Professor Yunlian

Meng
 Welcome to the classes of Histology and
Embryology. Actually the histology and embryology
are two courses. We′ll talk about histology firstly.

What is histology?
How to study it ?
Histology & Its Study
Histology is a branch of Anatomy.

Anatomy:
Gross anatomy
Microscopic anatomy

Histology is a science which study the microstructure

and the relationship between the structure and function
of human being.
Exactly histology is the study of cells, tissues and
organs as seen with a microscope. The human body
is made up of units called cells. Most of the cells
have certain features in common. Aggregations of
cells constitute tissues. Between the cells there are
some intercellular substances . Organs are made
up of combination of various kinds of tissue.
Cells
The cell is the functional unit of living organisms. There
are many kinds of cells in human body.

Tissues
Groups of cells and intercellular substances make up the
body tissues. There are four main types of tissue:
epithelium, connective tissue, muscle tissue and nervous
tissue.
Organs
Several different kinds of tissue are further organised
in particular ways to form organs(such as heart,
stomach or liver) .

System
Several organs with related functions together form
systems. For example, the kidneys, ureters, urinary
bladder and urethra constitute the urinary system.

Thus the human body may be examined at three

structural levels: cells, tissues and organs.
How to study histology?
Histology studies the microstructures. So, we should
have the aid of microscope to study. Several types of
microscopes are available. According to the light
source used, microscopes can be basally classified as:
Light microscope(abbreviate LM)
Electron microscope(abbreviate EM)
Light microscope
With the LM, stained specimens are usually examined
by means of light that passes through the specimens,
and the magnification of 1000 times can be achieved.

The maximal resolving power of LM is approximately

0.2um. That means that the smallest distances
between two particles at which they can be seen as
separate objects is 0.2um.
Um: micron
The EM uses an electron beam instead of light; and
electromagnetic fields in place of lenses. With the
EM magnification of 10 0000(hundred thousand)
times can be achieved. The structure of a cell or
tissue as seen with the EM is referred to as
ultrastructure.

The maximal resolving power is 0.2 nm under the

EM.
There are two kinds of EM:

The transmission electron microscope(abbreviate

TEM) and the scanning electron
microscope(SEM).

SEM can observe the surface appearances of a cell

and obtain three dimensional images.

The figures of TEM & SEM.ppt

Traditional Histological Methods
Preparation of tissue for LM

Before a tissue can be observed with a light

microscope, it must be made into a section. The
routine histological preparation for light microscope
examination is a paraffin sections stained with
haematoxylin and eosin. The procedures for making
a section are as follows.
Tissue collecting Fresh, small tissue blocks are get
from a organ. The size of a tissue block should be less
than 1.2cm×0.5cm×0.2cm. （ centimeter ）

Fixation Then put the tissue blocks into a fixative.

The process of fixation preserves a tissue by
denaturing its proteins. Numerous fixatives are
known, the most commonly used being 4%
formaldehyde (4% formaldehyde is also called
formalin).
Dehydration Then use ethyl alcohol to get rid of
water from the tissue blocks.

Clearing Then use xylene to get rid of alcohol.

*alcohol and xylene are embedding mediums

Embedding Before a tissue can be sectioned it has to

be given a firm consistency. One way of doing this is
embedding a tissue in paraffin wax .
The procedure of
embedding

Heat the paraffin

firstly, make it melt,
then spill it into a
box.

Put tissue block into

melted paraffin, allow
paraffin harden, the
tissue block is
embedded in.
Sectioning Usually sections 3 ～ 8μm thick are cut
with a microtome and mounted on glass slides. When
the sections are dry, they are ready for staining.

HE staining The sections are stained with

haematoxylin-eosin.
Haematoxylin as a basic dye binds to the acidic
components of cells and tissues, which then show a
blue colour. Such components, e.g. nuclei, are said to
be basophilic. HE stain.ppt

μm: micron
Eosin as an acidic dye binds to basic constituents and
give a pink colour. Such constituents, e.g.cytoplasm
and most of other components , are termed
acidophilic.

Numerous other staining methods are available for

demonstrating specific tissue elements.
In addition to the above routine paraffin sections, frozen
sections of fixed or unfixed tissues may also be cut with
a freezing microtome. Frozen sections can preserve
some chemical components and enzymes better.
Preparation of frozen sections is a fastest method of
examining a tissue. The technique allows the
examination of pieces of tissue removed by a surgeon,
while the patient is still on the operating table, making it
possible for the surgeon to plan his operation keeping in
mind the nature of the disease(a cancer or a benign
tumor).
Except sections we can also get a specimen as follows:

Smear preparation are sometimes used for examination of

blood or other secretions.

Spread preparations are used for examination of

connective tissue.

Grinding preparations are used for examination of bone

or tooth.
Histochemistry
Histochemistry or cytochemistry combine
histological methods with chemical and biochemical
methods and reveal the chemical composition of
tissues and cells in situ. This permits a precise
interpretation of the chemistry of cells and tissues in
relation to their structure. Many substances, such as
proteins, amino acids, nucleic acid, lipids,
carbohydrates, ions, catecholamines and enzymes,
can be detected in situ by histochemical and
cytochemical techniques.
PAS reaction

The periodic acid-Schiffs reagent (PAS) reaction is

the histochemical technique most extensively used to
localize polysaccharides, such as glycogen and
glycoprotein, in tissues and cells.
Immunocytochemistry
antigen-antibody reaction.

Specific molecules(antigen) within cells can be

identified by treating tissue sections with antibodies
specific to the molecules.
The technique enables chemical substances to be
localized in cells with great precision. Such
studies have greatly enhanced our knowledge of
chemical transformations taking place within
cells.
immunocytochemistry.ppt
In situ hybridization
In situ hybridization(abbreviate ISH), also termed in
situ hybridization histochemistry(ISHH), is a
method for detection of specific RNA or DNA
sequences directly in cells or tissue sections.
Observation of living tissue
Vital staining
Dyes used for vital staining are usually neither toxic to
living cells nor destroyed by them. When the dyes are
injected into living animals certain cells or structures
can be stained by the dyes or can be visualized by their
selective absorption of colouring substance. For
example, trypan blue injected into living animals is
removed from the blood by macrophages and
accumulated in the cytoplasm. This renders the
cytoplasm conspicuous.
Vital staining of trypan blue. Macrophages engulf trypan
blue granules in their cytoplasm.
Cell and tissue culture

Cell and tissue culture is a technique for the study of

living cells. Isolated cells or fragments of tissues are
cultured in a sterilized culture medium at the
appropriate temperature. Since the medium
contains essential nutrients, such as amino acids,
vitamins, et cetera, cells and tissues grow in vitro.
This technique is quite useful in detecting the effect
of various reagents on living cells.
Introduction to the cell
The cell is the functional unit of living organisms. The
simplest organisms such as bacteria and algae consist
of a single cell. More complex organisms consist of
many cells held together by connections between cells,
and between cells and extracellular matrix. The cells
of multicellular organisms, such as humans, show a
great variety of functional and morphological
specializations which have developed during the
process of evolution.
A cell includes three parts:
Cell membrane or plasma membrane
Cytoplasm
Nucleus
1. Cell membrane
The cell membrane separates cytoplasm of the cell
from surrounding structure.
(1) The structure of the cell membrane
you could not see the cell membrane clearly under
LM.
EM: average 7.5 nanometer( nm ) thick
It consists of two densely stained layers separated
by a lighter zone, thus creating a trilaminar
appearance.
Chemical components of cell membrane
Cell membranes are made up of lipids, proteins and
carbohydrates.

Sugar chain of glycoprotein

A. lipids
(predominantly phospholipids)
The trilaminar structure of membrane is produced by
the arrangement of lipid molecules that constitute the
basic framework of the membrane.
CELL MEMBRANE-1.ppt
Each phospholipid molecule
consists of an enlarged head
and two tails.
Head end: polar end
The head end is soluble
in water and is said to be
hydrophilic.
The tail end : non-polar end
The tail end is insoluble in
water and is said to be
hydrophobic.
B. proteins
In addition to molecules of lipids the cell membrane
contains several proteins. Most of them are embeded
within the thickness of the membrane and partly project
on one of its surfaces which are called integral proteins.
Some integral proteins occupy the entire thickness of the
membrane and may project out of both its surfaces, which
are called transmembrane proteins. However some
proteins looserly associate with membrane surfaces which
are called peripheral proteins.
cell membrane.ppt
The functions of the proteins in the membrane are as
follows:
a. They may be structural proteins that form an
essential part of the structure of the membrane.
b. Some proteins play a vital role in transport across
the membrane and act as pumps. Ions get attached to
the protein on one surface and move with the protein
to the other surface.
c. Some proteins are so shaped that they form passive
channels through which substances can diffuse through
the membrane. However these channels can be closed
by a change in the shape of the protein.
d. Other proteins act as recepters for specific hormones
or neurotransmitters.
e. Some proteins act as enzymes.
C. Carbohydrates

The carbohydrates are attached either to the proteins

forming glycoproteins, or to the lipids forming
glycolipids. The carbohydrate layer is specially well
developed on the external surface of the plasma
membrane forming the cell boundary. This layer is
referred to as the cell coat or glycocalyx.

CELL MEMBRANE-1.ppt cell membrane.ppt

The functions of the glycocalyx are as follows:
a. The glycocalyx as a special adhesion enable the cell to
adhere to specific types of cells, or to specific
extracellular molecules.
b. The glycocalyx contains antigens .
c. Most molecules in the glycocalyx are negatively
charged causing adjoining cells to repel one another.
However some molecules that are positively charged
adhere to negatively charged molecules of adjoining cells,
holding the cells together at these sites.
(2) The functions of the cell membrane
The cell membrane is of great importance in
regulating the activities of the cell as follows:
A. The membrane maintain the shape of the cell.
B. It controls the passage of all substances into or out of
the cell.
C. The cell membrane forms a sensory surface. This
function is most developed in nerve and muscle cells.
D. The surface of the cell membrane bears receptors that
may be specific for particular molecules. Stimulation of
such receptors can produce profound effects on the activity
of the cell. Receptors also play an important role in
absorption of specific molecules into the cell.
E. Membrane proteins help to maintain the structural
integrity of the cell by giving attachment to cytoskeletal
filaments. They also help to provide adhesion between
cells and extracellular materials.
F. Cell membrane may show a high degree of
specialization in some cells. for example, the membrane
of rod and cone cells in retina bear proteins that are
sensitive to light.
2. Cytoplasm
The cytoplasm (or cytosol ) of a typical cell contains
various structures that are referred to as organelles.
They include mitochondria, ribosomes, endoplasmic
reticulum(ER), Golgi complex, lysosomes,
peroxisomes, centrioles and various types of vesicles.
The cytosol also contains a cytoskeleton made up of
microtubules, microfilaments, and intermediate
filaments. There are cellular pigments, glycogen and
lipid droplets in cytoplasm.
A. Mitochondria
Mitochondria can be
seen with the LM in
specially stained
preparations. They
are so called because
they appear either as
granules or as rods of
0.5 to 2 micron(um) in
length.
The stomach inner covering
The cells show many round and
elongated mitochondra.
EM: Mitochondria are spherical or filamentous.
The mitochondrion is bounded
by an outer membrane and an
inner membrane, the two being
separated by an
intermembranous space. The
inner membrane is highly
folded forming incomplete
partitions called cristae. The
space bounded by the inner
membrane is filled by a
granular material called the
matrix which contain numerous
enzymes, DNA and RNA.
Function of mitochondria
Mitochondria provide energy for various cellular
functions. These facts can be correlated with the
observation that mitochondria are large in cells with
a high oxidative metabolism, and that within a cell
mitochondria tend to concentrate in regions where
energy requirements are greatest.
B. Ribosomes
Ribosomes are small electron-
dense particles, about
20×30nm in size. Each
ribosome is made up of two
subunits, and is composed of
rRNA and different proteins.
Ribosomes can be present in relation to
endoplasmic reticulum to form rough endoplasmic
reticulum(RER) . They also lie free in the
cytoplasm. They may be present singly, in which
case they are called mono-ribosomes; or in groups
which are referred to as polyribosomes.
rER.ppt
Function of ribosomes

Ribosomes are places that proteins are synthesized.

The proteins destined for cytoplasm, nucleus and
mitochondria are synthesized on free ribosomes.
The proteins destined for secreting are synthesized on
ribosomes bind to RER.
C. Endoplasmic reticulum
Endoplasmic reticulum consists of membranous tubules,
vesicles and flattened sacs which ramifies throughout
the cytoplasm to form an anastomosing network. Much
of its surface is studded with ribosomes, giving a rough
appearance leading to the name rough endoplasmic
reticulum(abbreviate RER). In contrast some of its
surface are devoid of ribosomes and constitute the
smooth endoplasmic reticulum(abbreviate SER).
RER-HE.ppt sER and rER.ppt rER.ppt sER &
rER.ppt
Function of ER

RER is represents the site at which the proteins that are

destined for export are synthesized. The attached
ribosomes play an important role in this process.

The principal functions of SER are lipid biosynthesis,

especially that of membrane phospholipids.
D. Golgi Complex (Golgi apparatus)
In light microscopic preparations suitably treated with
silver salts the Golgi complex can be seen as a small
structure of irregular shape, usually present near the
nucleus.
When examined with the EM the complex is seen to
be made up of membranes cisternae. The
membranes form the walls of a number of flattened
cisternae that are stacked over one another. Towards
their margins the cisternae are continuous with small
rounded vesicles. The cisterna of the Golgi complex
form an independent system. Their lumen is not in
communication with that of ER. Material from ER
reaches the Golgi complex through vesicles.
Golgi complex-1.ppt
Function of Golgi complex
From a functional point of view the Golgi complex is
divisible into three regions. The region nearest the
nucleus is the cis face. The opposite face (nearest the
cell membrane) is the trans face. The intermediate
part (between the cis face and the trans face) is the
medial Golgi.
Golgi complex-1.ppt
Materials synthesized in RER travels through the ER
lumen. Vesicles budding off from ER transport these
materials to the cis face of the Golgi complex. Some
proteins are phosphorylated here. From the cis face
all these materials pass into the medial Golgi. Here
sugar residues are added to proteins to form protein-
carbohydrate complexes.
Golgi complex.ppt
E. Lysosomes

These membrane bound vesicles contain acid

hydrolases that can destroy unwanted material
present within a cell. Such materials may have been
taken into the cell from outside (e.g., bacteria); or
may organelles that are no longer of use to the cell.
As many as 40 different lysosomal enzymes have
been identified.
lysosomes.ppt
Primary lysosomes
The enzymes in these vesicles are inactive because of
the lack of an acid medium.
Secondary lysosome
The vesicles fuse with other vesicles derived from
cell membrane (endosomes). These endosomes
possess the membrane proteins necessary for
producing an acid medium. The product formed by
fusion of the two vesicles is an endolysosome (or
secondary lysosome).
lysosomes.ppt
Phagolysosomes
A lysosome, containing appropriate enzymes, fuses
with the phagosome so that the enzymes of the former
can act on the material within the phagosome. These
bodies consisting of fused phagosomes and lysosomes
are refered to as phagolysosomes.
phagosome.ppt
 Multivesicular bodies
In a similar manner lysosomes may also fuse with
pinocytotic vesicles. The structures formed by such
fusion often appear to have numerous small vesicles
within them and are, therefore, called multivesicular
bodies.
lysosomes.ppt
Residual bodies
After the material in phagosomes or pinocytotic vesicles
has been ‘digested’ by lysosomes, some waste material
may be left. Some of it is thrown out of the cell by
exocytosis. However, some material may remain within
the cell in the form of membrane bound residual
bodies.
F. Peroxisomes
These are similar to lysosomes in that they are
membrane bound vesicles containing enzymes.
Peroxisomes contain catalase which destroys
hydrogen peroxide. Hydrogen peroxide is toxic to the
cell.
peroxisome.ppt
G. centrosome
All cells capable of division contain a pair of structures
called centrioles. With the light microscope the two
centrioles are seen as dots embedded in a region of
dense cytoplasm which is called the centrosome.

With the EM the

centrioles are seen to be
short cylinders that lie
at right angle to each
other.
A centriole is a short cylinder and
consists essentially of a series
microtubules arranged in a circle.
There are nine groups of tubules,
each group consisting of three
tubules .
 Centrioles play an important role in the formation
of various cellular structures that are made up of
microtubules. These include the mitotic spindles of
dividing cells, cilia, flagella, and some projections of
specialized cells.
H. The cytoskeleton
The cytoplasm is permeated by a number fibrillar
elements that collectively form supporting network. This
network is called the cytoskeleton. Apart from
maintaining cellular architecture the cytoskeleton
facilitates motility (e.g., by forming cilia), and helps
divide the cytosol into functionally discrete area. It also
facilitates transport of some constituent through the
cytosol, and plays a role in anchoring cells to each other.
The elements that
constitute the
cytoskeleton are:
microfilaments,
microtubules
intermediate filaments .

They can been seen under LM by useing Silver Stain.

a. Microfilaments
These are about 5 nm in diameter. They are made up
of the protein actin. Actin filaments form a meshwork
just subjacent to the cell membrane. This meshwork is
called the cell cortex. The cell cortex helps to maintain
the shape of the cell. The meshwork of the cell cortex
is labile. The filaments can separate, and can reform in
a different orientation. That is how the shape of a cell
is altered.
microvilli.ppt microfilaments.ppt
b. Microtubules
Microtubules are about 25
nm in diameter. The basic
constituent of microtubules is
the protein tubulin. Chains of
tubulin form protofilaments.
The wall of a microtubule is
made up of thirteen
protofilaments .
intermediate filamentsand
microtubules.ppt
The roles played by microtubules are as follows.
a) As part of the cytoskeleton, they provide stability to
the cell.
b) Microtubules facilitate transport within the cell.
c) In dividing cells microtubules form the mitotic
spindle.
c. Intermediate filaments
These are so called as their diameter (10nm) is
intermediate between that of microfilaments(5 nm) and
of microtubules (25 nm).
intermediate filamentsand microtubules.ppt
The roles played by intermediate filaments are as follows.
a) Intermediate filaments link cells together. The filaments
also facilitate cell attachment to extracellular elements .
b) In the epithelium of the skin the filaments undergo
modification to form keratin.
c) The neurofilaments of neurons are intermediate
filaments.
d) The nuclear lamina consists of intermediate filaments.
G.

There are cellular pigmemts, glycogen and

lipid droplets in cytosol.
3. The nucleus
The nucleus constitutes
the central, more dense
part of the cell. It is usually
rounded or ellipsoid.
Occasionally it may be
elongated, indented or
lobed. It is usually 4 ～
10µm in diameter. In HE
stain, the nucleus stains
dark purple or blue while
the cytoplasm is usually
stained pink.
With the EM the nucleus is seen to be surrounded by a
double layered nuclear membrane or nuclear envelope.
The space between the inner and outer membranes is
the perinuclear space. At several points the inner and
outer layers of the nuclear membrane fuse leaving gaps
called nuclear pores.
Deep to the inner
membrane there is a
layer containing
proteins and a network
of filaments: this layer
is called the nuclear
lamina.
The nucleus contains chromatin heterochromatin
in which the inherited
information is carried.
The chromatin is seen in the
form of irregular dark masses
that are called heterochromatin.
At other places the network is
loose and stains lightly: the
chromatin of such areas is
referred to as euchromatin.
euchromatin
In addition to the masses of heterochromatin, the nucleus
shows one or more rounded, dark staining bodies called
nucleoli.
cell.ppt nucleus-nucleolus.ppt

The nucleus also contains various small granules, fibres

and vesicles. The spaces between the various
constituents of the nucleus described above are filled by
a base called the nucleoplasm.
Chromatin is made up of a substance called
deoxyribonucleic acid (abbreviate DNA) and of
proteins. Heterochromatin represents areas where
chromatin fibres are tightly coiled on themselves
forming ‘solid’ masses. In contrast euchromatin
represents areas where coiling is not so marked.
During cell division the entire chromatin within the
nucleus becomes very tightly coiled and takes on the
appearance of a number of short, thick, rod-like
structures called chromosomes. Chromosomes are
made up of DNA and proteins. Proteins stabilize the
structure of chromosomes.
The number of chromosomes is fixed for a given
species and in man it is 46.
chromosome.ppt
The inherited information is stored in
chromosomes. This information is necessary for
the proper function of the cells.
Each chromosome bear on itself a very large
number of functional segments that are called
genes. A gene represent a unit of stored
information which guide the performance of
particular cellular function.
 QUESTIONS
1. What is HE stain?
2. What components can the PAS reaction detect?
3. Describe the structure and function of organelles.
4. What chemical component dose cell membrane
include?
5. What components dose the cytoskeleton include?