects electrons.

 The electron beam

scans the specimen
from end to end.

 The reflected electrons

are captured to produce
a pseudo-3D image of
the coated surface. Fig.6: Schematic drawing of SEM.
A SEM image of an ant.
Other methods of study
1) Autoradigraphy

 A radioactive substance (ions, amino acids,

sugars, etc…) is added to the tissue.

 The radioactive substance is taken up by the

tissue  The tissue will give off radiation.

 A film containing Silver Bromide is used to

detect the radiation.

 Areas of tissue with the radioactive substance

appear black in the film.
 Mouse lymph node injected with radioactive
thymidine. Note how it’s concentrated at one area of
the node
2) Histochemistry

 Chemical reactions occur throughout the body.

These reactions produce invisible, soluble
substances.

 In histochemistry, certain Markers are added to the

tissue that will convert the reaction products into
visible, insoluble substances that can be detected.
Lysosomes under electron microscope. A histochemical method
was used to localize areas with high acid phosphatase activity.
3) Immunocytochemistry

 Tagged antibodies
specific against a certain
part of a tissue are used.

 These bind to the tissue

causing their staining.

Adenocarcinoma of the intestine stained using an antibody

against a specific substance produced by the tumor. Cancer
cells are stained brown.