Histology: A Text and Atlas PDF

Chapter 1 Introduction 1  What is HISTOLOGY ? The study of microanatomy of cells, tissue and organs and how structure is related to function Cells – basic unit of life Techniques involved studying tissue Histochemistry, cytochemistry, immunocytochemistry H...

Chapter 1 Introduction 1  What is HISTOLOGY ? The study of microanatomy of cells, tissue and organs and how structure is related to function Cells – basic unit of life Techniques involved studying tissue Histochemistry, cytochemistry, immunocytochemistry Hybridization and autoradiography Organ and tissue culture Microscopic techniques  Tissue Group of cells with similar functions Can be specialized by grouping specific cells (e.g. hepatic cells → hepatic tissue) All biological problems arise from malfunctioning cells When a group of cells (tissue) or a single cell is damaged, it can affect neighboring cells and prevent original activities and may harm overall organism (e.g. Liver failure = Hepatic encephalopathy, Leukemia (WBC count is abnormal → cancer of the blood) Made of two interacting components: cells and extracellular matrix. The ECM consists of many kinds of molecules, such as collagen fibrils, there is, thus, an intense interaction between cells and matrix, with many components of the matrix.  Tissue Types Epithelium Connective Muscular Nervous  Microtechnique Factors for specimen to be observed by microscope Specimen must be sectioned to show structural details of tissue Specimen must be processed, stained, transparent, and mounted on a slide  TISSUE PREPARATION Step1 - Fixation Removal of small tissue sample as a block (less than 1 cm square). If removal is post-mortem, this must be done rapidly so as to avoid deterioration Fixation = immersion of small pieces of tissue in fixative solution (e.g., formaldehyde, alcohol, certain acids). Fixative solution used is determined by particular tissue and staining method. Formalin (37% formaldehyde) is by far the most common fixative. Wash to remove excess fixative FUNCTION = prevents deterioration (stops cell metabolism, prevents autolysis), hardens soft tissues, kill pathogenic microorganisms, can increase affinity for certain stains  TISSUE PREPARATION Step 2 - Embedding in paraffin to permit sectioning Dehydration = accomplished by passing tissue through successively stronger solutions of alcohol (removes water so hydrophobic embedding medium can be applied). Alcohol then removed with xylol or toluol (clearing - embedding agent is soluble in xylol). Time we submerge specimen in alcohol depends on size and density  Subject to non-aqueous fluid to make it transparent Egs of non-aqueous solutions – xylene, cedar wood oil, winter green oil (all lipid based) Specimen will float, since alcohol is less dense Alcohol is gradually replaced by lipid, specimen will sink and become transparent Subject specimen to hot wax (550C) Oil is replaced by the wax (note lipid is miscible with wax) Subject specimen to second round of pure wax Step removes any impurities left from previous step Allow specimen to cool and harden Specimen is mounted on a microtome for slicing Sliced to desired size  TISSUE/SLIDE PREP CONTD. Step 3 - Staining Stains are applied according to acidic or basic nature of specimen In order to stain, paraffin must be removed (most stains are insoluble in paraffin, but soluble in water). Slice mounted and passed through xylol, toluol or xylene to remove paraffin. Slice passed through 100% alcohol to remove xylol Finally, slice passed through decreasing strengths of alcohol and lastly through water -- ready to stain. Staining Procedure = stain applied to slice for varying amounts of time depending on stain and desired treatment effect. FUNCTION = to enhance contrast and make certain structures more apparent, different stains used for specific structures. Tissue Fixation and Processing for Light Microscopy Step 3 – Staining cont’d – once stained, specimen is mounted on slide and covered with coverslip Mounting a) excess stain washed away with water (or alcohol for some dyes, depending on dye solvent) b) tissue slice dehydrated by passing through increasing strengths of alcohol to absolute alcohol c) alcohol removed by a clearing agent (refractive index similar to tissue) so that unstained spaces appear transparent d) mounting medium is then added to tissue slice (medium with same refractive index as glass), covered with a coverslip and allowed to dry 1) Longitudinal = lengthwise along a structure (Sagittal section = lengthwise splitting structure into two halves) 2) Cross Section = perpendicular to longitudinal plane 3) Oblique Section = any angle between longitudinal and cross sections 4) Tangential Section (Grazing) = only a small portion of the surface FIGURE 1.1 Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections that demonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. a. This photomicrograph reveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structures that have a high affinity for the dye are most heavily stained—for example, the nuclear DNA and areas of the cell containing cytoplasmic RNA. b. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, that the nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin and then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost,  STAINS - CHEMISTRY Basic Dyes = carry positive charge, attracted to acidic components of cells (dye+,Cl-) Methyl green, Methylene blue, Pyronin G, Toluidine blue Cells/tissues that react to basic dyes are basophilic Acidic Dyes = carry negative charge, attracted to basic components of cells (Na+,dye-) Acid fuchsin, Aniline blue, Eosin, Orange G Cells/tissues that react to acidic dyes are acidophilic 3) Neutral Stains = anion (-) and cation (+) provide different colors PURPOSE of staining is to enhance contrast. This is accomplished in two ways:  Techniques used to detect specific components of tissue 1) Acid-Base Combinations = Most sections are stained with both acidic and basic dyes to enhance contrast by providing different colors. The most common combination is Hematoxylin and Eosin (H & E). Hematoxylin = basic dye, stains nuclear structures blue Eosin = acidic dye, stains cytoplasmic and intercellular structures pink 2) Trichrome Methods = provides 3 colors, allows differentiation between cytoplasmic and intercellular components 3) Specific Stains = stain certain structures or molecules specifically a) Iron hematoxylin = useful in distinguishing finer cytologic details (e.g., subcellular organelles) b) Mallory-Azan = trichrome method; stains collagen fibers and mucus blue, stains nuclei and cytoplasmic components red c) Mason = trichrome method; collagen fibers stain green, cytoplasmic components stain purplish-red d) Periodic-Acid Schiff = selectively stains carbohydrate-containing molecules/substances  Micrograph stained with hematoxylin and eosin (H&E). Columnar epithelium lining the small intestine Tissue stained with Tissue stained with the periodic hematoxylin and eosin acid-Schiff (PAS) reaction for  HISTOCHEMISTRY AND CYTOCHEMISTRY They are methods for localizing cellular structures in tissues using enzyme activity present in these structures. Usually applied to unfixed or mildly fixed tissue, sectioned on a cryostat or freezing microtome to avoid adverse effects of heat, paraffin and chemicals on enzymatic activity. 1. Tissue sections are immersed in a solution containing the substrate of the enzyme to be localized. 2. The enzyme is allowed to work on the substrate. 3. The section is put in contact with the marker compound. 4. This compound reacts with a molecule produced by enzymatic action on the substrate. 5. The final reaction product (insoluble and visible in LM or EM) only if it is colored of electron- dense, precipitate over the site that contains the enzyme. e.g. Phosphatases, split the bond between a PO4 group and an alcohol residue of phosphorylated molecules. The visible insoluble reaction product of phosphatases is lead phosphate or lead sulphide. At alkaline pH and acid phosphatase. Dehydrogenases, removes H from one substrate and transfer it to another. Mitochondria can be specifically identified by this method, dehydrogenases are key enzymes in the citric acid cycle.  Chemical Composition of Histologic Samples Chemical composition of a tissue ready for routine staining differs from living tissue Components that remain after fixation consist mostly of large molecules that do not readily dissolve, especially after treatment with the fixative. Macromolecular complexes, are usually preserved in a tissue section. E.g. nucleoproteins formed from nucleic acids bound to protein, intracellular cytoskeletal proteins complexed with associated proteins, extracellular proteins in large insoluble aggregates, bound to similar molecules by cross-linking of neighboring molecules, as in collagen fiber formation, and membrane phospholipid–protein (or carbohydrate) complexes.  Enzyme Histochemistry Histochemical methods are used to identify and localize enzymes in cells and tissues Mild aldehyde fixation is preferred Reaction product of enzyme activity, rather than the enzyme itself is visualized Capture reagent (dye or heavy metal) is used to trap or bind the reaction product of the enzyme by precipitation at the site of reaction To display hydrolytic enzyme, tissue is placed in a solution containing substrate (AB) and a trapping agent (T) that ppts one of the products as follows AB + T AT + B where AT is trapped end product and B is the hydrolyzed substrate Most common histochemical method uses HRP for enzyme mediated antigen detection FIGURE 1.3 Electron and light microscopic histochemical procedures. a. This electron micrograph shows localization of membrane ATPase in epithelial cells of rabbit gallbladder. Dark areas visible on the electron micrograph show the location of the enzyme ATPase. This enzyme is detected in the plasma membrane at the lateral domains of epithelial cells, which correspond to the location of sodium pumps. These epithelial cells are involved in active transport of molecules across the plasma membrane. X26,000. b. This photomicrograph shows macrophages stained with a histochemical method using peroxidase-labeled antibodies and DAB reagent. A paraffin-embedded section of mouse kidney with renal vascular hypertension disease was stained for presence of F4/80+ specific marker protein expressed only on the surface of macrophages. Initially, sections were exposed to primary rat anti- mouse F4/80+ antibodies followed by incubation with secondary goat anti-rat IgG antibodies labeled with horseradish peroxidase. The specimen was washed and treated with a buffer containing DAB. A  Immunocytochemistry Antigen and antibody reaction Ab is tagged with a fluorescent dye (Fluorescein, rhodamine, acridine orange etc) Fluorescent dye molecule emits light in the presence of UV light  Types of Ab used in Immunocytochemistry Polyclonal produced by immunized animals Monoclonal produced by immortalized (continuously replicating) antibody-producing cell lines. now widely used in immunocytochemical techniques conjugated with radioactive compounds are used to detect and diagnose tumor metastasis in pathology Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues Direct immunofluorescence Oldest technique Use a fluorochrome-labeled primary antibody (monoclonal or polyclonal) to react to antigen Indirect immunofluorescence Provides much greater sensitivity than direct methods Often referred to as the “sandwich” or “double-layer technique.” Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a secondary antibody directed against rat primary antibody (i.e., goat anti-rat antibody; FIGURE 1.5▲Direct and indirect immunofluorescence. a. In direct immunofluorescence, a fluorochrome- labeled primary antibody reacts with a specific antigen within the tissue sample. Labeled structures are then observed in the fluorescence microscope in which an excitation wave-length (usually ultraviolet light) triggers the emission of another wavelength. The length of this wavelength depends on the nature of the fluorochrome used for antibody labeling. b. The indirect method involves two processes. First, the specific primary antibodies react with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies. The visualization of labeled structures FIGURE 1.4 Confocal microscopy image of a rat cardiac muscle cell. This image was obtained from the confocal microscope using the indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter (MCT1) and is detected with a secondary antibody conjugated with rhodamine (red). The second primary antibody is directed against the transmembrane protein CD147, which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein (green). The yellow color is visible at the point at which the two labeled secondary antibodies exactly co-localize within the cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell, whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.)  MICROSCOPY I. LIGHT MICROSCOPE A) Allows visualization, magnifies 40-1000 times; magnification = Objective lens power X Ocular lens power B) High power oil immersion lens has refractive index (amount which it bends light) similar to that for oil, this is why oil must be used as the viewing medium C) Stains provide contrast which light microscope detects (color contrast and intensity contrast) Types Bright field microscopy Stained slides are examined by means of ordinary light that passes through Bright Field Microscope  Fluorescence Microscopy When certain substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength. This phenomenon is called Fluorescence. In Fluorescence Microscopy, tissue sections are irradiated with ultraviolet UV light and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant on a dark background. Thus, the microscope has a strong UV light source and special filters that select rays of different wavelengths by the substances. Fluorescent compounds with affinity for specific cell macromolecules may be used as Fluorescent stains. E.g. Acridine Orange (bind to DNA and RNA). e.g. Hoechst stain and DAP1 (4’,6-diamino-2-phenylindole) specifically bind to DNA giving a blue fluorescence under UV. Fluorescent Microscopy Kidney cells Culture of kidney cells DAPI (4’,6-diamino-2- acridine orange phenylindole)  Phase-contrast microscopy Can be used to look at unstained biological specimens (cells and tissues) Can be living or nonliving, fixed or unfixed Takes advantage of difference in refractive index in different sections of specimen Enhances contrast by sets of rings in the condenser lens (out of phase) The darker the color of the specimen the denser it is and vice versa Bright field Interference Phase contrast microscopy microscopy microscopy  Confocal microscopy Specimen can be seen in 3D via a computer Uses fluorescence dye with antibodies (Ab) Ab binds to actin and area bound is indicated by the fluorescent dye Laser scanning is used to scan one section at a time to detect the dye and compile the images to give a 3D image nciple of confocal microscopy FIGURE 1.4 Confocal microscopy image of a rat cardiac muscle cell. This image was obtained from the confocal microscope using the indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter (MCT1) and is detected with a secondary antibody conjugated with rhodamine (red). The second primary antibody is directed against the transmembrane protein CD147, which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein (green). The yellow color is visible at the point at which the two labeled secondary antibodies exactly co-localize within the cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell, whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.)  Polarizing microscopy Unpolarized light gives light in all directions Uses the polarizer to produce polarized light Thus is given in one direction/orientation Molecules with 4 different substituents have varying orientation This it is useful in determining the order (arrangement of certain element or molecules)  bright-field and polarizing microscopy Bright-field microscopy Polarizing microscopy Tissue appearance with bright-field and polarizing microscopy. Polarizing light microscopy produces an image only of material having repetitive, periodic macromolecular structure; features without such structure are not seen. Shown here is a piece of thin mesentery that was stained with red picrosirius, orcein, and hematoxylin, and was then placed directly on a slide and observed by bright-field and polarizing microscopy. (a): Under routine bright-field microscopy collagen fibers appear red, along with thin dark elastic fibers and cell nuclei. (b): Under polarizing light microscopy, only collagen fibers are visible and these exhibit intense birefringence and appear bright red or yellow; elastic fibers and nuclei lack oriented macromolecular structure and are not visible. II. ELECTRON MICROSCOPY - 2 types: Transmission (TEM) 100,000X magnification Specimen is dead Fixed in plastic Specimen is cut Dark regions indicate absence of electrons in that area Scanning (SEM) Similar to TEM  Other Microscopy Atomic Force Microscopy One newer microscope that has proved most useful for biologic studies is the atomic force microscope (AFM). It is a non-optical microscope that works in the same way as a fingertip, which touches and feels the skin of our face when we cannot see it. The sensation from the fingertip is processed by our brain, which is able to deduce surface topography of the face while touching it. (Fig 1.16) Virtual Microscopy Integrates conventional light microscopy with digital technologies. Using optical image acquisition systems with automatic focus, glass slides are scanned to create two- dimensional digital files that typically are stored in dedicated virtual