L1-2 Histology Techniques PDF

L1,2 HISTO Micro technique and Microscopy Histology is the study of the microscopic structure of tissues. So, it is the microscopic anatomy of the tissues. Microtechnique is the art of preparing objects for examination under the microscope (light and electron microscopes) and...

L1,2 HISTO Micro technique and Microscopy Histology is the study of the microscopic structure of tissues. So, it is the microscopic anatomy of the tissues. Microtechnique is the art of preparing objects for examination under the microscope (light and electron microscopes) and of preserving objects so prepared. Preparation of histological sections A) For light microscope (LM): 1. Paraffin technique. 2. Celloidin method. 3. Freezing technique. B) For electron microscope (EM) Paraffin technique (routine histological section) 1. Selection and obtaining tissue. 2. Fixation. 3. Washing. 4. Dehydration. 5. Clearing. 6. Infiltration and Embedding. 7. Microtomy and Sectioning. 8. Mounting. 10. Hydration. 12. Dehydration. 9. Deparaffinization. 11. Staining. 13. Clearing. 14. Mounting. 15. Cleaning and Labeling 1. Selection and obtaining tissue - Samples must be obtained from living tissue (biopsy) or very soon and as rapidly as possible after death (autopsy). - Samples are taken using very sharp razors to prevent distortion of tissue during cutting. - Samples should be small size to allow the penetration of fluid through it up to the most interior part. 2. Fixation - The tissue is put immediately in a suitable fixative. - Fixation is the process of tissue preservation through coagulation of the protoplasm. - The most commonly used fixative is formalin (buffered 10%). - Purpose of fixation: 1. Prevent autolysis as it destroys lysosomal enzymes. 2. Prevent putrefaction by killing bacteria. 3. Coagulate the proteins and harden the tissue making it easier for cutting into thin sections. 4. Some fixatives have mordating effect (increase the affinity of tissue for stains). 1|Page  L1,2 HISTO 3. Washing - After the tissue is fixed for the proper length of time, excess fixative is washed out - Washing also removes substances in the fixative which might interfere with the subsequent processing. - Since most fixatives are aqueous solutions, the washing is usually carried out for specific period of time in cold running tap water. 4. Dehydration - Removal of water is a necessity to embed the tissue in a wax or resin material to be ready for sectioning. - Since the embedding media are immiscible with water, dehydration (removal of water) must be undertaken to prepare the wax block. - Dehydration is usually done by ascending grades of ethyl alcohol (50, 70, 90, 100%). The gradual ascending concentrations are used to prevent sudden withdrawal of water which may cause distortion and damage of the tissue. 5. Clearing - Paraffin is not soluble in alcohol. - The alcohol is replaced by a substance in which paraffin is soluble, such as xylene, toluene, or benzene. 6. Infiltration and Embedding - The transformation of the cleared tissue into a paraffin block is termed embedding. - If the tissue is only surrounded with wax, sectioning will cause the tissue to be separated from its surrounding wax medium. Wax, thus, must be surrounding and passing into the inside of the tissue; the latter is termed infiltration. - Infiltration is done first by some sort of low molecular weight wax (soft paraffin). - Soft paraffin, thus, replaces the clearing agent and hard paraffin is used in the following step to conform the block around the tissue. 7. Microtomy and Sectioning - Sectioning is done using the microtome. - Microtomes in common use are usually the rotary types although other types are used in tissue preparation such as the sliding microtome usually used in sectioning of dense tissue (bone and skin). - Section thickness usually suitable for routine histological study is 4-7 um. 8. Mounting Sections are mounted onto a glass slide and left to dry 2|Page  L1,2 HISTO 9. Deparaffinization - Most stains in common use are natural salts having a basic and acidic radical. Such stains are usually in the form of aquous solutions. - Sections on the slide is surrounded and infiltrated with paraffin which immiscible with water. - Paraffin is removed by xylol 10. Hydration - Hydration of the tissue is a necessity to enable the stain reach and react with the tissue components. - Rehydration of the tissue is achieved by descending grades of alcohol (100, 90, 70, 50%), distilled water is then used to replace the alcohol. 11. Staining - The nucleus is stained first (by haematoxylin) because the use of acidic dyes (e.g. eosin) to stain the cytoplasm may impart some color which prevents perfect staining of the nucleus. So, haematoxylin (H or Hx) is used first then eosin (E). - Washing with water is done between the two steps and after eosin. 12. Dehydration by ascending grades of ethyl alcohol (50, 70, 90, 100%). 13. Clearing by xylene, it makes the stained tissue sections transparent. 14. Mounting to convert the slide into a permanent preparation used in the study of section for months or even years, the section is mounted (covered) with a very thin cover slip using a sticky mountant (Canda balsam or De Pe Xe) 15. Cleaning and Labeling Advantages of paraffin technique 1. It takes a short time. 2. It gives serial sections (ribbon). 3. It gives very thin sections. 4. The sections are easily stained. Disadvantages of paraffin technique 1. The fixatives and heat used may damage the tissues. 2. The fat contents of the cells are dissolved during preparation. 3. It is not suitable for histochemistry. 4. It can not used for large pieces of tissues with large cavities 3|Page  L1,2 HISTO Celloidin method - The tissue is embedded in celloidin block. - The sections are cut with a sliding microtome. Advantages of Celloidin method: 1. It preserves the relations of the tissues because no heating is used. 2. It can be used for cutting large organs with plenty of folds and lumina. Disadvantages of Celloidin method: 1. Time-consuming. 2. No serial sections can be obtained. 3. The sections are very difficult to be stained. Freezing technique - It is used to cut fresh specimen. - The specimens are frozen, hardened and cut by the cryostat (is an electrical freezing and cutting apparatus widely used to prepare frozen sections) Advantages: 1- It is used in hospitals to study specimens during surgical procedures. 2- Allows stained sections to be prepared rapidly (within a few minutes). 3- It is effective in the histochemical study of enzymes since freezing does not inactivate most enzymes. 4- It preserves lipids. Disadvantage: 1- Producing thick sections. 2- Does not give serial sections. 3- The section may fragment into small pieces Preparation of tissues for electron microscope Fixation: very small piece of tissue (not exceed 3mm) is obtained and fixed immediately in glutaraldehyde followed by osmium tetroxide. Clearing: in propylene oxide Embedding: using epoxy resin as araldite or epon Resin is resistant to the damaging effects of electron beam. It is much harder than paraffin so very thin section can be obtained. Sectioning: ultrathin sections are prepared (0.005-0.1 m) the epoxy resin block is cut by glass or diamond knives of the ultramicrotome. Mounting: sections are mounted in copper grids. NB: The presence of heavy metal salts as uranyl acetate and lead citrate enhance the contrast in EM 4|Page  L1,2 HISTO Staining methods - The sections must be stained to be studied microscopically. - With few exceptions, most tissues are colorless, so observing them unstained in the light microscope is useless. - Most of these dyes behave like acidic or basic compounds. Basophilic tissue: - The tissues components that stain with basic dyes because of acids in their composition (nucleic acids, acid glycoproteins) are known as basophilic. - Basic dyes as Hematoxylin and methylene blue. Acidophilic tissue: - The tissue components that stain with acidic dyes because of alkali (mitochondria, secretory granules and collagen) in their composition are known as acidophilic. - Acid dyes as eosin and acid fuchsin. Hematoxylin and Eosin (H&E) The combination of H&E is the most commonly used. Hematoxylin: is a basic dye, stains the cell nucleus and other acidic structures blue. Eosin: is an acidic dye , stains the cytoplasm and other basic structures pink. Vital staining: It is the staining of the living cells within the living animal (i.e. in vivo). It is done by injecting a nontoxic dye into living animals. e.g. staining of phagocytic cells with trypan blue or Indian ink Supravital staining: It is the staining of living cells outside the body (i.e. in vitro). e.g. staining of mitochondria with Janus green B Neutral stains: It is a mixture of acidic and basic stains. Are used to stain the blood elements in a blood film, e.g. Leishman’s stain. Trichrome stains: Three stains are used in combinations to give 3 colors to different tissue components. In addition to staining the nuclei and cytoplasm can differentiate collagen from smooth muscle. Examples: Mallory’s stain: stains collagen blue, smooth muscle yellow, cytoplasm red. Masson’s stain: it stains collagen green, nuclei blue and cytoplasm red. 5|Page  L1,2 HISTO Metachromatic staining It is the staining of certain cell components with a color which is different from that of the dye used. The phenomenon of altering the color is called metachromasia.It is due to the interaction between the dye and the cell component, producing a different compound with a different color. e.g. the staining of mucopolysaccharide granules of mast cells by toluidine blue which changes to the red or violet colors. Histochemistry & Cytochemistry They are methods for localizing substances in tissue sections. These methods produce insoluble colored or electron-dense compounds that enable the localization of specific substances by means of light or electron microscopy. Demonstration of Nucleic Acids a- Feulgen reaction: DNA can be identified in cell nuclei using the Feulgen reaction producing a red color. b- Methyl green Pyronin : DNA and RNA can be analyzed by staining cells or tissue sections with methyl green Pyronin stain. DNA stains green and RNA stains red. Demonstration of enzymes a- Phosphatases enzymes as : - Alkaline phosphatases: have activity at an alkaline pH. - Acid phosphatases: present in lysosomes b- Dehydrogenases enzymes as succinate dehydrogenase enzyme in the citric acid (Krebs) cycle present in mitochondria. c- Peroxidase: present in blood cells. Demonstration of glycogen a- Best’s carmine: It is a specific stain for glycogen, stains red. b- Periodic acid–Schiff (PAS) reaction produces a purple or magenta color. Polysaccharides, Oligosaccharides and Glycoproteins can be demonstrated by the PAS reaction and they are PAS positive. Demonstration of Lipids by fresh or frozen section and staining with a- Sudan III (orange color ) b- Sudan black (black color) c- Osmic acid (black color) Immunocytochemistry Is a technique for identifying cellular or tissue constituents (antigen) by means of antigen antibody interactions. 6|Page  L1,2 HISTO Microscopy Microscopy Is the science of studying subjects through a microscope Resolving power: is the smallest distance between two particles at which they can be seen as separate objects. -The human eye can recognize two objects if they are not closer than 0.1 mm at a normal viewing distance of 25 cm. Any finer detail than this can be resolved by the eye only if the object is enlarged by the use of microscopes. The maximal resolving power of light microscope is 0.2 µm. The resolving power improves as the wavelength of the illuminating light decreases. TYPES OF MICROSCOPES A) Depend on ordinary visible light 1. Conventional light microscope. 2. Polarizing microscope. 3. Phase contrast microscope. 4. Dark-field microscope. B) Depend on other than visible light 1. Fluorescent microscope. 2. X-ray microscope. 3. Laser microscopes. 4. Electron microscope Conventional light microscope Basically the ordinary light microscope performs its function through 2 systems mainly, the optical and illuminating systems. The illumination system Light, from the illumination source (electrical lamp) is directed and focused onto the specimen (mounted on a glass slide) through the aperture in the stage. This light then passes through the specimen and into the objective lens. The optical system consist of: Condenser: collects and focuses light, producing a cone of light that illuminates the object to be observed. Objective lenses: enlarge and project the illuminated image of the object in the direction of the eyepiece. Eyepiece lens: further magnifies this image. The total magnification is obtained by multiplying the magnifying power of the objective and eyepiece lenses. The resolving power of the light microscope is approximately 0.2µm. This power permits good images magnified 1000–1500 times. Objects smaller than 0.2 µm cannot be distinguished. 7|Page  L1,2 HISTO Polarizing microscope When light passes through certain substances e.g. crystals or body tissues as fibers it divides in a way that produce 2 light rays from one. This called double refraction or polarization (birefringence). In its simplest form, the polarizing microscope is a conventional microscope in what a Nicol prism (filter)is interposed in the light path below the condenser and is called polarizer which converts all light passing through the instrument into plane polarized light. A similar second prism (filter) termed the analyzer is placed within the parallel of the microscope above the objective lens. When the analyzer oriented so that its polarizing direction is parallel to that of polarizer, regular image was seen. However if the analyzer is rotated until its axis is perpendicular to that of the polarizer, no light can pass through the ocular lens and the field is black. The field will remain black if an isotropic or singly refractive object is placed on the stage. A birefringent object however will appear light upon a dark background when examined in this manner. Birefringent or an isotropy is exhibited by many biological structures e.g. muscle fibers and collagen fibers. Phase contrast microscope The principle is based upon the fact that the light changes its speed and direction when passing through cellular and extra-cellular substances with different refractive indices. These changes cause the structure to appear lighter or darker related to each other by using a phase contrast system (phase plate) that convert the difference in speed into difference in intensity. Dark-field microscope For darkfield illumination, the cone of light illuminating the specimen must not enter the microscope objective lens. Only light that is scattered by the specimen is detected by the objective lens. This is achieved by use special dark-field substage condensers called cardioid condensers. Fluorescence microscopy When exited by radiation of short wavelengths (non visible), some substances will emit light of a longer wavelength (visible). This phenomenon is called fluorescence. The usual light source for excitation radiation is ultraviolet (UV). The fluorescent images produced can be recorded on either black and white or color film. 8|Page  L1,2 HISTO X-ray microscope X-rays have a shorter wave length than visible or ultraviolet light so, it has a higher resolving power (Resolution is not particularly high and with the instruments available is still far from theoretical limits). The specimen placed upon a photpgraphic emulsion and exposed to soft X-irradiation. The small X-ray picture obtained is subsequently magnified optically. Laser microscopes - Confocal microscope - Multiphoton (Femto-second) microscope Electron Microscopes 2 types: Transmission (TEM) and Scanning (SEM) - In TEM the Specimens are examined by passing the electron beam through them, revealing more information of the internal structure of specimens. The resolution is very high, reaching up to about 0.2 nm and it can magnify up to 500.000 times - In the SEM the specimen is scanned with a focused beam of electrons which produce "secondary" electrons as the beam hits the specimen. These are detected and converted into an image on a television screen. The resolution power of SEM is 10 nm. 9|Page