HISTOLOGY &
HISTOCHEMISTRY
By
Professor Abdel-Majeed Safer
Professor Abdel-Majeed Safer Histology &
Histochemistry
1
Chapter 1
Professor Abdel-Majeed Safer Histology &
Histochemistry
2
What is HISTOLOGY ?
Histology is the study of the tissue of the body and how these tissues are
arranged to constitute organs. Histology involves all aspects of tissue
biology with the focus on how cells’ structure and arrangement optimize
functions specific to each organs.
Histochemistry & Cytochemistry is the study of the chemical
composition of the tissue and cell of the body. Or precisely, the
microscopic study of the chemical characteristics of tissues and cells,
through the use of substances (dyes etc.) producing identifying
chemical reactions.
Professor Abdel-Majeed Safer Histology
&Histochemistry
3
HISTOLOGY AND
HOW IT IS STUDIED
Professor Abdel-Majeed Safer Histology &
Histochemistry
4
HISTOLOGY and
Its Method of Study
Preparation of Tissues for Study
Fixation
Embedding & Sectioning
Light Microscopy
Bright field microscopy
Fluorescence microscopy
Phase-contrast microscopy
Interference microscopy
Confocal microscopy
Polarizing microscopy
Electron Microscopy
Transmission Electron Microscopy TEM
Scanning Electron Microscopy SEM
Professor Abdel-Majeed Safer Histology &
Histochemistry
5
HISTOLOGY and
Its Method of Study
Autoradiography
Cell & Tissue culture
Histochemistry & Cytochemistry
Detection Methods using specific Interactions between molecules
Immunohistochemistry
Hybridization Techniques
Problems in the study of Tissue Sections
Professor Abdel-Majeed Safer Histology &
Histochemistry
6
HISTOLOGY and
Its Method of Study
Histology is the study of the tissue of the body and how these tissues are
arranged to constitute organs. Histology involves all aspects of tissue
biology with the focus on how cells’ structure and arrangement optimize
functions specific to each organs.
Tissues are made of two interacting components: cells and
extracellular matrix. The ECM consists of many kinds of molecules, such as
collagen fibrils.
there is, thus, an intense interaction between cells and matrix, with
many components of the matrix .
Most organs are formed by an orderly combination of several
tissues, except the CNS, which is formed almost solely by nervous tissue.
The precise combination of these tissues allows the functioning of each
organ and of the organism as a whole.
The small size of cells and matrix components makes histology
Professor Abdel-Majeed Safer Histology &
depedent on the use of microscope.
Histochemistry
7
HISTOLOGY and
Its Method of Study
Preparation of Tissues for Study
Fixation see Fig 1
Is to avoid tissue digestion by enzymes within the cells (autolysis) or by
bateria and to preserve structure and molecular composition.
Chemical methods - the tissues are immersed in solution of
stabilizing or cross-linking agents called fixatives. or less frequently
Physical methods (deep freeze).
In routine LM, formalin ; a buffered isotonic solution of 37% formaldehyde.
Formaldehyde and glutaraldehyde are commonly used and known to react
with amine groups NH2- of tissue proteins. Glutaraldehyde, is reinforced
by virtue of being a dialdehyde which croos-links proteins.
In EM, where high resolution is require, a buffered glutaraldehyde followed
by a second fixation in buffered OsO4 for fine structural studies. OsO4 is
used to preserve and stain lipids and proteins.
Professor Abdel-Majeed Safer Histology &
Histochemistry
8
Tissue Fixation and Processing for Light Microscopy
HISTOLOGY and
Its Method of Study
Dehydration
To extract the water from the tissue, through graded series of alcohol (70% 100% ethanol).
Clearing
Ethanol is then replaced by a clearing agent that is miscible with both alcohol
and the embedding medium. E.g. xylene
Paraffin
Melted paraffin 52- 60oC in an oven.
Embedding and Sectioning
Tissues are embedded in a solid medium to facilitate sectioning. To obtain thin
sections with the microtomes, tissues must be infiltrated after fixation with
embedding solutions that impart a rigid consistency to the tissue. Such as
paraffin and plastic resins.
Professor Abdel-Majeed Safer Histology &
Histochemistry
10
HISTOLOGY and
Its Method of Study
Tissues to be embedded in plastic resin are also dehydrated in ethanol, cleared
and embedded in plastic solvent, which are then hardened by means of crosslinking polymerizes.
Sectioning and Microtomy see Fig.2
The hard blocks containing the tissues are placed in microtome. Cut 1-10 um
(ium= 1/1000 mm.
1nm = 0.001um = 10-6 mm. 1A = 0.1nm 10-4um.
Sections are are floated on water and then transferred to glass slides to be
stained .
Frozen sections are also performed by rapid freezing (physical , not chemical
fixation), using a freezing mictotome or cryostat. This is mainly for rapid
invistigation in hospitals and for histochemical studies, as enzymes and small
molecules and lipids are very sensitive.
Professor Abdel-Majeed Safer Histology &
Histochemistry
11
Ultramicrotome
Columnar epithelium lining the small intestine
• Micrograph stained with hematoxylin and
eosin (H&E).
Tissue stained with hematoxylin
and eosin (H&E).
Tissue stained with the periodic acidSchiff (PAS) reaction for glycoproteins.
Its Method of Study
Histological Stains
For routine histological work it is customary to use two dyes, one stains
certain components, and the other stains different ones (counterstain).
Toluidine blue
Basophilic and Acidophilic Staining
Basophilic substance is the one that takes up basic stain, acidophilic is the
one that takes up an acidic stain.
Hematoxylin is a basic stain or cationic stain, while Eosin is an acidic stain or
anionic stain.
What do the color seen in Hematoxylin sections
indicate?
Ordinary stains such H&E provide only general information about the
chemical composition of the components that they color.
Professor Abdel-Majeed Safer Histology &
Histochemistry
14
Light Microscopy
Bright field microscopy
Stained slides are examined by means of ordinary light that passes through the
specimen.
See Fig.1-3. for details.
Flourescence Microscopy
When certain substances are irradiated by light of a proper wavelength, they emit
light with a longer wavelength. This phenomenon is called Flourescence. In
Flourescence Microscopy, tissue sections are irradiated with ultraviolet UV light
and the emission is in the visible portion of the spectrum. The fluorecent
substances appear brilliant on a dark background.
Thus, the microscope has a strong UV light source and special filters that select
rays of different wavelengths by the substances.
Fluorescent compounds with affinity for specific cell macromolecules may be
used as Fluorescent stains. E.g. Acridine Orange (bind to DNA and RNA). See
Fig.1-4a
e.g..Hoechst stain andProfessor
DAP1
specifically bind to DNA giving a blue
Abdel-Majeed Safer Histology &
15
Histochemistry
flourecence under UV .
Bright Field Microscope
Fluorescent Microscopy
Kidney cells
acridine orange
Culture of kidney cells
DAPI (4’,6-diamino-2-phenylindole)
Phase-contrast microscopy and Interference microscopy
Unstaned biological specimens are usually transparent and difficult to view in
detail, beacause all parts of the specimen have the same optical density. Phasecontrast microscopy uses a lense system that produces visible images from
transparent objects (Fig. 1-5).
Principle:
Light changes its speed when passing through cellular and extracellular
structures with different refractive indices. Thses changes are used by Phasecontrast system to cause the strctures to appear lighter or darker in relation ro
each other. So, living cells and tissue cultures can be observed ( no need for
fixation or staining). Phase-contrast microscope is essential tool in tissue culture
labs.
also, differential Interference microscopy, which produces images with a more
apparent 3-D aspect than routine Phase-contrast microscopy (Fig. 1-5).
Professor Abdel-Majeed Safer Histology &
Histochemistry
18
Bright field microscopy
Interference microscopy
Phase contrast microscopy
Confocal microscopy
With a regular bright-field microscopy the beam of light is relatively large and
fills the specimen.
Stray light reduces contrast within the image; confocal microscopy avoids stray
light and achieves greater resolution by using:
1. A small point of high intensity light providing by a laser .
2. A plate with a pinhole aperture infront of the image detector.
the point light source, the focal point of the lens, and the detector’s pinpoint
aperture are all optically conjugated or alighned to each other in the focal plane
(CONFOCAL) and unfocused light does not pass through the pinhole. This
greatly improves resolution of the object in focus and allows the localization of
specimen components with much greater precision than with bright-filed
microscope. Fig. 1-6.
Professor Abdel-Majeed Safer Histology &
Histochemistry
20
Principle of confocal microscopy
• Figure 1–6.
• Principle of confocal microscopy. Although a very small spot
of light originating from one plane of the section crosses
the pinhole and reaches the detector, rays originating from
other planes are blocked by the blind. Thus, only one very
thin plane of the specimen is focused at a time. The
diagram shows the practical arrangement of a confocal
microscope. Light from a laser source hits the specimen
and is reflected. A beam splitter directs the reflected light
to a pinhole and a detector. Light from components of the
specimen that are above or below the focused plane is
blocked by the blind. The laser scans the specimen so that a
larger area of the specimen can be observed.
Polarizing microscopy
Allows the recognitio, in of structures made of highly organized molecules.
When normal light passes through a polarizing filter, it exists vibrating in only
one direction.
If a second filter is placed in the microscope above the first one, with its main
axis perpendicular to the first filter, no light passes through. If, tissue structures
containing oriented macromolecules are located between the two polarizing
filters, their repetitive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark background Fig.17.
So, crystalline substances or substances containing highly oriented molecules
such as cellulose, collagen, micromolecules and microfilaments have the
property of birefringes.
Professor Abdel-Majeed Safer Histology &
Histochemistry
23
• bright-field and
polarizing microscopy
Bright-field microscopy
Plarizing microscopy
• Figure 1–7.
• Tissue appearance with bright-field and polarizing microscopy.
Polarizing light microscopy produces an image only of material having
repetitive, periodic macromolecular structure; features without such
structure are not seen. Shown here is a piece of thin mesentery that
was stained with red picrosirius, orcein, and hematoxylin, and was
then placed directly on a slide and observed by bright-field and
polarizing microscopy. (a): Under routine bright-field microscopy
collagen fibers appear red, along with thin dark elastic fibers and cell
nuclei. (b): Under polarizing light microscopy, only collagen fibers are
visible and these exhibit intense birefringence and appear bright red
or yellow; elastic fibers and nuclei lack oriented macromolecular
structure and are not visible.
Electron microscopy
TEM and SEM are based on the interaction of electrons and tissue components.
The wavelength in electron beam is much shorter than of light. Allowing a
thousand-fold increase in resolution.
Transmission Electron Microscopy TEM
with a resolution of 0.3nm (Fig.1-8a). Which allows magnification of up to
800,000 times to be viewed with details. (1nm = 0.001um = 10-6 mm. 1A =
0.1nm 10-4um).
Principle:
A beam of electrons can be deflected by electromagnitic fields. E beam is
produced by a cathode and passes down the column in a vacuum. The beam can
be focused by means of electric coils (electromagnetic lenses).
First lens is Condenser lens, which focus beam on the specimen section.
Second lens is Objective lens, when electrons pass the specimen without
interacting with it, it reaches the Objective lens. This form a focused, magnified
image, and reach a viewing screen.
The image is white and black and gray. (bright areas indicate electron passing
through, darker areas – electron dense- indicate electrons were absorbed or
Professor Abdel-Majeed Safer Histology &
26
deflected).
Histochemistry
Schematic view of a transmission electron microscope (TEM)
TEM requires very thin sections (40-90 nm), so plastic embedding blocks are
cut with glass or diamond knives. Sections are collected on small copper grids
and transferred into the microscope to be analyzed.
Freeze-fractyre, cryofracture, freeze etched combined with TEM have been
useful for examining membrane structure.
Scanning Electron Microscopy SEM
It permits 3-D views of the surfaces of cells, tissues, and organs.
Beam does not pass through the specimens (Fig.1-8a). The sample is dried and
coated with a very thin layer of metal atoms through which electrons do not
pass readily. When the beam is scanned from point to point across the
specimen it interact with the metal atoms and produces reflected electrons or
secondary electrons emitted from the metal. These are captured by a detedtor
and the resulting signal is processed to produce a black-and-white image on a
monitor (Fig.1-8b).
Professor Abdel-Majeed Safer Histology &
Histochemistry
28
Schematic view of a scanning electron microscope (TEM)
Autoradiography
Is a method of localizing newly synthesized macromolecules (DNA, RNA,
protein, glycoproteins, and polysaccharides) in cells or in tissue sections.
Radioactively labled metabolites (nucleotides, amino acids) incorporated into
the macromolecules emit weak radiation that is restricted to the cellular
regions where the molecules are located. Radiolabel cells or mounted tissue
sections are coated in a darkroom with photographic emulsion containing
silver bromide crystals, which acts as microdetectors of this radiation in the
same way that they respond to light in common photographic film. After some
times, the slides are developed photographically. The silver bromide crystals
reduced by the radiation are reduced to small black grains of metallic silver,
indicating location of radiolabled macromolecules in the tissue.
This tech can be used for both LM and EM (Fig.1-9).
Practically, it is possible to know which cells is a tissue produce more protein
and which cell produce less, because the number of silver grains formed over
the cells is proportional to the intensity of protein synthesis.
Professor Abdel-Majeed Safer Histology &
Histochemistry
30
Autoradiographs are tissue preparations in which particles called silver grains indicate the regions
of cells in which specific macromolecules were synthesized just prior to fixation
Black “silver grains” are visible over regions with secretory granules and the duct
indicating glycoprotein locations. X1500.
Autoradiographs are tissue preparations in which particles called silver grains indicate the regions
of cells in which specific macromolecules were synthesized just prior to fixation
The same tissue prepared for TEM autoradiography shows silver grains with a coiled
or amorphous appearance again localized mainly over the granules (G) and in the
gland lumen (L). X7500.
Cell & Tissue Culture
Live cells and tissues can be maintained and studied outside the body. cell culture has been
very useful in isolating the effects of single molecules on specific types of cells. It also
allows the direct observation of the behavior of living cells under the phase contrast
microscope. Many experiments that cannot be performed in the living animal can be
accomplished in vitro.
The cells and tissues are grown in complex solutions of known composition (salts, amino
acids, vitamins) to which serum components or specific growth factors are added.
1. Cells must be dispersed mechanically or enzymatically.
2. The cells are then cultivated in a clear dish to which they adhere, as single-layer cellss
(Fig.1-5). This is called primary cell culture, which makes cell line. Normal or
pathologic tissue have been maintained in vitro ever since they have immortalized and
now constitute a permanent cell line.
Cell culture has been widely used for study of the metabolism of normal and cancerous
cells and for the development of new drugs.
Professor Abdel-Majeed Safer Histology &
Histochemistry
33
Histochemistry & Cytochemistry
They are methods for localizing cellular structures in tissues using enzyme
activity present in these structures. Usually it is applied to unfixed or mildly
fixed tissue, sectioned on a cryostat or freezing microtome to avoid adverse
effects of heat, paraffin and chemicals on enzymatic activity.
1. Tissue sections are immersed in a solution containing the substrate of the
enzyme to be localized.
2. The enzyme is allowed to work on the substrate.
3. The section is put in contact with the marker compound.
4. This compound reacts with a molecule produced by enzymatic action on the
substrate.
5. The final reaction product (insoluble and visible in LM or EM) only if it is
colored of electron- dense, precipitate over the site that contains the enzyme.
e.g. Phosphatases,
split the bond between a PO4 group and an alcohol residue of
phosphorylated molecules. The visible insoluble
reaction product of
phosphateses is lead phosphate or lead sulphide. At alkaline pH and cid
phosphatese. (Fig.1-10).
Professor Abdel-Majeed Safer Histology &
Histochemistry
34
Dehydrogenases
Remove H from one substrate and transfer it to another. Mitochondria can be
specifically identified by this method, dehydrogenases are key enzymes in the
citric acid cycle.
Peroxidases
Present in several types of cells, promotes the oxidation of certain substates with
the transfer of H ions to H2O2, forming H2O.
Fixed sections are incubated in a solution of H2O2 and 3,3”-diaminoazobenzinedine DAB. DAB is oxidized in the presence of peroxidaze, resulting
in an insoluble , brown, electron-dense ppt w permit the localization of
peroxidase activity by LM and EM.
Professor Abdel-Majeed Safer Histology &
Histochemistry
35
Problems in the study of Tissue Sections (Fig.1-15)
Artifacts
Kind of flaw that is due to faulty technique:
Postmortem degeneration
Shrinkage
Precipitants
Wrinkles and Folds
Nicks in the microtome knife
Rough Handling
Professor Abdel-Majeed Safer Histology &
Histochemistry
36
END of Chapter 1
Professor Abdel-Majeed Safer Histology &
Histochemistry
37