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Histology Lecture Notes on ALL systems...

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Lecture Notes on General Histology For Medical Students

Naama Abdulgader, MD, PhD, Professor Department of Histology and Medical Genetics Faculty of Medicine Tripoli University

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CONTENTS

1. Introduction and Microscopy …………………

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2. Cytology and Cell Biology …………………… 22 3. Epithelial Tissue ……………………………...

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4. Connective Tissue ……………………………

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5. Blood …………………………………………

115

6. Cartilage ……………………………………… 138 7. Bone …………………………………………..

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8. Muscles ………………………………………

161

9. Nervous Tissue ……………………………… 178 10. Circulatory System ………………………...

205

11. Immune System and Lymphoid Organs …… 222 12. Integumentary System …………………….. 249

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INTRODUCTION Histology means the study of tissue components by the use of micros copes

(light and electron) and stained sections of tissues. The main objective in studying histology is to identify the mammalian tissues quickly and accurately. The human body is composed of systems, which are made of organs. Organs are made of tissues which are composed of cells. The cell size is measured by special units and examined by microscopes. The main idea in the study of histology is to know the general and specific features of tissues and organs and memorize them very well. In addition to understanding the histology and ultrastructure of cells and tissues, it is also important to correlate the morphological features with an understanding of the biochemistry and physiology of these structures. Remember that histology is one of the most useful courses, because it is the core subject in the study of function, macroscopic and morbid anatomy, and cell and molecular biology. Moreover, most medical researchers depend on histology in their research.

Units of measurements: 1 centimeter (cm) = 10 millimeters (mm) 1 millimeter (mm) = 1000 micrometers (um) 3

1 micrometer (1um) = 0.001mm 1 nanometer (1nm) = 0.001um 1 Angstrom (1 Å) = 0.1nm = 0.0001um

MICROSCOPY The basic type is the light microscope.

Other types of microscopes are

modifications of the light microscope. 1. The Light Microscope (LM) The light microscope is the most important machine in medical laboratory. It uses a visible light source with a system of condenser lenses to send the light through the object to be examined. The light microscope is used to enlarge small objects and to reveal their fine details. The light microscope is composed of: 1. Frame and mechanical parts 2. Optical (magnification) system 3. Illumination system 1. The frame supports the different parts of the optical system; it consists of base, arm, stage with a central hole for the light to pass through and a body tube carrying the optical system.

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2. The magnifying system is composed of:  Eyepiece (ocular) with 8, 10, or 12 X of magnification.  The objectives: four lenses with different magnifications: 3.5, 10, 40, 100 (oil immersion lens); they are carried on an objective nose piece. Usually the oil immersion lense is kept separately. The objective lenses collect the light transmitted through the section from the condenser, magnify the image and direct it to the ocular lens. The eyepiece (ocular lens) magnifies the image and directs it to the viewer eye or to a screen or a camera. The property of making things to appear larger is called magnification. The total magnification = ocular power x objective power The property of disclosing the fine details is called resolution (R). Resolution is the smallest distance between two particles that can be distinguished from each other.

Resolution is more important than

magnification, since it separates clearly between two points located close together. Resolution power of:  Human naked eye: 0.1-0.2 mm  LM is 0.1-0.2 um  EM: 0.1-0.2 nm The resolution (R) is directly proportional to the wavelength of light (‫ )גּ‬and indirectly proportional to the numerical aperture (NA) of lenses. The NA of 5

the lens is the sine of the angle of light entering between the middle and the edge of the lens. Lenses with larger NA have better resolution. Resolution (R) = constant x wavelength Numerical aperture R = 0.61 x ‫גּ‬ NA The only way to improve the resolving power of a microscope (resolution) is to reduce substantially the wavelength of the light. This was achieved by the electromagnetic beam of the electron microscope. 3. The illumination system consists of: light source, condenser and iris diaphragm to regulate the amount of light. How to take care of your light microscope? 1. Do not touch lenses with fingers 2. Do not take objective lenses apart 3. Oculars should be cleaned with lens tissue 4. Carry the microscope by its arm with other hand under its base 5. Turn off the microscope and carefully cover it with its plastic cover. 2. The Electron Microscope (EM) The Electron Microscope (EM) is better suited to study the details of cells than LM. The detailed morphology revealed by EM may be called fine or submicroscopic structures or ultrastructure. There are two types of the electron microscopes, the transmission and scanning. 6

A. Transmission Electron Microscope (TEM): In this microscope, the ordinary light source is replaced by a beam of electrons, which are emitted by heated tungsten gun or filament (cathode) in a vacuum controlled system. There is a voltage difference between the cathode and the anode which accelerates the electron beam and attracts the electrons to the anode where they pass through its central hole. While passing in the microscope tube, the electron beam is subjected to electric coils with magnetic field, which deflect the electrons and change their path, thus called electromagnetic lenses. The image is viewed directly on a fluorescent screen because the human eye is not sensitive to electrons. The tissue is treated with a double fixation method to preserve the ultrastructural details. Buffered gluteraldehyde is used as a first fixative, and then a special second fixative such as osmium tetra-oxide is used to stain lipids and proteins. The tissue, then, embedded in resin or plastic in vacuum oven. The resulting blocks are very hard; they are cut into very thin sections (40-90nm) by the use of ultra -tome and a diamond or glass knife. The thin sections are mounted on a special 100-400 mesh copper grids, and stained with heavy metals such as lead and uranyl acetate. The image is seen on a screen as a black and white and recorded on photographs. The electron microscope requires a vacuum-enclosed system, high voltage (60-120KV), and mechanical stability.

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The high resolving power of EM made possible to study the details of the interior of cells with a final magnification of 400,000 times. The use of high voltage (500,000-1,000,000V) allowed the use of relatively thicker sections to be examined to get 3-D images. B. Scanning Electron Microscope (SEM): This microscope provides a three dimensional image of the surface of fixed and dehydrated tissues; it has less resolving power than TEM. The sample is coated with gold, which emits secondary electrons after being hit with a primary electron beam. The electrons do not pass through the specimen, but scan sequentially the different surface points of the specimenand the resulting image will be in black and white color. Comparison between light (LM) and electron microscopes (EM)

Light Microscope

Electron Microscope Presented on a screen in shades of green. In photographs, image appears in grey scale or in black and white. High, up to X 400,000 (2,000,000?)

Resolution

Presented directly to the eye. Image keeps the colors given by staining. Up to X 1500 (times), wider field of sample view; good orientation. 0.1 – 0.2 μm

Time

By frozen section

Tissue prcessing takes one day at

Image

Magnification

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High, 0.1nm

Artefacts

sample can be prepared in 20 min. Many

least. Frozen tissue for cryofructure and histochemistry Fewer

Section

1 – 10 μm

Very thin 40 – 90 nm

processing

thickness

Stain

Can be constructed by Obtained by thicker section of serial sections high-voltage EM, freezefructured techniques, and SEM Routine H&E Heavy metals

Specimen size

Can be large and alive Very small samples

Light source

Visible light (electric) Beam of electron

Lenses

Glass

3-D image

Electromagnetic

3. The Phase-Contrast Microscope This type is used to study the living and unstained tissues. Its idea is based upon the fact that light passing through the media of different refractive indices changes direction and speed, thus creating contrast. It is equipped with a lens system that enables it to convert the phase variation into intensity variations, which are perceived as differences in brightness, and the unstained object becomes visible. 4. The Interference Microscope This microscope uses the same principle of the phase contrast microscope, but with two beams of lights, which interfere with one another in a way to

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provide precise information on the density of cellular region even in the living state.

5. Fluorescence Microscope This microscope depends on the fact that certain substances present in nature emit light of longer visible wavelength on exposure to ultraviolet or lase light. This character is known as fluorescence and the molecules having this character will appear colored or bright in a dark field.

Some of these

substances are used to stain the cell components such as acridine orange which stain specifically nucleic acids. With DNA, the acridine orange emit yellowish green light and with RNA, acridine orange emits reddish orange light. This microscope is used to localize nucleic acids in the cells, to localize antigen-antibody complexes and to trace the pathway of nerve fibers. 6. The Polarizing Microscope This type uses two filters, one of them is located below the condenser, called the polarizer; the other filter is localized between the objective lens and the eye piece and called the analyzer. When the axes of the two polarizers become perpendicular on each other, no light passes resulting in a dark field scene. This type is used to examine a crystalline substance or well ordered fibrous molecule, such as collagen, microtubules and microfilaments which appear light against the dark field. These substances alter the plane of the entering polarized light and rotate the axis of the emerging light (birefringence).

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7. The Confocal Microscope This microscope uses a very small beam of laser light for illumination, and a highly computerized system. Its main principle is based on the fact that a very small laser beam originating from one thin plane of the section passes through a pinhole of a plate, while the rest of beams coming from other planes are blocked by that plate.

The small beam scans other planes of the

section, and thus able to collect serial optical sections from thick specimens. Using filters to eliminate the unfocused images and the photomultiplier detectors within a highly computerized system, made obtaining of a high quality sharp three-dimensional image is feasible.

It is used to optically

dissect the specimen and study the structure of biologic material after fluorescent labeling of the area of interest, thus cannot be used as a routine procedure. Methods of study in histology The human or animal tissue can be studied as: smears, spreads, sections or teased material. Several methods are applied for this purpose, such as: 1. Microtechniques: using fixed, stained material, examined by light microscope. 2. Electron microscopy: a fixed material is studied and high magnification is obtained. 3. Autoradiography: A method used to study the tissue synthetic activities by the use of radioactive isotopes by the use of light or electron microscopy. Tissues or small animals are treated with radioactive isotopes of certain molecules (precursor molecules) such as amino acids, nucleotides or sugars 11

to synthesize larger molecules of proteins, nucleic acids or polysaccharides. Tissue sections are prepared and covered with photographic emulsion containing silver bromide and kept in the dark. When silver bromide is exposed to radioactivity, it is transformed into metallic silver precipitating on the sections as silver grains.

Presence of silver grains and their number on

certain cells denote their synthetic activity and the path of their secretion. 4. Cell and tissue culture to study the living cells and tissues in vitro (outside the body) and the effect of single molecule or substance on one type of them. All the steps of culture are performed under strict sterile conditions. Primary cell culture: Before cultivation, the cells should be dispersed mechanically or by enzymes, and then suspended in nourishing medium containing salts, amino acids, vitamins and serum components or spread out on glass slides or petri dish. The resulting cell types are kept in vitro for reuse and are known as a cell line. The cultured cells are genetically programmed for a limited life span, but some of them show transformation changes and become immortal, changing from normal to cancer cells. 5. Cell fractionation: By this method, the cell components and organelles are separated according to their sedimentation coefficient. This method is used to study the chemical composition and function of the separated components in vitro. 6. Histochemical and cytochemical techniques: These methods are used to localize substances in tissue sections based on the specific chemical reactions between macromolecules and production 12

of insoluble colored substances that could be seen by the microscope (LM or EM). Examples of the substances that can be studied: a. Ions: such as calcium, iron, and phosphate. b. Nucleic acids: DNA and RNA are studied by the use of basic dyes. Feulgen reaction is specific to study and quantify the amount of DNA. c. Proteins: to study the enzyme activity in the tissue by localising the action of the enzyme with its substrate by producing colored insoluble substance that can be seen by the microscope (LM or EM). The most common enzymes to be studied:  Phosphatases: acid phosphatase (lysosomes in macrophages) and alkaline phosphatase (liver).  Dehydrogenases: a group of enzymes that remove hydrogen from one substrate to another that receives and precipitates it as an insoluble

colored

substance

e,g.

succenic

dehydrogenase

ofmitochondria.  Peroxidase enzyme present in liver and kidney cells; it promotes the oxidation of its substrates. It is used as a marker enzyme in diagnosis of leukemia and other diseases. d. Polysaccharides and oligosaccariades: the carbohydrates are present either free (glycogen) or as combined with lipids as glycolipids or with proteins as glycoproteins. The carbohydrate part can be demonstrated by the use of PAS reaction.

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PAS (Periodic Acid Schiff reagent): this method is used to stain glycosamino-glycan [GAG], glycoproteins, and glycogen (salivary amylase is used to break down glycogen), then counterstained by hematoxylin. e. Lipids: since lipids dissolve in routine preparations, it is better to use dyes that dissolve in lipids.

Frozen sections are dipped into

alcohol saturated with Sudan IV or Sudan black which stains lipid droplets red or black respectively. 7. Freeze-fracture methods: Where the cell components are frozen to a very low temperature to minus 170°C, then cleaved at the phospholipids bilayer. The fractured cells are coated with carbon and platinum then examined by EM. 8. Molecular high affinity interactions: these methods depend on specific interaction between the molecules that have great affinity towards one another. Labeled molecules with fluorescent compounds, enzymes, gold particles or radioactive atoms are used to identify the specific interacted molecules.

Phalloidin (to demonstrate actin filaments), protein A

(immunoglobulins for staph aureus) and lectins (binds sugars in glycoproteins, glycolipids, proteoglycans) are commonly used. methods are used to detect sugars, proteins and nucleic acids. a. Immunocytochemistry: this is a highly specific reaction between an antigen (foreign nonself protein) and its antibody (immunoglobulin).

Cells or tissues are incubated with

antibodies to their contents of proteins so that each antibody 14

These

binds specifically to its protein (antigen) denoting the protein location in the cell that can be seen by microscope. Polyclonal antibodies when the antibodies produced and collected contain a mixture of several groups of antibodies for different parts of proteins.

Monoclonal antibodies when the antibodies for

different parts of proteins are collected separately. The methods of immunocytochemistry could be direct or indirect. The direct method is one step but less sensitive; the tissue section is incubated directly with the antibody. The indirect method is more sensitive, more expensive and needs longer time. If rat protein (actin) is injected into another animal of different species such as rabbit, the latter will produce antibodies in his blood for rat actin (antirat actin antibodies). Antibodies from a normal non-injected rabbit must be injected into another animal of third species like a goat.

Goat will

produce antibodies againt rabbit antibodies. Rat tissue sections are incubated in solution containing antibodies produced by rabbit against rat proteins, then washed and reincubated with labeled goat antibodies against rabbit antibodies which already recognized the rat protein. b. Hybridization techniques: these techniques depend on binding between two single strands of nucleic acids that recognize each other if they are complementary.

This method allows

identification nucleic acid sequences.

In Situ Hybridization

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when this technique is applied directly to cells, chromosomes or tissue sections. c. Polymerase chain reaction (PCR): This is an in vitro method widely used in cell biology and diagnostic laboratories; it is used to synthesize specific DNA sequences using two oligonucleotide primers on both sides of DNA.

Tissue preparation for light microscopy To prepare tissue for light microscopic examination, living or dead tissue can be used.

Living tissues such as tissue cultures in transparent dishes are

examined by phase-contrast microscopes. Samples of dead tissue are used in the form of:  Sections on glass slides  Smears on glass slides for blood, mucus, urine, and bone marrow  Spreads for areolar connective tissue  Teased samples for muscle tissue Microtechnique: To prepare histology sections and get the tissue in its final shape on a microscopic slide, the tissue must pass through the following steps: 1. Obtaining the tissue in small pieces to facilitate penetration of the fixative into the tissue. 2. Fixation:

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Fixation could be done by physical (freezing) or chemical means (formaldehyde and gluteraldehyde). The tissue is immediately placed in a fixative such as buffered isotonic solution of 4% formaldehyde for routine light microscopy. The functions of a fixative are:  To stabilizes or cross-links proteins, and protein conjugates for firmer structures of the cell.  To prevent autolysis (tissue digestion by enzymes present within their cells).  To preserve tissue and molecula...