[MLS 2024] [Book-Junqueira\'s] Histology- Chapter 1 v PDF

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[JUNQUEIRA’S BASIC HISTOLOGY] / [MESCHER][BOOK] CHAPTER 1: HISTOLOGY AND ITS METHODS OF STUDYDe Castillo [CSU BS MLS 2B] 1OUTLINEI. Introduction to Histology II. Preparation of tissues for study a) Steps in tissue preparation i. Fixation ii. Embedding & Sectioning iii. Staining III. Microscopy a...

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[JUNQUEIRA’S BASIC HISTOLOGY] / [MESCHER] [BOOK] CHAPTER 1: HISTOLOGY AND ITS METHODS OF STUDY OUTLINE I. Introduction to Histology II. Preparation of tissues for study a) Steps in tissue preparation i. Fixation ii. Embedding & Sectioning iii. Staining III. Microscopy a) Light Microscopy i. Bright-field Microscopy ii. Fluorescence Microscopy iii. Phase-contrast Microscopy iv. Confocal Microscopy v. Polarizing Microscopy b) Electron Microscopy i. Transmission Electron Microscopy ii. Scanning Electron Microscopy IV. Autoradiography V. Cell & Tissue Culture VI. Enzyme Histochemistry a) Histochemical (cytochemical) procedures VII. Visualizing specific molecules a) Immunohistochemistry i. Direct Immunohistochemistry ii. Indirect Immunohistochemistry b) Hybridization techniques i. In situ hybridization VIII. Interpretation of structures in tissue sections

Dehydration Clearing

Infiltration Embedding Trimming

Basophilic

Acidophilic

INTRODUCTION TO HISTOLOGY • Histology o Study of tissues of the body o How tissues are arranged to constitute organs • Tissues o Two interacting components: o Cells: produce ECM locally, influenced by matrix molecule o Extra-cellular Matrix (ECM): supports the cell, contains fluid transporting nutrients to the cell, carry away wastes

Basic Dyes

Acidic Dyes

PREPARATION OF TISSUES FOR STUDY • Section/Preparation of tissue slides: most common procedure in histologic research. o Can be examined by transmitted light o Uses thin translucent section

STEPS IN TISSUE PREPARATION Fixation

Fixation • Preserve tissue structure • Prevent degradation of enzymes • Fixative: solution that cross-link proteins and inactive degradative enzyme o Light microscopy: Formalin o Electron microscopy: Glutaraldehyde

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Hematoxylin and Eosin (H&E)

Embedding and sectioning • Removal of water by transfer of increasing ethanol solutions. • Alcohol is removed in organic solvents where paraffin and alcohol are miscible • Tissues have translucent appearance • Tissue is placed with melted paraffin until fully infiltrated • Paraffin-infiltrated tissue is placed in a small container allowing it to harden. • Paraffin-block is trimmed • Exposes the tissue for sectioning on a microtome. • Paraffin sections are cut: o Light microscopy: 3-10 µm thick; placed on glass slides o Electron microscopy: 1 µm thick; placed on metal grids Staining • Cell components with net negative charge (anionic) with affinity for basic dyes. • Ex. Nucleic Acids • Cell components with positive charge (cationic) has an affinity with acidic dyes. • Ex. Proteins Types of dyes • Reacts with tissue components with acids in their composition: DNA, RNA, glycosaminoglycans o Toluidine blue o Alcian blue o Methylene blue o Hematoxylin: behaves like a basic dye; stains basophilic components • Stains acidophilic components: mitochondria, secretory granules, collagen o Eosin o Orange G o Acid Fuchsin Staining methods • Most commonly used • Hematoxylin o Stains DNA, RNA, Matrix of cartilage; Produces dark blue or purple color • Eosin o Stains cytoplasmic structures and collagen pink o Counterstain: single dye applied separately to distinguish additional features of a tissue o Trichrome strain (Masson’s trichrome): greater distinction of extracellular cells

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JUNQUEIRA’S BASIC HISTOLOGY: Histology and its method of study

Periodic acid-Schiff (PAS) reaction Metal impregnation

Slide preparation

Parts and functions of Bright-field microscope Condenser • Focuses light on the object being studied Objective • Enlarges and projects the image of the object toward the eyepiece. • Magnifications used in Histology: o X4: large area of tissue at low magnification o X10: smaller field at medium magnification o X40: more detailed areas at high magnification Eyepiece • Magnifies the image by another X10 • Only enlarge the image; does not improve resolution • Note: The total magnification is obtained by multiplying the magnifying power of the object and ocular lens. • Total magnifications are: X40, X100, or X400

• Hexose rings of polysaccharides and carbohydrate • Stains macromolecules • Purple or magenta • Feulgen reaction: Stains DNA • Solution of silver salts to visualize ECM fibers

Slide preparation • Final step before microscopic observation • Includes mounting of protective glass cover slip on the side with clear adhesive.

Medical Applications • Biopsies: tissue samples removed during surgery. o Fixed in vials of formalin o Rapid method: Frozen in liquid nitrogen. o Cryostat: microtome used to section the block with tissue.

MICROSCOPY LIGHT MICROSCOPY

• Based on the interaction of light with tissue components • Used to study tissue features • Categorized into: o o o o o

Bright-field Microscopy Fluorescence Microscopy Phase-contrast Microscopy Confocal Microscopy Polarizing Microscopy

• Resolving power: smallest distance between two structures at which they can be seen as separate objects. o Maximum resolving power of BF microscope: 0.2 µm; permit clear images magnified 1000-1500 times. o Smaller than 0.2 µm cannot be distinguished by this instrument. Ex. Single ribosome, cytoplasmic microfilament o Determines the quality of image, clarity and richness of detail. • Virtual Microscopy o Includes the conversion of microscopic stained tissues to high resolution images o Study of tissues using a computer or digital device without an actual stained slide or a microscope.

Bright-field Microscopy

Fluorescence Microscopy

• Stained tissue is examined with ordinary light • Includes an optical system that moves and focus a specimen.

• Fluorescence: light with a longer wavelength; emitted through irradiation by light of a proper wavelength • Tissue selections are irradiated with Ultraviolet (UV) • Substances appear bright on a dark background • Has an affinity for specific cell macromolecules o Acridine orange: binds DNA and RNA; emits different fluorescence allowing them to be localized separately. o DAPI and Hoechst: stains and bind DNA, and cell nuclei; emits blue fluorescence. o Coupling compounds (Fluorescein): binds to cellular components that allows identification of structures. o Antibodies stained with fluorescent: important in immunohistologic staining.

Phase-contrast Microscopy Used to view unstained cells and tissue collections. Uses a lens system that produce visible images Can be used with living, cultured cells. Principle: Light changes its speed when passing through cellular and extracellular structures with different refractive indices. • Allow the examination of cells without fixation or staining. • • • •

Figure 1-1. Components and light path of a bright-field microscope De Castillo [CSU BS MLS 2B]

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JUNQUEIRA’S BASIC HISTOLOGY: Histology and its method of study

• Differential Interference contrast microscopy: modification of phase-contrast microscopy with Nomarski optics; produces 3D aspect.

Confocal Microscopy • Achieves high resolution and sharp focus by: o a small point of high intensity light (laser) o a plate with a pinhole aperture in from of the image detector • The point light source, focal point of the lens, and the detector’s pinpoint aperture are confocal. • Unfocused light does not pass through the pinhole. • More precise that with the bright-field microscope. • Includes a computer-driven mirror system (beam splitter) that moves the point of illumination • The diagram below includes the principle of confocal microscopy.

Transmission Electron Microscopy

• Imaging system that permits resolution around 3 nm. • Allows magnification as much as 400,000 times • Studies very thin resin-embedded tissue sections. • Principle: a beam of electrons using electromagnetic lenses passes through the tissue section to produce and image with black, white, and intermediate shades of gray. • Heavy metal ions o Improves contrast and resolution in TEM o Added to the fixative or dehydrating solution in tissue preparation o Ex. Osmium Tetroxide, Lead Citrate, Uranyl Compounds: bind to cellular macromolecules that increases their electron density and visibility

• Cryofracture and freeze etching o

Techniques that allow TEM study of cells without fixation or embedding.

Scanning Electron Microscopy • Provides a high-resolution view of the surface of cells, tissues, and organs. • Produces beam of electrons but the beam does not pass through the specimen. • The surface of the specimen is dried and spray-coated with a heavy metal (gold) that reflects electrons. • Reflected electrons are captured by a detector that produces black and white image. • Presents a 3D view

AUTORADIOGRAPHY

Figure 1-2. Principle of confocal microscopy

Polarizing Microscopy • Recognizes stained or unstained structures made of highly organized subunits. • 1st Filter: Polarizing filter o Allows the exit of light in only one direction • 2nd Filter: Filter above the 1st filter o No light will pass through • Birefringence o Ability to rotate the direction of vibration of polarized light o Feature of crystalline substances or highly oriented molecules

ELECTRON MICROSCOPY

• Interaction of tissue components with beams of electrons. • The wavelength of electron beams is shorter than that of light.

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• Localizes cell components synthesized using radioactive precursors by detecting silver grains produced by weakly emitted radiation in a photographic emulsion. • Silver grains o Indicate the region of cells where macromolecules were synthesized just prior to fixation. • Example: Tritium-labeled thymidine o Radioactive precursor of DNA o Used to know which cells in a tissue are replicating DNA

CELL AND TISSUE CULTURE • In vitro: outside the body • In vivo: inside the body • Cell Culture o Allows the direct observation of cellular behavior. o Grown in complex solutions (salts, amino acids, vitamins) o Dispersed mechanically or enzymatically • Primary cell culture o Culture of cells that re maintained in vitro for long periods o Constitutes a cell line • Transformation o Changes that promote cell immortality o Example: Normal cell to cancer cell

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JUNQUEIRA’S BASIC HISTOLOGY: Histology and its method of study

ENZYME HISTOCHEMISTRY

Direct Immunohistochemistry

• Method in localizing cellular structures using specific enzymatic activities.

HISTOCHEMICAL (CYTOCHEMICAL) PROCEDURES • Use unfixed or mildly fixed tissues sectioned on a cryostat.

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Steps in Enzyme Histochemistry Tissue sections are immersed in solution containing the substrate of the enzyme to be localized The enzyme is allowed to act on its substrate The section is put in contact with a marker compound that reacts with the product of the enzymatic reaction The final product precipitates over the site of the enzyme, identifying their location

Enzymes that can be detected histochemically Phosphatases Removes phosphate group form macromolecule Dehydrogenases Transfer hydrogen ions from one substrate to another Peroxidase Promotes oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide

Medical Applications • Perlsb’ Prussian Blue o Reaction with Iron: Diagnosis of iron storage disease, hemochromatosis and hemosiderosis o Reaction with Polysaccharides: Detect glycogenosis and mucopolysaccharides o Reaction with Lipids and Sphingolipids: Detect sphingolipidosis

VISUALIZING SPECIFIC MOLECULES • Some substances specifically bind certain target in cells Molecules that interact with other molecules Phalloidin • Extracted from mushrooms (Amanita phalloides) • Interacts with actin protein microfilaments Protein A • Purified from Staphylococcus aureus • Binds to the Fc region of antibody molecules • Localize applied antibodies bound to cell Lectins • Glycoproteins derived from plant seeds • Bind to carbohydrates

IMMUNOHISTOCHEMISTRY • Identifies and localize specific proteins, not just those with enzymatic activity • Based on specific reactions between an antigen and antibody labeled with visible markers o Markers in light microscopy: Fluorescent compounds and Peroxidase o Markers in TEM: Gold particles De Castillo [CSU BS MLS 2B]

• The cell or tissue antigen of interest is detected by directly binding a labeled primary antibody specific for that antigen.

Indirect Immunohistochemistry • Uses an unlabeled primary antibody that is detected bound to its antigen with labeled secondary antibodies. • More commonly used because the added level of antibody binding amplifies the signal detected and provides greater technical flexibility.

HYBRIDIZATION TECHNIQUES • Specific binding between two single strands of nucleic acids. • Grater the similarity; the more complementary “hybrid” doublestrand molecules. Specific antigen with diagnostic importance Antigens Diagnosis Specific Tumors of epithelial origin Cytokeratin Protein and Certain endocrine tumors polypeptide hormones Carcinoembryonic Glandular tumors, mainly of digestive antigen (CEA) tract and breasts Steroid hormone Breast duct cell tumors receptors Antigens Specific virus infections produced by viruses

In situ Hybridization • A technique used in the detection of specific gene sequences or mRNAs of cells using labeled cDNA probes. • Ideal for: o Determining if a cell has a specific sequence of DNA, such as gene or part of a gene o Identifying the cells containing specific mRNA o Determining the localization of a gene in a specific chromosome

INTERPRETATION OF STRUCTURES IN TISSUE SELECTION DIFFICULTY IN STUDYING HISTOLOGIC SECTIONS • Microscopic preparations are the end results of tissue collecting to mounting a coverslip on the slide. o Artifacts: minor structural abnormalities not present in the living state caused by the distortion of tissues during microscopic preparations. o Causes shrinkage that creates an artificial space between cells and other tissue components. o Spaces can result from loss of lipid or lowmolecular-weight substances. o May include small wrinkles in the section (confused as linear structures) and precipitates from the from the stain (confused as cellular structures)

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JUNQUEIRA’S BASIC HISTOLOGY: Histology and its method of study

• Differentially staining all tissue components on one slide o A single stain can seldom demonstrate well tissue components. • Sections of cells/ tissues are essentially 2D planes (length and width) through 3D structures. Viewer must keep in mind that components are missing in front of and behind of what is seen.

REFERENCES Mescher, A. L. (2018). Junqueira’s Basic Histology Text and Atlas. Fifteenth Edition. p.1-16.

REMINDER QUICK QUOTES Don’t limit your challenges. Challenge your limit. Padayon Future RMT!

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