Histological Techniques and Microscopy PDF

HISTOLOGICAL TECHNIQUES AND MICROSCOPY Lecture 1 Topic Outline: Introduction General Architecture of the Body Units of Measurement Used in Histology Interpretation of a Section Processing of Tissues for Light Microscopy (Paraffin Wax Embedding) Staining Procedure Mic...

HISTOLOGICAL TECHNIQUES AND MICROSCOPY Lecture 1 Topic Outline: Introduction General Architecture of the Body Units of Measurement Used in Histology Interpretation of a Section Processing of Tissues for Light Microscopy (Paraffin Wax Embedding) Staining Procedure Microscopy Review INTRODUCTION Histology ○ Greek words: Histos - web (tissue) Logos - the study of histology is used not only for the study of tissue alone but also for the study of cells and fine structure of organs and can collectively be called microscopic anatomy. provides a structural basis for functional correlation of an organ or tissue is a necessary prerequisite to the study of the abnormal tissue (pathology) GENERAL ARCHITECTURE OF THE BODY Tissues composed of: 1. specialized cells 2. Intercellular matrix There are four basic types of tissues in the body and each one performs a specific function: a. Epithelial tissue – protection b. Connective tissue – support c. Muscular tissue – contraction d. Nervous tissue – conduction UNITS OF MEASUREMENT USED IN HISTOLOGY 1. For Light Microscopy a. The term micrometer (μm) is being used nowadays instead of micron (μ). b. 1 micrometer or micron = 0.001 mm or 10–6 m. 2. For Electron Microscopy a. The term nanometer (nm) is being used nowadays instead of angstrom (A°). b. 1 nanometer = 0.001 (μm or 10–9 m. c. 1 angstrom = 0.1 nm or 10–10 m. INTERPRETATION OF A SECTION 3D -> 2D Serial sectioning of the tissue is prepared and studied in a sequential order to get information about the three-dimensional architecture of the structures. PROCESSING OF TISSUES FOR LIGHT MICROSCOPY (PARAFFIN WAX EMBEDDING) Tissues are processed by the following procedure to obtain thin translucent sections so that they can be examined under microscope by transillumination. a. Fixation b. Dehydration c. Clearing d. Embedding e. Section Cutting (Microtomy) Fixation and Fixatives 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) Fixation and Fixatives Small pieces of fresh tissues are placed in common fixatives like 10% neutral formal saline for 24 hours. The purpose of fixation is a. to preserve the morphology and chemical composition of the tissue, – to prevent autolysis and putrefaction, b. to harden the tissue for easy manipulation, c. to solidify colloidal material, and d. to influence staining. Decalcification 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: ○ 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: a. paraffin wax, celloidin, gelatin, plastic resins (for EM), etc. Paraffin is the routinely used embedding medium for light microscopy. Embedding involves two steps a. impregnation b. casting or block making. Embedding 1. Impregnation a. 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. 2. Casting or block making a. After impregnation, the tissue is placed in ‘L’ moulds containing molten paraffin. b. 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 is done routinely by using a basic and an acidic dye that stain tissue components selectively. Basophilic: ○ Tissue components that stain more readily with basic dyes and are blue in colour Acidophilic: ○ affinity for acid dyes and are pink/orange in colour. Examples of stains: ○ Hematoxylin & Eosin (H&E) - most commonly used ○ Periodic Acid Schiff (PAS) reagent ○ Osmic acid ○ Mallory and Masson’s ○ Trichrome stain STAINING PROCEDURE 1. Deparaffinization 2. Hydration 3. Staining 4. Dehydration 5. Clearing and Mounting STAINING PROCEDURE 1. Deparaffinization: a. To remove the paraffin from the section, the slides are treated with xylol. Three changes are necessary, each for 3–5 minutes. 2. Hydration: a. 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 PROCEDURE 3. Staining a. For differential staining (the commonly used technique), following steps are involved: A staining with haematoxylin for 5–7 minutes. i. Washing well in running tap water until the section becomes blue. ii. Differentiation with 1% acid alcohol for 5 seconds. iii. Washing in running tap water again, until the section becomes blue. iv. Staining with 1% eosin for 1 minute. STAINING PROCEDURE 4. Dehydration a. The stained sections are dehydrated in the following series: i. 50% alcohol – 10 sec ii. 70% alcohol – 10 sec iii. 90% alcohol – 30 sec iv. Absolute alcohol – 5 min (with 2 changes) 5. Clearing and Mounting a. The sections are cleared in xylene and mounted in DPX. Examples of Dyes BASIC DYES ACIDIC DYES Toluidine Blue Eosin Alcian Blue Orange G Methylene Blue Acid Fuchsin Hematoxylin dyes *acidic dyes stains the structures with positive charge or basic components in the tissue (ex: *basic dyes stains tissue components with net mitochondria, secretory granules, and collagen) negative or acidic structures (ex: DNA, RNA, Glycosaminoglycans) *basic structures are attracted to acidic stains = ACIDOPHILIC *acid structures are attracted to basic stains = BASOPHILIC (opposite attracts :)) Special Stains SPECIAL STAIN STRUCTURES BEING COLOR STAINED Feulgen stain DNA DNA - Red Background - Green Periodic Acid Schiff Stain Glycogen Glycogens - Purple/ Magenta Sudan Black Lipids Lipids - Blue to Black Gram Stain Bacteria Gram Negative Bacteria - Pink Gram Positive Bacteria - Purple/ Blue 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 Basic Principles of Some Special Microscopies 1. Dark Ground (Dark Field) Microscope a. is a modified light microscope where the objects are examined by dark ground illumination. b. Dark ground illumination is obtained simply by inserting a small circle of black paper in the centre of the filter carrier of the condenser. c. This microscope is used to examine extremely minute particles (colloid suspension) or large transparent objects (e.g. living protozoa, crystals, etc.) Basic Principles of Some Special Microscopies 2. Phase-contrast Microscope based on the refractive index 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. used to view any transparent living biological specimens. (There is no need to stain the specimen.) Basic Principles of Some Special Microscopies 3. Polarizing Microscope Modified LM with 2 filters: 1. Polarizer 2. Analyzer 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. birefringency - capacity to rotate the direction of the vibration of the polarized light, present in crystalline substances. Electron Microscopes it uses shorter wavelengths of electrons instead of light rays to achieve a very high resolution, as low as 3Å. This enables one to view fine structural details of cells and organelles. The electrons are deflected/scattered by a series of electromagnetic lens in a manner similar to light deflection by glass lens of optical microscope. TEM vs. SEM Transmission electron microscope scanning electron microscope utilizes the transmitted electrons that the electrons do not pass through the specimen because of its thickness and penetrate the specimen and are because of a coating formed by heavy produced due to scattering of incidental metals (e.g. gold). primary electrons. utilizing only the reflected (backscattered) These transmitted electrons are focused and secondary electrons which are by an objective coil or lens. The image deflected back at varying angles as a result of interaction between the gold coated obtained is further enlarged by one or surface and the primary incident beam of two projector coil or lens and is finally electrons falling on it. projected on a fluorescent screen or These electrons are collected by special photographic film to produce electron detectors that make electrical signals to a micrograph. television tube which gives a 3-dimensional image of the specimen surface effective tool to study the surface topography of a specimen has less resolution than TEM REVIEW While processing the tissues for paraffin embedding, dehydration is done by immersing the tissue in (a) alcohol only (b) xylol only (c) mixture of alcohol and xylol (d) formalin REVIEW Haematoxylin is a basic dye and it stains (a) the basic components of a cell only (b) the acidic components of a cell only (c) both basic and acidic components of a cell (d) none of these REVIEW The optical part(s) of a light microscope involved in magnification is the (a) condenser and filter (b) eyepiece only (c) objective only (d) both objective and eyepiece REVIEW State whether the following statements are true (T) or false (F): 1. Tissue is a collection of cells specialised to perform a specific function 2. The purpose of fixing a tissue is to prevent autolysis and putrefaction 3. Melting point of paraffin wax is 80 °C 4. Deparaffinization is done by treating the section with xylene 5. While staining a section, the microslides are stained first with eosin