Human_Histology

x Textbook of Human Histology

Chapter 15: Digestive System: Esophagus, Stomach, and Intestines

General structure of GIT	174
The esophagus	176
Microscopic features	176
The stomach	176
Microscopic features	177
Gastric glands	178
The small intestine	182
Microscopic features	183
Distinguishing features of duodenum, jejunum, and ileum	187
The large intestine (colon)	188
The colon	189
The vermiform appendix	189
The rectum	190
The anal canal	190
Chapter 16: Hepatobiliary System and Pancreas
The liver	193
Microscopic features	193
Bile	196
Blood supply of liver	197
Functions of liver	197
Extrahepatic biliary apparatus	198
The gallbladder	198
The extrahepatic ducts	198
The pancreas	200
The exocrine pancreas	200
The endocrine pancreas	202
Blood supply	203
Nerve supply	203
Chapter 17: The Urinary System
The kidneys	204
The ureters	213
The urinary bladder	215
Chapter 18: Male Reproductive System
Testis	217
Spermatogenesis	221
Accessory urogenital organs	224
Epididymis	224
Ductus deferens	225
The seminal vesicle	227
Prostate	228
Penis	228

	Contents xi
Chapter 19: Female Reproductive System
The ovaries	231
Oogenesis	231
Uterine tubes	236
The uterus	239
Vagina	242
Female external genitalia	243
Mammary gland	243
Placenta	246
Function	246
Components	246
Formation of Placenta	247
Placental Circulation and Barrier	247
Microscopic Structure	247
Chapter 20: Endocrine System
Hormones	249
Distribution of endocrine cells	249
Hypophysis cerebri	249
Subdivisions of the hypophysis cerebri	249
Adenohypophysis	251
Neurohypophysis	253
Blood supply of the hypophysis cerebri	253
Control of secretion of hormones of the adenohypophysis	253
Thyroid gland	254
Structure of thyroid gland	255
Parathyroid glands	257
Structure of parathyroid glands	257
Cells of parathyroid glands	257
Suprarenal glands	259
Structure of suprarenal glands	259
Pineal gland	261
Microscopic features	261
Some other organs having endocrine functions	262
Paraganglia	262
Para-aortic bodies	262
Carotid bodies	262
Chapter 21: Special Senses: Eye
Structure of eyeball	265
Outer fibrous coat	265
Vascular coat or uvea	266
Retina	268
Lens	274
Accessory visual organs	276

xii Textbook of Human Histology

Chapter 22: Special Senses: Ear

The external ear	281
Middle ear	282
The internal ear	282
Specialized end organs in the membranous labyrinth	288
Some elementary facts about the mechanism of hearing	290
Index	293

Chapter

1 Light Microscopy and

Tissue Preparation

—Sabita Mishra

A light microscope is an optical device that uses visible light for illumination and lenses to magnify a specimen or tissue section for detailed visualization. Simple microscopes utilize a single lens while compound microscopes have a number of lenses in combination. In light microscopy the tissue is visualized against a bright background so it is also referred to as bright field microscopy and is best suited to view stained tissue sections. In contrast, in dark field microscopy unstained specimens, e.g. living cells, are observed. The light enters from the periphery and scattered light enters the objective lens showing a bright specimen against a dark background. All routine histological techniques involve the bright field microscopy.

Antonie Philips van Leeuwenhoek in 1673 invented the concept of using combination of convex lenses to magnify small structures and visualize them. His invention of this primitive microscope detected small structures like bacteria, yeast and blood cells. The invention of microscope led to the discovery and description of cell by Robert Hooke, an English scientist in 1655. Modern microscopy started with the invention of achromatic lens by Lister in 1829.

COMPONENTS OF A LIGHT MICROSCOPE (FIG. 1.1)

A light microscope has optical parts and non-optical parts.

Optical Parts

The functioning of the microscope is based on the optics of the lenses.

Illuminating Device

Most of the advanced microscopes come with a built-in illuminator using low voltage bulbs for transmitted light. The brightness of light can be adjusted. Older monocular microscopes used mirrors to reflect light from an external source.

Condenser: Collects and focuses light from the light source onto the specimen being viewed. The condenser is close to the stage and has an aperture (iris dia­ phragm) that controls the amount of the light coming up through the condenser.

Fig. 1.1: Binocular light microscope

Aperture closed	Aperture open
Light comes in center	Image is brighter
Contrast high	Contrast low

Objective lenses: These lenses are attached to the nosepiece of the microscope. The objective lens is responsible for magnifying the specimen/section to be visualized. The type and quality of an objective lens influences the performance of a microscope. A standard microscope has three, four, or five objective lenses that range in power from 4× to 100×. The objective lens collects maximum amount of light from the object to form a high quality magnified real image.

Eye piece: The viewer looks through the eyepiece to observe the magnified image. Eye pieces may be monocular, binocular or combined photo binocular (trinocular). It is the final stage of the optical path of the microscope and produces a magnified virtual image which is seen by the eye. The eye piece has a power of 10×. In a binocular microscope diopter adjustments are present to adjust the focusing of the eye piece.

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Non-Optical Components of Microscope

Body Tube (Head)

A cylindrical tube that connects the eyepiece to the objective lenses. Standard length 160 mm.

Arm: The arm connects the body tube to the base of the microscope.

Coarse adjustment: Mechanical knobs that bring the specimen into general focus.

Fine adjustment: Fine tunes the focus and increases the detail of the specimen. Individual user has to adjust according to his/her own vision to observe the tissue section.

Nosepiece: A rotating turret that houses the objective lenses. The viewer rotates the nosepiece to select different objective lenses.

Stage: The flat platform where the slide is placed and lies perpendicular to the optical pathway. It has Stage clips that hold the slide in place. Stage Control Knobs move the stage left and right or up and down. The stage also has a vernier caliper attached to it so that the viewer can come back to any reference point by the help of the caliper. An aperture in the middle of the stage allows light from the illuminator to reach the specimen.

Base: The base supports the microscope. The illumin-

ator with its power switch is located on the base. Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. Stained tissue sections are mounted on a glass slide with a coverslip placed over it. This allows the slide to be easily inserted or removed from the microscope. It also allows the specimen to be labelled, transported, and stored without any damage. The slide is placed on the stage for viewing.

PRINCIPLES OF A CONVENTIONAL

BRIGHT FIELD MICROSCOPE

The word compound refers to the fact that two lenses, the objective lens and the eyepiece (or ocular), work together to produce the final magnified image that is projected onto the eye of the observer.

Magnification

Magnification (M final) is calculated by the formula M final = M (objective) × M (eyepiece).

Resolution

Given sufficient light an unaided eye can distinguish 2 points lying 0.2 mm apart. This distance is called resolution

of a normal eye. By assembling a combination of lenses the distance can be increased and the eye can visualize objects closer than 0.2 mm.

Resolution of a microscope is dependent on

a.Wave length of light 400-800 l

b.Numerical aperture (NA): collecting power of the objective and condenser lens NA= n × sin u

Where n = refractive index of media between cover slip and objective lens

sin u = Angle between optical axis of lens and outer most ray (r/h)

	NA = n × r/h (Fig. 1.2)
		Fig. 1.2: Numerical aperture
	Refractive index of air =1
	Refractive index of oil
	immersion 1.51
	Resolution of microscope
	(r) = 0.6l/NA
	Resolution of light
	microscope is 0.2 µ

Working of a Light Microscope

(Flowchart 1.1 and Fig. 1.3)

To view a section/specimen under a light microscope: Light from the light source enters the condenser, passes through specimen and is magnified by the objective lens. The real magnified image formed by the objective lens is further magnified by the eyepiece. Thus the viewer observes a magnified virtual image.

Axial Aberrations

When light passes through the lens it suffers a number of aberrations which result in image degradation. The optical parts are the condenser, objective, and eyepiece. The best (and most expensive) lenses have the least aberrations.

Commonly seen aberrations are:

Chromatic aberration: Production of a colored spectrum of white light. Different lenses corrected for chromatic aberrations are listed in Table 1.1.

Spherical aberration: A defect of single lenses due to their curved surface. Light passing through the periphery of the lens is refracted to a greater extent than through the central part. This is corrected by using a compound lens.

Chapter 1 Light Microscopy and Tissue Preparation 3

Flowchart 1.1: Working of a light microscope

Table 1.1: Different lenses corrected for chromatic aberrations

Achromatic	Fluorite	Apochromatic
Corrected for red and	Spread of	Fully corrected for
blue focused at same	secondary	three colors
length	spectrum reduced
Green shorter focus	Type of correction	In combination
Violet longer focus	though similar to	with fluorite
	achromatic	causes elimination
		of all colors

Illumination

Critical Illumination

This is used with simple equipment and a separate light source. Light source is focused in the same plane as the object, when the object is in focus.

Kohler Illumination

High intensity microscopes have a small light source that is insufficient to fill the whole field with light and are usually supplied by an auxiliary lens and iris which increases the apparent light source. With Kohler illumination auxiliary lens of the lamp focuses the enlarged image on to the iris diaphragm of the sub-stage condenser. The resolving power of critical and Kohler are similar, but Kohler illumination provides an evenly illuminated view and displaces critical illumination.

PRACTICAL TIPS IN USING A

BRIGHT FIELD MICROSCOPE

Mount the specimen with the coverslip facing up on the stage

Optimize the lighting

Fig. 1.3: Working of a light microscope (Schematic representation)

Adjust the condenser

Focus, locate, and center the specimen

Adjust eyepiece separation and focus

Select an objective lens for viewing

Move up the magnification in steps

TYPES OF MICROSCOPES

Dark­field microscopy: The specimen is illuminated from the side and only scattered light enters the objective lens which results in bright objects against dark background. Images produced by dark-field microscopy are low resolution and details cannot be seen. Dark-field microscopy is especially useful for visualization of small particles, such as bacteria.

Phase contrast microscopy and differential­interfe­ rence­contrast allow objects that differ slightly in refractive index or thickness to be distinguished within unstained or living cells.

Fluorescence microscopy: A fluorochrome is excited with ultraviolet light and the resulting visible fluorescence is viewed. This produces a bright image in a dark background. There are some natural fluorescence substances which fluoresce when ultraviolet light falls on them, called primary fluorescent substances. Certain fluorescent dyes when added to the tissue lead to a secondary fluorescence which are visualized by a fluorescent microscope.

Confocal microscopy: The confocal scanning optical microscope is designed to illuminate an object in a serial fashion, point by point, where a small beam of light (from a LASER) is scanned across the object rapidly in an X-Y raster pattern. The images are digitized and stored.

4Textbook of Human Histology

Electron microscopy: The property of accelerated electrons in vacuum to behave like light and travel in a straight line has been exploited in the invention of the electron microscope. Instead of glass lenses here one uses electromagnetic lenses. The wave length of the electrons in vacuum is 10,0000 times less than light. The resolving power of an electron microscope is 0.2 nm. In transmission electron microscopy the beam of electron passes through the tissue which is a thin section less than 100 nm. To prepare such thin section one uses an ultramicrotome and instead of steel blades, the sections are cut with laboratory prepared fresh glass knives. The sections are picked up in grids and are stained with uranyl acetate and lead citrate before viewing. Transmission electron microscope is used for ultrastructural studies. In scanning electron microscope the beam of electrons are reflected back from the surface, thus giving us the surface view.

TISSUE PROCESSING

To visualize the microstructure of any tissue under a light microscope, the specimen has to go through a thorough protocol of tissue fixation, tissue processing, sectioning and staining.

STEPS INVOLVED IN TISSUE PREPARATION

Tissue collection: Commonly tissue is obtained from, autopsy, surgical procedures, experimental animals (rabbit, rats, mice, etc.) either perfused or decapitated (Guideline of ethics are always observed in any experimental study).

Fixation: The primary objective of fixation is that stained section of any tissue must maintain clear and consistent morphological features to almost that what was existing during life.

Effects of fixation: It coagulates the tissue proteins and constituents, thus minimizing their, loss during tissue processing, hardens the tissue and makes it insensitive to hypotonic or hypertonic solution.

Commonly used fixative is formaldehyde and glutaraldehyde.

Formaldehyde is a cross-linking fixative which acts by creating a covalent bond between proteins in the tissue. Formaldehyde is a gas and is soluble in water to an extent of 40% by weight. Ten percent methanol is added to it as a stabilizer. Paraformaldehyde is a polymer of formaldehyde available as a white crystalline powder.

Tissue processing: Principle of tissue processing involves replacement of all extracellular water from the tissue and replacing it with a medium that provides sufficient rigidity to enable sectioning without any damage or distortion to the tissue.

STEPS IN TISSUE PROCESSING

Dehydration: Removal of water by a dehydrating agent. Commonly used dehydrating agent is alcohol in descending grades (e.g. 100%, 90%, 70%, 50%, 30%).

Clearing: Making the tissue clear by removing the dehydrating agent, e.g. xyline, chloroform.

Infiltration: Permeating the tissue with a support media.

Embedding: Paraffin wax is routinely used as an embedding media. It has a melting point of 45–55 degree. Other embedding media used are celloidin and resins.

The tissue is embedded and orientated in the media and forms a solid block at room temperature. The tissue blocks are ready for sectioning.

Sectioning: A rotary microtome is used to cut sections of 5–7 µ thick for routine histology. The sections are cut and picked up on clean glass slides under a water bath. They are dried before staining.

Staining: Hematoxylin and eosin is routinely used for all teaching slides. Morphological identification becomes easier. Hematoxylin is a basic dye and stains the nucleus blue while eosin is an acidic dye and stains the cytoplasm pink. Once the sections are stained they are mounted and are ready for viewing under a light microscope.

Cell Structure

INTRODUCTION

Cell is the fundamental structural and functional unit of the body. A cell is bounded by a cell membrane (or plasma membrane) within which is enclosed a complex material called protoplasm. The protoplasm consists of a central more dense part called the nucleus; and an outer less dense part called the cytoplasm. The nucleus is separated from the cytoplasm by a nuclear membrane. The cytoplasm has a fluid base (matrix) which is referred to as the cytosol or hyaloplasm. The cytosol contains a number of organelles which have distinctive structure and functions. Many of them are in the form of membranes that enclose spaces. These spaces are collectively referred to as the vacuoplasm.

THE CELL MEMBRANE

The membrane separating the cytoplasm of the cell from surrounding structures is called the cell membrane or the plasma membrane.

It has highly selective permeability properties so that the entry and exit of compounds are regulated. The cellular metabolism is in turn influenced and probably regulated by the membrane. Thus the membrane is metabolically very active.

Basic Membrane Structure

When suitable preparations are examined by electron microscope (EM), the average cell membrane is seen to be about 7.5 nm thick. It consists of two densely stained layers separated by a lighter zone; thus creating a trilaminar appearance.

Membranes are mainly made up of lipids, proteins and small amounts of carbohydrates. The contents of these compounds vary according to the nature of the membrane.

Lipids in Cell Membrane

The trilaminar structure of membranes is produced by the arrangement of lipid molecules (predominantly

Fig. 2.1: Structure of a phospholipid molecule (phosphatidyl choline) seen in a cell membrane (Schematic representation)

phospholipids) that constitute the basic framework of the membrane.

Each phospholipid molecule consists of an enlarged head in which the phosphate portion is located; and of two thin tails (Fig. 2.1). The head end is called the polar end while the tail end is the non-polar end. The head end is soluble in water and is said to be hydrophilic. The tail end is insoluble and is said to be hydrophobic.

When such molecules are suspended in an aqueous medium, they arrange themselves so that the hydrophilic ends are in contact with the medium; but the hydrophobic ends are not. They do so by forming a bi-layer which forms the basis of fluid mosaic model of membrane (Singer and Nicolson 1972) (Fig. 2.2).

The dark staining parts of the membrane (seen by EM) are formed by the heads of the molecules, while the light staining intermediate zone is occupied by the tails, thus giving the membrane its trilaminar appearance.

Because of the manner of its formation, the membrane is to be regarded as a fluid structure that can readily reform when its continuity is disturbed. For the same reasons, proteins present within the membrane can move freely within the membrane.

6Textbook of Human Histology

Fig. 2.2: Fluid mosaic model of membrane (Schematic representation)

Added Information

As stated above phospholipids are the main constituents of cell membranes. They are of various types including phosphatidylcholine, sphingomyelin, phosphatidylserine, and phosphatidyl ethanolamine.

Cholesterol provides stability to the membrane.Glycolipids are present only over the outer surface of

cell membranes. One glycolipid is galactocerebroside, which is an important constituent of myelin. Another category of glycolipids seen are ganglionosides.

Fig. 2.3: Some varieties of membrane proteins (Schematic representation)

Proteins in Cell Membrane

The proteins are present in the form of irregularly rounded masses. Most of them are embedded within the thickness of the membrane and partly project on one of its surfaces (either outer or inner). However, some proteins occupy the entire thickness of the membrane and may project out of both its surfaces (Fig. 2.2). These are called transmembrane proteins (Fig. 2.3).

The proteins of the membrane are of great significance as follows:

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.

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.

Some proteins are so shaped that they form passive channels through which substances can diffuse through

Fig. 2.4: Glycolipid and glycoprotein molecules attached to the outer aspect of cell membrane (Schematic representation)

the membrane. However, these channels can be closed by a change in the shape of the protein.

Other proteins act as receptors for specific hormones or neurotransmitters.

Some proteins act as enzymes.

Carbohydrates of Cell Membranes

In addition to the phospholipids and proteins, carbohydrates are present at the surface of the membrane. They are attached either to the proteins (forming glycoproteins) or to the lipids (forming glycolipids) (Fig. 2.4). 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.

Chapter 2 Cell Structure 7

Fig. 2.5: Three stages in the absorption of extracellular molecules by endocytosis (Schematic representation)

Added Information

The glycocalyx is made up of the carbohydrate portions or glycoproteins and glycolipids present in the cell membrane. Some functions attributed to the glycocalyx are as follows:Special adhesion molecules present in the layer enable

extracellular molecules.

The layer contains antigens. These include major histocompatibility complexes (MHC). In erythrocytes, the glycocalyx contains blood group antigens.

Most molecules in the glycocalyx are negatively charged causing adjoining cells to repel one another. This force of repulsion maintains the 20 nm interval between cells. However, some molecules that are positively charged adhere to negatively charged molecules of adjoining cells, holding the cells together at these sites.

Functions of Cell Membrane

The cell membrane is of great importance in regulating the following activities:

The membrane maintains the shape of the cell.

It controls the passage of all substances into or out of the cell.

The cell membrane forms a sensory surface. This function is most developed in nerve and muscle cells. The plasma membranes of such cells are normally polar- ized—the external surface bears a positive charge and the internal surface bears a negative charge, the potential difference being as much as 100 mv. When suitably stimulated, there is a selective passage of sodium and potassium ions across the membrane reversing the charge. This is called depolarization. It results in contraction in the case of muscle, or in generation of a nerve impulse in the case of neurons.

The surface of the cell membrane bears receptors that may be specific for particular molecules (e.g. hormones or enzymes). Stimulation of such receptors (e.g. by the specific hormone) can produce profound effects on the activity of the cell. Receptors also play an important role in absorption of specific molecules into the cell as described below.

Cell membranes may show a high degree of specialization in some cells. For example, the membranes of rod

and cone cells (present in the retina) bear proteins that are sensitive to light.

Role of Cell Membrane in Transport of

Material into or out of the Cell

It has already been discussed that some molecules can enter cells by passing through passive channels in the cell membrane. Large molecules enter the cell by the process of endocytosis (Fig. 2.5). In this process the molecule invaginates a part of the cell membrane, which first surrounds the molecule, and then separates (from the rest of the cell membrane) to form an endocytic vesicle. This vesicle can move through the cytosol to other parts of the cell.

The term pinocytosis is applied to a process similar to endocytosis when the vesicles (then called pinocytotic vesicles) formed are used for absorption of fluids (or other small molecules) into the cell.

Some cells use the process of endocytosis to engulf foreign matter (e.g. bacteria). The process is then referred to as phagocytosis.

Molecules produced within the cytoplasm (e.g. secretions) may be enclosed in membranes to form vesicles that approach the cell membrane and fuse with its internal surface. The vesicle then ruptures releasing the molecule to the exterior. The vesicles in question are called exocytic vesicles, and the process is called exocytosis or reverse pinocytosis (Fig. 2.6).

In a Nutshell:

Cell membrane controls the passage of substance in and out of the cell.

Small molecules pass through passive channels.Large molecules enter the cell by the process of

endocytosis.

If the engulfed material is a foreign body (e.g. bacteria), the term phagocytosis is used.

When the vesicles release the molecule to the exterior, the process is then referred to as exocytosis or reverse pinocytosis.