Lecture 1 Introduction to Histology and Types of Tissues - General Histology

Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 Subject: General Histology for Second year Students of Dentistry Department Credits Units: 4 Theory Lecture: 2hrs Practical Lab: 2 hrs Dr....

Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 Subject: General Histology for Second year Students of Dentistry Department Credits Units: 4 Theory Lecture: 2hrs Practical Lab: 2 hrs Dr. Ibtesam Radhi Thammer PhD in Applied Embryology 1 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 Course Overview The principal objective of this course is to provide students with an understanding of the structural and functional organization of the human body at the cellular and subcellular levels. Introduction to Histology: Histology is the branch of anatomy that focuses on the study of tissues of animals and plants. The term tissue refers typically to a collection of cells. In humans, organs comprise two or more tissue types, including epithelial, connective tissue, nervous, and muscular. The word “histology” stems from the Greek word “histos,” meaning web or tissue, and “logia,” meaning branch of learning. In brief, histological processing involves obtaining fresh tissue, preserving it (i.e., fixing it) in order to allow it to remain in as life-like a state as possible, cutting it into very thin sections (3–8 microns), mounting it on glass microscopic slides, and then staining the sections so that they can be observed under a microscope to identify different histological components within the tissue. Techniques and Microscopes Uses in Histology The microscope allows us to visualize images and objects that are too small to be seen by the naked eye. Microscopy has evolved tremendously since its inception in the late 1500’s, and has proven an invaluable tool for the advancement of biomedical knowledge. The microscope is an essential tool for any histology course and even though technology has given us digital microscopes on our computers, it is important to learn the basics of light microscopy and to understand other microscopic instruments. 2 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 A- Different Kinds of Microscopes & Their Uses There are several types of microscopes including:- 1- Light Microscopy 2- Dissecting Microscopy 3- Fluorescence Microscopy 4- Phase-Contrast Microscopy (Inverted Microscopy) 5- Confocal Microscopy 6- Electronic Microscopy 1- Light Microscope Light microscopes are extremely versatile instruments. They can be used to examine a wide variety of types of specimen, frequently with minimal preparation. Light microscopes can be adapted to examine specimens of any size, whole or sectioned, living or dead, wet or dry, hot or cold, and static or fast-moving. Figure 1: Light Microscope. 3 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 2- Dissecting Microscopy These are also known as stereoscopic microscopes. This is a type of digital optical microscope designed with a low magnification power (5x-250x), by use of light reflected from the surface of the specimen, and not the light reflected the specimen. Its primary role is for dissection of specimens and viewing and qualitatively analysing the dissected samples. Figure 2: Dissection Microscope. 4 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 3- Electronic Microscopy Electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it.... Its resolution & magnification is about 10,000,000x. There are two main types of electron microscope – the transmission electron microscopy (TEM) and the scanning electron microscopy (SEM). The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc) through which electrons can pass generating a projection image. Figure 3: Scanning Electron Microscope. 5 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 4- Inverted Microscope Inverted microscopes are almost similar to a simple microscope, but all the components are placed in the inverted condition. The Inverted microscope comes with its own light source and a condenser lens. Which are located at the top portion of the microscope and is pointing down. Whereas the objectives and the turret are located below the stage, pointing upwards. In this microscope, we observe the specimen from down, (upwards) instead of from the up. Inverted Microscopes are useful for:- 1- The observation of living specimens or tissues. 2- Cell division process can be observed by using this microscope which is not possible by using a conventional compound microscope. Living cells can be observed for a long time by using this microscope, which makes it more useful. Figure 4: Inverted Microscop 6 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 5- Fluorescence Microscopy Fluorescence microscopy is a basic requirement in cell biology, molecular biology and biotechnology. Advancements over the years has helped scientist to trace molecules in live cells and understand the basis of cell metabolism, exchange, mutation and toxicity. Fluorescence microscopy is an optical microscopy technique that utilizes fluorescence, which is induced using fluorophores. A fluorophore is a type of fluorescent dye used to mark proteins, tissues, and cells with a fluorescent label for examination by fluorescence microscopy. A fluorophore works by absorbing energy of a specific wavelength region, commonly referred to as the excitation range, and re-emitting that energy in another specific wavelength region, commonly referred to as the emission range. Figure 5: Fluorescence Microscopy 7 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 6- Confocal Microscope. Confocal microscopy is broadly used to resolve the detailed structure of specific objects within the cell. Similar to wide-field fluorescence microscopy, various components of living and fixed cells or tissue sections can be specifically labeled using immunofluorescence, for example, and then visualized in high resolution. As a distinctive feature, confocal microscopy enables the creation of sharp images of the exact plane of focus, without any disturbing fluorescent light from the background or other regions of the specimen. Therefore, structures within thicker objects can be conveniently visualized using confocal microscopy. Furthermore, by stacking several images from different optical planes, 3D structures can be analyzed. The sample penetration depth is limited, however, when using confocal microscopy. Thicker objects, like large spheroids, organoids, tissue, and small animals, should instead be optimally imaged using two-photon microscopy. 8 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 Figure 6: Confocal Microscope. 9 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 B- Types of stain In the modern age of histology there have been significant improvements in histological stains and techniques. Advanced histological techniques are immunohistochemistry, antibody binding and electron microscopy. In the same line, advanced stains include: immunohistochemical (IHC), routine hematoxylin eosin (H&E) and the in situ hybridization. Modern stains used are;  Masson's Stain used in connective tissues  Golgi Stain used in neuronal fibres  Toluidine Blue  Immunological labeling that have fluorescent or enzymatic stains  Kluver-Barrera Stain used in Lipofuscin  Mallory's CT Stain  Periodic Acid-Schiff (PAS) Stain used in carbohydrates Preparation of Tissues for Study The most common procedure used in histologic research is the preparation of tissue slices or “sections” that can be examined visually with transmitted light. Tissue processing procedure 1. Obtaining a fresh specimen Fresh tissue specimens will come from various sources. It should be noted that they can very easily be damaged during removal from the patient or experimental animal. It is important that they are handled carefully and appropriately fixed as soon as possible after dissection. Ideally, fixation should take place at the site of removal, perhaps in the operating theatre, or, if this is not possible, immediately following transport to the laboratory. 10 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 2. Fixation The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps. 3. Dehydration Most of the water in a specimen must be removed before it can be infiltrated with wax. This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached. A typical dehydration sequence for specimens not more than 4mm thick would be: 1. 70% ethanol 15 min 2. 90% ethanol 15 min 3. 100% ethanol 15 min 4. 100% ethanol 15 min 5. 100% ethanol 30 min 6. 100% ethanol 45 min 4. Clearing Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax because wax and ethanol are largely immiscible. We, therefore, have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax. This solvent will displace the ethanol in the tissue, then this, in turn, will be displaced by molten paraffin wax. This stage in the process is called “clearing” and the reagent used is called a “clearing agent”. A popular clearing agent is xylene, and multiple changes are required to completely displace ethanol. 5. Wax infiltration The tissue can now be infiltrated with a suitable histological wax. Although many different reagents have been evaluated and used for this purpose over many years, the paraffin wax- based histological waxes are the most popular. A typical wax is liquid at 60°C and can be infiltrated into tissue at this temperature then allowed to cool to 20°C, where it solidifies to a consistency that allows sections to be consistently cut. 11 Dentistry Department General Histology Dr. Ibtesam Radhi Kut University College Lecture 1 6. Embedding or blocking out Now that the specimen is thoroughly infiltrated with wax, it must be formed into a “block” which can be clamped into a microtome for section cutting. This step is carried out using an “embedding centre” where a mold is filled with molten wax and the specimen placed into it. The specimen is very carefully orientated in the mold because its placement will determine the “plane of section”, an important consideration in both diagnostic and research histology. A cassette is placed on top of the mold, topped up with more wax, and the whole thing is placed on a cold plate to solidify. When this is completed, the block with its attached cassette can be removed from the mold and is ready for microtomy. Figure 7: Histological tissue processing steps. 12