2.2 PREPARATION OF TISSUES FOR STUDY-DR. MILIMO.pptx

Introduction to Anatomy & Physiology Preparation of tissues for study DR. MILIMO BSc.HB, MBChB, MPH, MSc.EB*,DTM, AdvCert.Psy. PREPARATION OF TISSUES FOR STUDY  Fixation: Small pieces of tissue are placed in solutions of chemicals that cross-li...

Introduction to Anatomy & Physiology Preparation of tissues for study DR. MILIMO BSc.HB, MBChB, MPH, MSc.EB*,DTM, AdvCert.Psy. PREPARATION OF TISSUES FOR STUDY  Fixation: Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure. Dehydration: The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water. Clearing: Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible. Infiltration: The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance. Embedding: The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden. Trimming: The resulting paraffin block is trimmed  Fixation and Fixatives Chemical substances like formalin, mercuric chloride, acetic acid, picric acid and glutaraldehyde are used as fixatives to preserve tissues. All fixatives have both desirable and undesirable effects. A combination of these fixatives is often prepared to get the maximum desirable effect. Such combinations commonly used are: 1. Bouin’s fluid (formalin, acetic acid and picric acid) 2. Formal sublimate (formalin and mercuric chloride) 3. Helly’s fluid (formalin, mercuric chloride and potassium dichromate) 4. Zenker’s fluid (acetic acid, mercuric chloride and potassium dichromate) Small pieces of fresh tissues are placed in common fixatives like 10% neutral formal saline for 24 hours.  The purpose of fixation is – To preserve the morphology and chemical composition of the tissue, – To prevent autolysis and putrefaction, – To harden the tissue for easy manipulation, – To solidify colloidal material, and – To influence staining. After fixation, some hard tissues like bone and tooth, which contain large amount of calcium salts, require an additional step called decalcification before they are subjected for dehydration. Decalcification makes the hard tissues soft, enabling them to be cut with microtome. For decalcification, several decalcifying agents are used, namely 10% nitric acid, 5% trichloroacetic acid and ethylene diamine tetra acetic acid (EDTA).  Dehydration Water from the tissues is removed in a gradual manner by immersing the tissues in ascending grades of alcohol, viz. 50%, 70%, 90% and absolute alcohol, in order to embed it in paraffin wax which is not miscible in water. Tissue remains in each of these grades for 30–60 minutes.  Clearing After dehydration the tissue is treated with a paraffin solvent (clearing agent) like xylene or toluene for 2-3 hours. These agents penetrate and replace the alcohol from the tissue and make it translucent (clear)  Embedding In order to obtain thin sections with microtome, tissue is infiltrated with embedding medium which gives a rigid consistency to the tissue. The various embedding media are paraffin wax, celloidin, gelatin, plastic resins (for EM), etc. Paraffin is the routinely used embedding medium for light microscopy. Embedding involves two steps, namely, impregnation and casting or block making. A. Impregnation: After clearing, the tissue is impregnated with molten paraffin wax (at 58°–60 °C) in a hot air oven for 2 hours with three changes. The melting point of paraffin wax is 56 °C. B. Casting or block making: After impregnation, the tissue is placed in ‘L’ moulds containing molten paraffin. The molten wax cube with the tissue is allowed to cool and the paraffin block is then removed from the mould.  Section Cutting (Microtomy) 5–7 μm-thick sections are cut with a rotary microtome. The cut paraffin sections are affixed to albuminised glass microslides after flattening the sections over warm water. The microslides with sections are either air dried or dried in an incubator overnight at 37 °C and stored for staining at room temperature.  STAINING PROCEDURE Staining is done routinely by using a basic and an acidic dye that stain tissue components selectively. Tissue components that stain more readily with basic dyes (e.g. anionic cell components) are termed basophilic and are blue in colour and those with an affinity for acid dyes (cationic components) are termed acidophilic and are pink/orange in colour. The basic dyes are haematoxylin, toluidine blue and methylene blue. The acidic dyes are eosin, orange G and acid fuchsin. Of these dyes, the combination of haematoxylin and eosin (H&E) is most commonly used in histological staining procedure. However, special stains like periodic acid Schiff reagent (PAS), osmic acid, Mallory and Masson’s, trichrome stains are being used to selectively identify certain tissue components. Haematoxylin usually stains the acid component (nucleus) of the cell, blue or black, whereas eosin stains the basic components present in the cytoplasm, pink. Deparaffinization To remove the paraffin from the section, the slides are treated with xylol. Three changes are necessary, each for 3–5 minutes. Hydration The slides are passed through the following series to hydrate the sections: – Absolute alcohol – 5 min (with 2 changes) – 90% alcohol – 3 min – 70% alcohol – 3 min – 50% alcohol – 3 min [Wash in] Distilled water – 3 min Staining For differential staining (the commonly used technique), following steps are involved: A staining with haematoxylin for 5–7 minutes. – Washing well in running tap water until the section becomes blue. – Differentiation with 1% acid alcohol for 5 seconds. – Washing in running tap water again, until the section becomes blue. – Staining with 1% eosin for 1 minute. Dehydration The stained sections are dehydrated in the following series: – 50% alcohol – 10 sec – 70% alcohol – 10 sec – 90% alcohol – 30 sec – Absolute alcohol – 5 min (with 2 changes) Clearing and Mounting The sections are cleared in xylene and mounted in DPX (Mixture  MICROSCOPY Once the paraffin sections are stained with haematoxylin and eosin (H&E) or with some special stains, it can be viewed through a light microscope. Bright-Field Microscopy With the bright-field microscope, stained tissue is examined with ordinary light passing through the preparation. The microscope includes an optical system and mechanisms to move and focus the specimen.  Dark-ground (Dark field) microscope Dark-ground microscope is a modified light microscope where the objects are examined by dark ground illumination. Dark ground illumination is obtained simply by inserting a small circle of black paper in the centre of the filter carrier of the condenser. The central rays which would normally pass through the object and into the objective are cut off and the peripheral rays from the condenser pass through the object, but do not enter the objective; the only light entering the objective will be that scattered (refracted) by the object, which makes the object bright and self- luminous against a dark background with a high degree contrast. This microscope is used to examine extremely minute particles (colloid suspension) or large transparent objects (e.g. living protozoa, crystals, etc.) which are otherwise invisible with ordinary light microscope.  Fluorescence Microscopy When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength— a phenomenon called fluorescence. In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear bright on a dark background. For fluorescent microscopy, the instrument has a source of UV or other light and filters that select rays of different wavelengths emitted by the substances to be visualized.  Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains. Acridine orange, which binds both DNA and RNA, is an example. When observed in the fluorescence microscope, these nucleic acids emit slightly different fluorescence, allowing them to be localized separately in cells. Other compounds, such as DAPI and Hoechst stain, specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV. Another important application of fluorescence microscopy is achieved by coupling compounds such as fluorescein to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures under the microscope. Antibodies labeled with fluorescent compounds are extremely important in immunohistologic staining.  Phase-Contrast Microscopy This microscope has been developed based on the fact that light passing through any transparent object mounted in a medium of a different refractive index slows down and changes its direction. Within the cell, different organelles exhibit different refractive indices and consequently alter the phase of the light that passes through them to different extents. These phase differences are transformed into differences of light intensity (by means of a special optical system) so that structures within the cells become visible in high contrast and with good resolution. So this microscope is being used to view any transparent living biological specimens. (There is no need to stain the specimen.) Unstained cells and tissue sections, which are usually transparent and colorless, can be studied with these modified light microscopes. Phase-contrast microscopy uses a lens system that produces visible images from transparent objects and, importantly, can be used with living, cultured cells.  Because they allow the examination of cells without fixation or staining, phase-contrast microscopes are prominent tools in all cell culture laboratories. A modification of phase-contrast microscopy is differential interference contrast microscopy with Nomarski optics, which produces an image of living cells with a more apparent three-dimensional (3D) aspect. Confocal Microscopy With a regular bright-field microscope, the beam of light is relatively large and fills the specimen. Stray (excess) light reduces contrast within the image and compromises the resolving power of the objective lens. Confocal microscopy avoids these problems and achieves high resolution and sharp focus by using (1) a small point of high-intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confocal), and unfocused light does not pass through the pinhole. This greatly improves resolution of the object in focus and allows the localization of specimen components with much greater precision than with the bright-field microscope. Confocal microscopy involves scanning the specimen at successive focal planes with a focused light beam, often from a laser, and produces a 3D reconstruction from the images.  Polarizing Microscope Polarizing microscope is a modified light microscope with two Polaroid filters. The first filter is placed below the condenser and is called polarizer and the second filter is placed between the objective and the eyepiece and is called analyser. When both polarizer and analysers are kept with their main axes at right angle to one another, no light passes, resulting in a darkfield effect. However, when structures oriented in a linear (e.g. bones, muscle, collagen, nerve fibres) or radial fashion (e.g. lipid droplets, starch granules) are examined, they appear as bright structures against a dark background because they are able to rotate the direction of the vibration of polarized light. The capacity to rotate the direction of the vibration of the polarized light is called birefringency and is present in crystalline substances or biological materials containing oriented molecules Electron microscopy QUIZ Identify Parts A – F Identify the epithelium and give 2 reasons why Give an example of organ where it is found