Introduction of Animal Histology Methods used in Histology Histology is the study of tissue. To examine the tissue components, it is necessary to process the tissue. The steps of processing involve: fixation, sectioning, and visualization. 1. 2. 3. 4. Making histological slide Histochemistry Immunohistochemistry Microautoradiography 1. Making histological slide a. Fixation--prevent decomposition, cell metabolism is stopped b. Dehydration—For LM and TEM samples b. Embedding– LM using paraffin, TEM using plastic monomers c. Sectioning—LM 5-10µm, TEM using 0.02-1.0µm thick section d. Staining—LM sections with dyes or fluorescent tags, TEM treating the section with heavy metal salts e. Cover slipping 2.Histochemistry • The branch of science that deals with the chemical composition of the cells and tissues of the body , including cytochemistry 3.Immunohistochemistry • Immunohistochemistry is a method of analyzing and identifying cell types based on the binding of antibodies to specific components of the cell. It is sometimes referred to as immunocytochemistry. 4. Microautoradiography • Technique for the study of surfaces of solids by monochromatic-radiation (such as x-ray) contrast effects shown via projection or enlargement of a contact radiograph. B. Vital(live) tissue study 1.Tissue culture(cell preparation) 2.Vital staining(Isotope) A technique in which a harmless dye is used to stain living tissue for microscopical observation. The stain may be injected into a living animal and the stained tissue removed and examined (intravital staining) or the living tissue may be removed directly and subsequently stained (supravital staining). Microscopic organisms, such as protozoa, may be completely immersed in the dye solution. Vital stains include trypan blue, vital red, and Janus green, the latter being especially suitable for observing mitochondria. Read more: vital staining - intravital staining, supravital staining - Living, Tissue, and Dye http://science.jrank.org/pages/20597/vital-staining.html#ixzz1ApqVcrFj 3.Cell fusion(cloning) 4. Cell electrophoresis C. New technology used in Histology study • Freeze-fracture-etch overcomes the inherent limitation of thin specimen sections to real details of the internal structure of membranes and intercellular junctions. • Cell morphometry • Flow cytometry(FCM) • Microspectrophotometry D. Microscope 1.Light microscope(LM) a. Biomicroscope b. Fluorescence microscope c. Phase contrast microscope d. Darkfield microscope e. Polarizing microscope f. Confocal laser scanning microscope 2.Electron microscope, EM • Transmission electron microscope (TEM) • Scanning electron microscope(SEM) • Ultrahigh voltage electron microscope Staining reaction • • Acidophila-tissue components that binds acidic dyes(eosin, orange C), + Erythrocyte cytoplasm, collagen fibers, mitochondria, lysosomes Basophila- tissue binds basic dyes(hematoxylin, methylene blue), Nuclei,RER,extracellular matrix • Metachromasia-change in color, is a property of certain dyes(usually basic dyes,(low-blue), (high-purple) • Hematoxylin-Eosin (HE) Reaction • Periodic Acid Schiff Reaction (PAS) This an all-around useful stain for many things. It stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi. A predigestion step with amylase will remove staining for glycogen. PAS is useful for outlining tissue structures--basement membranes, capsules, blood vessels, etc. It does stain a lot of things and, therefore, can have a high background. It is very sensitive, but specificity depends upon interpretation. • Silver staining Hematoxylin and Eosin: The most common stain used in histology is hematoxylin and eosin or H&E. Hematoxylin stains acidic tissue components such as the RNA in these nuclei purple. Acidic tissue components stained with this basic dye are referred to as basophilic structures. Eosin stains components pink to orange; components which react with this acidic dye are acidophilic. H and E staining Hematoxylin is the oxidized product of the logwood tree known as hematein. Since this tree is very rare nowadays, most hematein is of the synthetic variety. In order to use it as a stain it must be "ripened" or oxidized. This can be done naturally by putting the hematein solution on the shelf and waiting several months, or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution. Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus. Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing. Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues. Microscopy The microscope is an important tool in your study of histology. You need to understand and have a working knowledge of the Parts of the microscope and what objective engravings mean so you can use this tool most efficiently. Also, the principles of image formation are important. As you work through your slide set, you will be using slides stained with hematoxylin and eosin plus a variety of special stains that reveal different structures within the tissue. In addition, you must be able to recognize artifacts of preparation. Parts of the microscope The image on the left indicates the major parts of the microscope. Objective engravings The engravings on the microscope objective provide information about the use of the objective. The objective below is a flat field objective which means the image is in focus across the entire field. Magnification is the number of times the image of the original specimen is increased in size by the objective lens. The numerical aperture is related to resolution; the higher the N.A., the higher the resolving power of the objective. Tube length is the distance from the objective lens to the eyepiece objective. Objectives should be used with a microscope body having matched tube length. Coverslip thickness indicates the thickness of the glass which should be used to cover the specimen. Image formation through the microscope Scroll to the bottom of this diagram and note that the light source emits light which travels through the condenser lens and then the specimen. The objective lens projects an inverted and reverted primary image. The eyepiece lens, in concert with the lens of the eye, forms a secondary image which is again inverted and reverted (or normal in orientation). The brain interprets the secondary image and projects it back to the level of the stage as a virtual image which is once again inverted and reverted. Thus, the image that you view through the microscope is "upside down and backwards." Special stains: Special stains, other than the standard hematoxylin and eosin (H&E) stain, can give you more information about the tissue. To the left is a PAS stain which emphasizes the basement membrane (black arrow) and the brush border (blue arrow) of the cells. The stain colors carbohydrates in these regions. These structures would not be as heavily stained with H&E. This trichrome stain helps differentiate muscle fibers (red) from connective tissue proper (blue). With H&E, both of these tissue would stain pink with eosin. Gridley's reticulum stain emphasizes fine black reticular fibers which would not stand out in a standard H&E preparation. Artifacts in tissue preparations: The processing of tissues sometimes results in artifacts in the final tissue section. Some more common artifacts are shown in the image to the left. (1) Extraneous fiber on the coverslip. Note how it is out of focus. (2) Tissue fold the tissue has folded on top of itself. (3) Chatter - cracks in the tissue section which occur during cutting. The images on the left show the tissue and debris (blue arrow) in focus on the far left. The fiber (black arrows) is out of focus and thus, above the plane of the tissue. When the fiber is in focus (right image), the plane of the tissue is out of focus. You can use this focusing principle to determine if a structure is within the tissue or not. Basic Cell Biology The cell is the basic structural unit of the tissue and organs of the body. Various Cell shapes include spherical, stellate, spindle, polyhedral, squamous, cubodial Or columnar. Cell sizes range from a single micrometer to several centimeters in diameter Cell Membrane (1) • Each cell in the body is bounded by a cell membrane (plasma lemma) which provides a barrier and controls movement of substances into and out of the cell. The cell membrane is a lipid bilayer with embedded proteins. • Integral proteins are tightly bound within the membrane and often extend across it as transmembrane proteins. These transmembrane proteinss frequently form ion channels or carrier proteins that transport molecules across the cell membrane. Cell Membrane (2) • Peripheral proteins, located on the cytoplasmic surface of the cell membrane, are more loosely bound to other membrane proteins or lipids. • A glycocalyx, comprised of carbohydrates on the outer surface of the cell membrane, functions in cell recognition, adhesion, absorption and antigenicity Nucleus • More than one nucleus can be present in a cell. Within this spherical structure, deoxyribonucleic acid (DNA) is transcribed and ribonucleic acid (RNA) is synthesized. The surrounding nuclear envelope is formed by two adjacent bilaminar lipid membranes with embedded proteins. Scattered nuclear pores, which perforate the envelope, regulate passage of substances between the cytoplasm and the nucleus. Nucleus • Chromatin, primarily comprised of DNA, is located within the nucleus. The inert form of chromatin, heterochromatin, stains intensely while euchromatin, which is actively involved in protein production, stains lightly. Nuclear chromatin condenses to form chromosomes during cell division. Also within the nucleus is the nucleus is the nucleolus that is the site of rRNA synthesis. The number and size of cell nucleoli are related to the amount of protein synthesis occurring within the cell. Cytoplasm • The cytoplasm, which surrounds the nucleus and organelles of the cell, varies in composition of water, protein, carbohydrates and salts. • A cytoskeleton of microfilaments, intermediate filaments and microtubles provides structure for cell shape and movement. Organelles and inclusions (1) • Granular endoplasmic reticulum (rough endoplasmic reticulum, rER) is comprised of membranous cisternae with attached ribosomes. The ribosomes, made up of ribosomal RNA, translate messenger RNA from nucleus. As a result of the translation, specific proteins are formed within the lumen of the cisternae and are subsequently packaged for export outside the cell. Free ribosomes may also be present in the cytoplasm and function in the production of intracellular proteins. Organelles and inclusions (2) • Agranular endoplasmic reticulum (smooth endoplasmic reticulum, sER) lacks ribosomes and is associated with glycogen synthesis, steroid production and cell detoxification. • The Golgi comples, a curved stack of membranes in the cytoplasm, receives proteincontaining transport vesicles from the rER on the convex surface and releases secretory vesicles from the concave surface. While passing through the Golgi complex, proteins are glycosylated, phosphorylated or sulfated in preparation for export. Organelles and inclusions (3) • Mitochondria have a smooth outer membrane while the inner membrane is folded into characteristic cristae. Matrix granules are also present. Mitochondria produce chemical energy for the cell through the tricarboxylic acid cycle, oxidative phosphorylation and fatty acid oxidation. • Many different types of vesicles containing material being transprted through the cell are also presnet in the cytoplasm. These vesicles may be coated with materials such as clathrin or coatomer. The vesicles shuttle between the cell surface, Golgi apparatus, granular endoplasmic reticulum or lysosomes depending on the transport pathway. • Other inclusions within the cell often include secretory granules, nutrients such as glycogen and lipid, and pigments. Cell Surface Modifications • Depending on cell shape and function, the surface membrane may be modified to form special stuctures. Microvilli are short, finger-like projections of the cell membrane which are non-motile, but dramatically increase cell surface area for absorptive functions. • Stereocilia are long microvilli limited to the epididymis and ear. • Cilia and longer flagella contain microtubules arranged in a characteristic pattern. Nine pairs of microtubules occupy the periphery of the cilium or flagellum while one pair is found in the center. Both cilia and flagella are motile. Another cilium-like structure, the kinocilium, is found in the ear. The cell---Overview • The cell is the basic structural unit of tissues • A lipid bilayer with embedded proteins forms the cell membrane • The nucleus controls cell function • Granular endoplasmic reticulum and associated ribosomes produce proteins • Agranular endoplasmic reticulum is assocaited with steroid production and detoxification • The Golgi complex packages protein for export • Mitochondria provide energy for the cell • Cell surface modifications include microvilli, cilia, flagella and stereocilia.