Professor Yunlian Meng

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 embedding

of

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 electrondense 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 rER.ppt

sER and rER.ppt

rER.ppt

sER &

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 proteincarbohydrate 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 lipid droplets in cytosol.

pigmemts, glycogen

and

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 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.

heterochromatin

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?