1st ppt - asmph cell lecture

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ASPMH

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  • CELLULAR STRUCTUREAriel Jose U. Ampil, MD, FPSAInternist Diabetologist

  • Cell- smallest functional and structural unit of the human body Cytology- study of the individual cell and the structure it containsTissue- group of cells similar in structure, function and intercellular substancesHistology- science that deals with the study of tissuesMicroscopic anatomy- study of the human body with the aid of a magnifying object

  • MicroscopyMicrocopy is the aid in the study of the human body structure by the use of a magnifying instrument called the microscope. The microscope renders the details of the cells, tissues and organs.

  • MicroscopyThe useful magnification of an ordinary light microscope is about 1200X.The magnification of lens is the number of times the image of an object is enlarged compared with the actual size of the object.The total magnification of the object is solving the product of the separate magnification powers of the objective lens and ocular lens.

  • MicroscopyThe usefulness of any microscope is not only dependent on its ability to magnify but also on its ability to resolve details.The resolving power (resolution) is its capacity to give images of objects close together. It is measured as the smallest distance between two particles that can be distinguished from each other.

  • Compound MicroscopeA compound microscope is also called a light microscope.The principle involved is the refraction of light.The resolving power of the best light microscope is approximately 0.2 um.The magnifying image is about 1,500 times or 0.25 um.

  • Electron MicroscopeThis also known as the transmission electron microscope.The electron microscope functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses.

  • Electron MicroscopeElectrons are produced by high temperature heating of a metallic filament (cathode) in a vacuum.Its resolving power is very high and objects can be magnified over a hundred thousand times.It uses extremely thin preparations of tissues because of the low penetration of electrons.

  • Advantages of Electron MicroscopesVery high resolutionVery high magnificationHas greatly extended the understanding of the ultrastructure or molecular structure of cells

  • Disadvantages of Electron MicroscopesRequires a vacuum enclosed systemRequires ultra thin sections of specimensRequires a high voltageRequires the service of highly-trained personnelVery complex system Very expensive

  • Differences between light microscopes and electron microscopesLight microscopes use light while electron microscopes use electron beams emitted by tungsten filaments or electron particlesLight microscopes use glass lens while electron microscopes use electron magnetic fields or electric fieldsLight microscopes have a low resolving power while electron microscopes have a higher resolutionLight microscopes are open systems while electron microscopes are vacuum enclosed systemsLight microscopes offer direct viewing of objects with the naked eye, while electron microscopes use a fluorescent screen for viewingLight microscopes less expensiveLight microscopes do not require high voltage while electron microscopes do

  • Differences between light microscopes and electron microscopesLight microscopeElectron microscope

  • Other MicroscopesScanning electron microscope a variant of the electron microscope which permits a three dimensional analysis of surfaces of fixed and dehydrated cells, organs or small organisms. The image is formed indirectly by accumulation from the specimen point by point. The electron beams do not have to pass thru the specimen so that there is no need to cut ultrathin sections but there is less resolution than the transmission electron microscopePhase contrast microscope a modification of the light microscope, particularly useful for the study of unstained cells, either living or fixed. The different protoplasmic constituents produce phase changes because they vary in thickness and refractive index. The phase microscope converts phase variants into intensity variations and thereby enables the eyes to detect more contrast between different structures. The microscope is useful in the study of mitosis that renders the chromosomes and other cell organelles darker than the surrounding cytoplasmInterference microscope utilizes the principle of sending two separate beams of light thru the specimen, which are then recombined in the image plane. One beam is focused in a neutral area beside, above or below the observed structure. This provides tridimensional images of living cells and its components

  • Fluorescent microscope a microscope that uses selected wavelengths of light to illuminate the biological specimen. Specific molecules within the tissue absorb light and emit light and other wavelengths. The exiting wavelengths are absorbed with filters and emitted wavelengths are viewed in the microscope objectives. It is useful in localizing antigen-antibody complexes within tissuesPolarizing microscope microscopes that detects linearly oriented structures of living cells in tissue cultures or in fixed stained preparations. It restricts the light to preferred directions and orientations making it possible to detect the presence or orderly arrangements of fibrous proteins or arrays of long molecules. It is useful in viewing the spindle fibers of dividing cells or the bonding pattern of striated musclesUltraviolet microscope this makes of the ultraviolet light by passing visible light thru quartz or reflecting lenses. It is used more for the natural contrast it provides owing to the absorption of nucleic acids and proteins in the ultraviolet regionsDark field microscope this utilizes oblique light that do not enter the objective lens. A special dark field is employed and a vacant field of view shows merely a dark background while the object appears brightX-ray microscope x-rays have shorter wavelengths and therefore have greater penetration and theoretically a higher resolving power. Penetration technique allows the specimen to be placed upon a photographic emulsion and exposed to soft irradiation. The small x-ray picture obtained is enlarged or magnified optically

  • Microtechnique Microtechnique is also known as Histotechnique.Microtechnique is a method of preparation of tissue for microscopic examination.

  • Methods of MicrotechniqueParaffin wax methodIt uses paraffin for impregnation of tissue at room temperature.It is the most common procedure used in the study of tissue in the laboratory and hospital set-upFrozen section methodIt is a quick method in which the tissues are hardened at low temperature by the use of cryostat or freezing microtomeCelloidin methodIt uses celloidin as embedding medium, which is useful for large objects (eg. whole brain) and for hard and brittle specimens (eg. cartilage)

  • Steps in Paraffin MethodProcurement of tissue specimenFixation submerging the specimen in a chemical substance in order to preserve the tissuePurpose:To preserve tissue morphologyTo act as disinfectantTo kill microorganismsTo harden the tissueTo permit better staining reaction

  • Steps in Paraffin MethodFixationFixative used:10% neutral formalin (most commonly used)10% formol salineBouins solutionZenkers solutionPotassium bichromateOsmic acidGlutaraldehyde

  • Steps in Paraffin MethodDehydration immersing the tissue in increasing concentration of alcoholPurpose: to remove water from the tissueDehydrating agent: graded concentration of ethyl alcohol (65 to 100% alcohol) Clearing (dealcoholization) replacing the alcohol with clearing agentsPurpose: to impregnate tissue with paraffin solvent, since alcohol is insoluble with paraffin; it must be replaced by a clearing agent so that it can be impregnated with paraffin wax

  • Steps in Paraffin MethodClearingClearing agentsXylene (Xylol) most commonly usedChloroformBenzeneCedar wood oilEtherCarbon tetrachlorideInfiltration of tissue with melted paraffin wax

  • Steps in Paraffin MethodEmbedding placing the tissue in paraffin blocksPurpose: paraffin penetrates all intercellular spaces and even into the cells making the tissues more resistant in sectioningSectioning cutting of tissue by use of microtome about 3 5 um in thickness. The cut tissue, paraffin ribonettes, are placed in a basin of waterPlacing the cut sections in clean glass slides. Egg albumin is used as adhesive

  • Steps in Paraffin MethodDissolving or melting the embedding medium (paraffin wax) by passing the slide over a flame (alcohol lamp or bunsen burner)Rehydration immersing the tissue in decreasing concentration of alcoholStaining with appropriate stainHematoxylin and Eosin (H&E stain) is usually used. Hematoxylin, a basic dye, gives a bluish or purple color tothe nucleus. Eosin, an acid dye, imparts a reddish or pinkish color to the cytoplasm

  • Steps in Paraffin MethodDehydration immersing again the tissue in increasing concentration of alcoholClearing with clearing agentsMounting placing cover slip with a few drops of Canada balsam as a mounting medium on processed tissueLabelling label the organ on the prepared slides

  • CellComprise the greater part of the body Basic functional and structural unit of living organisms2 basic cell typesEukaryotic cellCell type that has a true nucleus surrounded by a nuclear membrane or envelopeProkaryotic cellLacks a nuclear envelope and the nuclear substance is mixed or is in direct contact with the rest of the protoplasm

  • Principal Parts of the CellCell membraneForms the outer boundary of all eukaryotic cellsAlso called plasma membrane or plasmal