Histology & Genetics Epithelial Tissues PDF

HISTOLOGY & GENETICS T01 08...

HISTOLOGY & GENETICS T01 08/12/2024 HG201A| Dr. Ronnie Mayo | PSU School of Medicine Year 1 | Batch 2028 EPITHELIAL TISSUES (LEC) HISTOLOGY BASICS (LAB) OUTLINE FUNCTION Examples I. Epithelial Tissue ○ Gastrointestinal tract A. Function Protects through what we eat B. Characteristics II. Classification of Epithelia ○ Genitourinary tract A. Simple squamous ○ Respiratory tract B. Simple cuboidal ○ Lumen of blood vessels C. Simple columnar Epithelia have 6 main functions: protection, D. Stratified squamous absorption, sensation, secretion, filtration, and E. Pseudostratified columnar F. Transitional exchange. III. Epithelial Surface Specializations Protection A. Microvilli ○ Covering and lining B. Cilia ○ Protection from external environment C. Stereocilia ○ Prevents desiccation (dryness) D. Keratinized surface of skin IV. Glandular Epithelium A. Signaling mechanisms Peyer’s patches are very large clusters of lymphoid follicles B. Classification of exocrine glands located in the wall of the ileum which allow close monitoring of C. Mechanisms of secretion microorganisms in the gut. (Junqueira) V. Question and Answer VI. Histology Basics (Laboratory) Bacteria in the large intestine produce vitamin K. Newborns A. Tissue preparation have a sterile gut microbiome, which does not produce vitamin B. Stains K. Therefore are injected with vitamin K that is essential for VII. References clotting factors and prevent bleeding. EPITHELIAL TISSUE Absorption Tissue - Group of cells with shared characteristics, ○ Ex: Intestine organized to perform one or more specific function Large intestine - where final absorption 4 Basic Types: occurs ○ Connective Tissue Sensation connects one structure to another; underlies ○ Ex: Olfactory epithelium, Tastebuds or supports other tissues, structurally and Secretion functionally ○ Ex: Glands ○ Epithelial Tissue Filtration covers body surfaces, lines body cavities, ○ Ex: Lining of kidney tubules forms glands Exchange ○ Muscular Tissue ○ Ex: Alveoli made up of contractile cells and is responsible for movement TYPES OF EPITHELIA ○ Nervous Tissue Lining Epithelia receives, transmits, and integrates ○ Covers free surfaces of the body information from outside and inside the body Glandular Epithelia to control the activities of the body ○ Produce and secrete substances Epithelial Tissue ○ Avascular CHARACTERISTICS ○ It covers surfaces of the body exposed to Avascular (do not contain blood vessels) external environment (skin) Cells are closely apposed and adhere to one another ○ Found in lining of cavities, lining of internal by specialized structure cavities and lumen of bodies (abdominal cavity, ○ Tight Junctions- impermeable thoracic cavity), and blood vessels ○ Anchoring Junctions- anchors one cell to ○ Resting in basement membrane another ○ Channel-forming Junctions 85% of human cancers originate from cells of epithelial origin Ingestion of probiotics gives broad spectrum antibiotics that provides good bacteria - HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 1  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 CLASSIFICATION OF EPITHELIA BASED ON # OF LAYERS Simple ○ Composed of 1 layer only Stratified ○ 2 or more cell layers BASED ON SHAPE OF THE SURFACE CELLS Surface cell = pinakataas Squamous ○ Flattened or like scales nucleus ○ Width of the cell is greater than its height Figure 1.1 Characteristics of Epithelia-Cell Junctions Cuboidal ○ Rounded nucleus Exhibit functional and morphological polarity ○ Width, depth, and height are approx. the same Epithelial layers are attached to the extracellular Columnar matrix of the basal lamina ○ Elongated/pahaba nucleus Cell surface can be highly modified ○ Height of cell exceeds the width ○ Microvilli - for absorption ○ Cilia - for movement SPECIAL CATEGORIES found in trachea, bronchioles ○ Vibrissae Pseudostratified Epithelium Ex: hair in horse ○ Looks stratified but is a single layer ○ Stereocilia aka Pseudocillia Transitional Epithelium ○ Mucus - top of epithelial lining ○ Can change shape, particularly the surface cells ○ Can assume 2 different shapes depending on the physiological stage Squamous metaplasia frequently occurs in the pseudostratified Ex: gallbladder has 2 physiological stage respiratory epithelium of the trachea and bronchi in response to depende kung empty siya or full prolonged exposure to cigarette smoke. Cigarette contains hundreds of chemicals and 20 are carcinogens. The cilia are Specialization of the apical cell surface domain affected first, paralyzing it which irritates the respiratory tract and ○ Presence of cilia, microvilli causes smoker’s cough. The cilia affected takes 2 years to ○ Keratinized or nonkeratinized regenerate from this condition. Table 1.1 Classifying Epithelial Cells BASEMENT MEMBRANE # of Shape of Epithelial Domain Main function is the anchoring of epithelial cells to layers surface Surface connective tissue below cells Specializ Acts as mechanical barrier ation Important in angiogenesis Combination of basal lamina and reticular lamina ex. Simple Columnar Ciliated Epithelium ○ Basal lamina 40-120 nm thick = Simple Columnar Ciliated Epithelium Underlies all epithelia Strong, flexible foundation Meshwork formed by interaction between protein fibers When in doubt, always check the shape of the Laminin nucleus. Type IV collagen Entactin Look for the surface, what type of epithelium it is Perlecan since there are instances where the inner layer is ○ Reticular lamina cuboidal but the outside layer is stratified. Mainly composed of type III collagen Mnemonic: 3ticularRe3cular Below basal lamina; anchor epithelial SIMPLE SQUAMOUS EPITHELIUM tissues to connective tissues composed of a single layer of thin wide cells Act as mechanical barrier Function Important in angiogenesis ○ Absorption Movement of substances will be easy; Blood brain barrier keeps harmful substances from reaching simple so madaling daanan the brain. Some antibiotics are blocked due to the barrier, but Ex: lining of cavities there are certain antibiotics used to penetrate the blood brain ○ Filtration barrier. Specialized ○ Diffusion Passive diffusion in the lungs HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 2 EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Lines surfaces in which metabolites & gasses can pass through For blood vessels & kidney tubules ○ Barrier Lines surfaces that require minimal protection Central nervous system (CNS) ○ Exchange Figure 1.3 Simple Cuboidal Epithelium ○ Lubrication Location SIMPLE COLUMNAR EPITHELIUM ○ Endothelium Composed of single layer of tall, narrow cells Usually found at the Inner lining of blood & Some cells may have cilia (e.g. bronchioles of the lymphatic vessels lungs) or microvilli (intestines) Blood vessels have vascular Goblet Cells – mucus secreting cells positioned endothelium among other columnar cells. Capillaries Function ○ Mesothelium ○ Protection Lining of an internal body cavity ○ Lubrication Abdominal & Pleural Cavities ○ Absorption ○ Alveoli (respiratory spaces in Lungs) ○ Secretion ○ Bowman’s Capsule (Kidney) Locations ○ Lining of the intestine ○ Gallbladder ○ Bronchioles of the lungs ○ Auditory tubes ○ Uterus ○ Uterine tubes ○ Stomach lining ○ Ventricles of the brain Figure 1.2 Simple Squamous Epithelium SIMPLE CUBOIDAL EPITHELIUM Composed of single layer of cube-shaped cells Some cells may have microvilli (kidney tubules) or cilia (terminal bronchioles of the lungs) Their greater thickness allows cytoplasm to be rich in mitochondria and other organelles for high levels of active transport across the epithelium. Function Figure 1.4 Simple Columnar Epithelium ○ Absorption ○ Secretion STRATIFIED SQUAMOUS EPITHELIUM ○ Transport or conduit ○ Barrier or covering Its cells form many layers, with the less differentiated Locations cuboidal cells near the basement membrane. ○ Small ducts of exocrine glands These cells have many desmosomes and become ○ Surface of ovary (germinal epithelium) more irregular in shape. ○ Kidney tubules Flatten as they accumulate keratin in the process of ○ Thyroid Follicles keratinization. ○ Choroid plexus of the brain ○ Lining of the terminal bronchioles of the lungs KERATINIZED Packed with Keratin Filaments Functions ○ Barrier ○ Protection Location ○ Epidermis (Skin) ○ Anus HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 3  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Location ○ Anorectal junction ○ Largest duct of exocrine glands ○ Conjunctiva lining the eyelids Figure 1.5 Stratified Squamous Keratinized NON-KERATINIZED With relatively sparse keratin. Lines moist internal cavities where water loss is not a problem. Functions Figure 1.8 Stratified Columnar Epithelium ○ Barrier ○ Protection PSEUDOSTRATIFIED COLUMNAR EPITHELIUM Locations ○ Oral Cavity (Mouth) Appear stratified but is actually a simple epithelium ○ Upper throat Some of its cells do not reach the free surface ○ Esophagus All rest on the basement membrane ○ Vagina Has motile cilia; ○ to sweep mucus and other trapped foreign matter towards the oropharynx and; ○ to propel sperm to the epididymis Function ○ Absorption ○ Protection Location ○ Upper respiratory tract ○ Trachea Figure 1.6 Stratified Squamous Non-Keratinized ○ Bronchial Tree ○ Ductus deferens STRATIFIED CUBOIDAL EPITHELIUM ○ Efferent ductules of epididymis Relatively rare. A unique form of epithelial tissue made up of cuboidal cells stacked in several layers. Functions ○ Barrier ○ Transport Locations ○ Anorectal junction Figure 1.9 Electron micrograph of pseudostratified columnar (L); Photomicrograph of stained specimen (R) ○ Sweat gland ducts ○ Large duct of exocrine glands TRANSITIONAL EPITHELIUM “Urothelium” A stratified epithelium The epithelium lining the urinary tract Changes shape due to contraction and relaxation; cuboidal when relaxed, and squamous when stretched Function Figure 1.7 Stratified Cuboidal Epithelium ○ Barrier ○ Distensible property Location ○ Bladder STRATIFIED COLUMNAR EPITHELIUM ○ Renal Calyces Relatively rare ○ Ureters Made up of multiple layers of epithelial cells, ○ Urethra wherein the apical layer is composed of columnar cells and the deeper layer can be columnar or cuboidal. Functions ○ Barrier ○ Transport HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 4  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 deferens Efferent ductules of epididymis Transitional Barrier Bladder Distensible Renal calyces property Ureters Urethra EPITHELIAL SURFACE SPECIALIZATIONS The apical domain of many epithelial cells can exhibit special structural surface modifications or surface specializations. MICROVILLI Figure 1.10 Electron micrograph of transitional epithelium Surface specialization of the GI tract when stretched and relaxed Finger-like projections Cytoplasmic processes containing a core of Table 1.2 Summary Table Classification of Cell cytoskeletal proteins (20 to 30 actin filaments) Type Function Position Massively increase surface area for absorption Simple Absorption Capillaries squamous Filtration Alveoli Diffusion Abdominal & Barrier pleural cavities Exchange Bowman’s Lubrication capsule Simple cuboidal Absorption Glands and Secretion ducts Figure 1.11 Electron micrograph of microvilli (L); Transportation Kidney tubules Photomicrograph of stained specimen (R) Surface of ovary CILIA Thyroid Surface specialization of the respiratory tract and follicles fallopian tube Hair-like extensions Simple Absorption Digestive tract Cytoplasmic process containing microtubules columnar Protection Gallbladder (central and peripheral) Barrier Transport secretions, foreign bodies, and cells on the Secretion surface, example: ○ In the oviduct or fallopian tube, cilia transport ova and fluid toward the uterus Stratified Barrier Skin ○ In the tracheobronchial tree, cilia sweep mucus squamous Protection Oral cavity and trapped material toward the oropharynx Upper throat Esophagus Vagina Stratified Barrier Sweat gland cuboidal Transport ducts Large ducts of exocrine Anorectal junction Figure 1.12 Ciliated epithelium Pseudostratifie Absorption Upper d columnar Protection respiratory tract Trachea & Bronchial tree Ductus HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 5  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Figure 1.13 Cross-section of the cilium Figure 1.16 Keratinized surface of skin SPECIALIZED EPITHELIAL SURFACE GLANDULAR EPITHELIUM Lining epithelium of upper respiratory tract/ trachea - Develop from epithelial cells that extend from the ciliated pseudostratified columnar surface into the underlying connective tissue Involved in the production and release of secretory products in the body Typically classified into two major groups according to how their products are released - exocrine and endocrine glands EXOCRINE GLANDS Connected to the surface epithelium by excretory ducts, into which their secretory products pass to the external surface Secretions → unaltered form or as a modified secretion Figure 1.14 Ciliated pseudostratified columnar epithelium of URT/ Trachea ENDOCRINE GLANDS Goblet cell - unicellular gland; secretes mucus No duct system The accumulation of the mucus flattens the cilia and Lose their attachment to the surface epithelium triggers coughing, to clear the mucus out of the Secretory products delivered directly into the airway capillaries of the connective tissue Coordinated beating of cilia is the primary Products of endocrine glands → hormones mechanism for clearance of mucus in the airway. STEREOCILIA Surface specialization with limited distribution Unusually long, immotile microvilli Facilitate absorption in the male reproductive system (epididymis, proximal part of ductus deferens) Serve as sensory mechanoreceptors in the sensory epithelium (hair) of the inner ear Figure 1.17 Endocrine Gland SIGNALING MECHANISMS PARACRINE SIGNALING Figure 1.15 General Structure, Cross-section, and Motion Secretory activity that does not reach the Trajectory of Stereocilia bloodstream but affects other nearby cells Paracrine substances are released into the KERATINIZED SURFACE OF SKIN subjacent extracellular matrix Reaches the target cells by diffusion The skin is covered by keratinised squamous Endothelial cells of the blood vessels impact the epithelium vascular smooth muscle cells by releasing multiple Keratin provides inert protective layer factors that cause either contraction or relaxation of Phospholipid around the upper layer of cells allows the vascular wall for water proofing, as it prevents evaporation. HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 6  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Figure 1.20 Photomicrograph of mucus-secreting surface cells of stomach CLASSIFICATION OF MULTICELLULAR EXOCRINE Figure 1.18 Signaling Mechanisms GLANDS AUTOCRINE SIGNALING Table 1.3 Classification of Multicellular Glands Cells secrete molecules that bind to receptors on the same cell that release them Signaling molecules (autocrines) initiate negative feedback pathways to modulate their own secretion Frequently used by cells of the immune system and involves the family of interleukin signaling molecules. CLASSIFICATION OF EXOCRINE GLANDS Either unicellular or multicellular Have three basic release mechanisms for secretory products UNICELLULAR EXOCRINE GLANDS Simplest in structure Individual cells remain in the epithelium of origin and secrete onto that surface Goblet cell, a mucus-secreting cell positioned among other columnar cells → located in the surface lining and glands of the intestines and in certain passages of the respiratory tract MECHANISMS OF SECRETION Three basic release mechanisms Figure 1.19 Photomicrograph of intestinal epithelium showing single goblet cells (arrows) dispersed among absorptive cells MULTICELLULAR EXOCRINE GLANDS Composed of more than one cell, exhibiting varying degrees of complexity Allows subclassification according to the arrangement of the secretory cells (parenchyma) and the presence or absence of branching of the duct Figure 1.21 Mechanism of Secretion elements Lining of the stomach and its gastric pits is a sheet of mucus-secreting cells HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 7  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 MEROCRINE SECRETION ○ Cilia helps move mucus and trapped particles Secretory product delivered to apical surface in out of the respiratory tract. membrane-bounded vesicles Most nuclei are in contact with the basal membrane Vesicles fuse with the plasma membrane and extrude their contents by exocytosis Why columnar? Pancreatic acinar cells Nuclei are elongated. APOCRINE SECRETION Secretory product surrounded by a thin layer of cytoplasm within an envelope of plasma membrane Lactating mammary gland HOLOCRINE SECRETION Secretory product accumulates within the maturing cell Simultaneously undergoes destruction orchestrated by programmed cell death pathways Sebaceous glands of skin, tarsal (Meibomian) glands of the eyelid Why simple? QUESTION AND ANSWER Number of layer: 1 layer (simple) Why cuboidal? The pointed nucleus is somewhat elongated, but most nuclei are circular. Also, consider that this slide includes tissues from areas known to contain simple cuboidal epithelium, such as proximal renal tubules or gland ducts. [Tip: know the common structures and the type of epithelium found there] Simple squamous because the: Number of layer (look at the center only): 1 layer (simple) Nuclei shape: round or oval (squamous) Other features to notice: ○ The presence of red blood cells Simple squamous epithelium is known to line the interior surface of blood vessels (If it lines the blood vessels, it is now called the endothelium). Answer: Microvilli. The one cell layer thick allows for the close Lining of small intestine where absorption of proximity of RBCs to the epithelial cells, nutrients happen facilitating efficient gas exchange. Notable feature: forms a "brush border" that appears as a dense band or fringe of projections TISSUE PREPARATION Why pseudostratified? Number of layer: looks stratified but only 1 layer (Pseudostratified) Figure 1.22 Diagram of the steps in tissue preparation Apical layer has modification (cilia on top) HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 8  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 FIXATIVES SECTIONING Most common chemical formulas for fixatives and its structure LIGHT MICROSCOPY Tissues are sectioned after fixation and embedding FORMALDEHYDE Paraffin-embedded tissue sectioning is performed on Formaldehyde is a single carbon, monoaldehyde a rotary microtome Reacts with primary amino groups to cross-link and stabilize a protein Formaldehyde is used in a solution called formalin Figure 1.23 Chemical structure of formaldehyde reacting to Figure 1.26 Rotary microtome an amino group Tissue embedded in paraffin (tissue block) is GLUTARALDEHYDE secured in a holder attached to a moveable arm of Glutaraldehyde is a dialdehyde the microtome Also reacts with amino groups to cross-link and stabilize proteins It provides superior fixation since it is bi-functional Glutaraldehyde is used for electron microscopy and often used in combination with formaldehyde Formaldehyde and glutaraldehyde are primarily protein fixatives (a) (b) Figure 1.27 Tissue block secured on the microtome; (a) tissue block, (b) knife blade Figure 1.24 Chemical structure of glutaraldehyde reacting Section thickness is generally between 5-20 to 2 amino groups microns for light microscopy The sections form a ribbon as each one is OSMIUM TETROXIDE generated. Collected sections are placed on a glass Osmium tetroxide is used for stabilization and slide for staining retention of lipids It reacts with unsaturated double bonds in fatty acids Since osmium is a heavy metal, it adds contrast to tissues. Osmium fixation is routinely used in electron microscopy (a) (b) Figure 1.25 Chemical structure of osmium tetroxide Figure 1.28 Sectioned paraffin-embedded tissue result; (a) reacting to unsaturated double bonds sectioned tissue, (b) section thickness wheel HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 9  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 ELECTRON MICROSCOPY Electron microscopy sectioning process is analogous to that for light microscopy Sectioning is performed on a highly precise rotary microtome called ultramicrotome Figure 1.32 Oblique section of skeletal muscles, 400x Tangential section is a slice that superficially cuts through the surface of a structure. Figure 1.33 Tangential section of skeletal muscles, 400x Figure 1.29 Ultramicrotome STAINING Tissues are mostly colorless and lack contrast Ultramicrotome is able to produce ultrathin sections 30-60 nm thick SECTION PLANES Histological sections are 2D slices of a 3D piece of tissue Appearance of the tissue and its structures will depend on the angle of the section plane Cross sections, also called transverse sections, Figure 1.34 Unstained paraffin section pass in a plane perpendicular to the long axis of the structure Stains provide contrast and help in differentiating components Stains are often used in combination (a) (b) Figure 1.30 Cross section of skeletal muscles, 400x Figure 1.35 Paraffin section stained by hematoxylin and eosin; (a) hematoxylin shows bluish, (b) eosin shows Longitudinal sections pass in a plane parallel to the pinkish color long axis of the structure TYPES OF STAINS Stains are grouped into 4 major categories: ○ Conventional ○ Histochemical ○ Enzyme histochemical ○ Immunohistochemical CONVENTIONAL STAINS Conventional stains bind to tissue elements primarily based on charges between the stain molecule and Figure 1.31 Longitudinal section of skeletal muscles, 400x tissue structure. Example: While sections are often depicted in either cross or ○ Nucleic acids while acidic dyes bind to longitudinal section, oblique and tangential sections positively-charged elements, e.g., collagen fibers are also used to further interpret the structure 3-dimensionally Oblique sections are sliced through the structure in an angle HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 10  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Figure 1.36 Examples of conventional stains Figure 1.39 Glycogen is seen using the periodic acid Schiiff (PAS) stain LIGHT MICROSCOPY STAINS Hematoxylin and eosin are the most commonly ENZYME HISTOCHEMISTRY STAINS used conventional stains for light microscopy Enzyme histochemistry detects presence of a Hematoxylin is a basic dye that leaves a blue color specific enzyme in the tissue Eosin is an acidic dye that leaves a pinkish-orange This method is often employs substrate analogs to color produce a visible reaction product ○ E.g. Acid phosphatase reacted with its substrate (a) (b) Figure 1.37 (a) hematoxylin stain shows bluish imprint, (b) Figure 1.40 Acid phosphatase is seen using the reaction to eosin stain shows pinkish imprint its substrate ELECTRON MICROSCOPY STAINS IMMUNOCYTOCHEMISTRY STAINS Heavy metals are used for conventional stains for Immunocytochemistry uses antibodies that electron microscopy specifically bind to unique proteins The heavy metal blocks the passage of electrons Sites of antibody binding are detected by through the tissue which imparts contrast to the fluorescent dyes that are fused to the antibody tissue Dark areas in the images are called electron dense due to blockage made by metals bonded to the tissue elements Light areas in the images are called electron lucent due to no encounter with the metals Figure 1.41 Actin is detected using antibodies fused with fluorescent dye MICROSCOPY Microscopes allow us to look more closely at objects (a) (b) and to see beyond what is visible to the naked eye Figure 1.38 (a) Electron dense areas, (b) Electron lucent There are 2 major types, Light and Electron areas microscopes, that are fundamentally differentiated by the type of illumination used HISTOCHEMICAL STAINS They differ in their effective magnification ranges that Histochemical staining involves a chemical reaction correspond to most histological applications between the components and staining reagents This staining is more specific than conventional staining and used to detect specific chemical group ○ E.g. Glycogen is seen by using periodic acid Schiff (PAS) stain HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 11  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Figure 1.42 Magnification ranges of the 2 microscopes Figure 1.45 The compound, bright-field microscope (L) Illumination pathway through the lenses of the microscope (R) Light microscopes have an effective range of magnification from 10x to 1000x. Tissue structure ELECTRON MICROSCOPE and cells can be seen in this range. Larger The optics of TEM are formed by the illumination of intracellular structures like mitochondria are the specimen using a beam of electrons focused by visible electromagnetic lenses Electron microscopes have an effective range of This type of electron microscope is referred to as a magnification from 500x to 200,000x. Greater detail transmission electron microscope of cell and tissue structures can be seen. High performance microscopes can provide images at the molecular level. MAGNIFICATION AND RESOLUTION The final image produced by the microscope entails both magnification and resolution Magnification refers to the amount of enlargement of the specimen Figure 1.46 Transmission electron microscope (TEM) (Left) Figure 1.43 Example of magnification principle Pathway of the beam of electrons inside TEM (Right) Resolution refers to the ability of a microscope to STAINS distinguish closely spaced structures as distinct Stains are necessary to visualize the histological objects structure in a tissue Stains provide contrast and color to the section and reveal chemical information about the structures HEMATOXYLIN AND EOSIN (H&E) Most commonly used for routine histology and pathology section Figure 1.44 Example of resolution principle Referred to as conventional stains because they bind to tissue elements based on charge interaction LIGHT MICROSCOPE The most commonly used light microscope (compound bright-field microscope) It uses 2 lenses to form the final image, hence called compound The lenses are made of glass Figure 1.47 Pancreas stained with H&E, under 1000x PARTS SEEN IN THE IMAGE a) Basophilia HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 12  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 ○ Structures such as nucleus and rough ER stain with hematoxylin and are referred to as basophilic or base loving b) Nuclei ○ Hematoxylin is positively charged, basic dye, and stains structures with negative charge purple to blue ○ Nucleic acid inside nucleus have negative charge thus binds to the hematoxylin c) Rough endoplasmic reticulum ○ Rough ER also has negative charge and binds to the hematoxylin, creating purple stain Figure 1.49 Toluidine blue stained in peripheral nerve, d) Eosinophilia under 1000x ○ Eosinophilia refers to the property of a structure that stains with acidic dye, like eosin PARTS SEEN IN THE IMAGE ○ Eosin is negatively charged a) Nuclei ○ Eosinophilic structures include collagen fibers, b) Blood vessel mitochondria, and some secretory granules c) Axons (arrowed as d) d) Myelin e) Cytoplasm e) Metachromasia (Mast cells) ○ An eosinophilic structure that stains with ○ Metachromasia results from structures with negatively-charged eosin abundant polyanions binds with toluidine blue ○ Due to high density of negative charges, the dye METAL STAINS molecules are bound in close proximity and form aggregates with different absorptive Metals stained in tissues are usually lead and properties uranium ○ Secretory granules demonstrates Heavy metal stains are used for electron metachromasia since they have high density of microscopy proteoglycans These metals block the passage of electrons produced by transmission electron microscope, resulting in dark and light areas in the image OSMIUM Heavy metal staining is a conventional stain Osmium tetroxide is both a stain and a fixative for lipids Reaction of osmium with lipid produces brown color Osmium is also routinely used to preserve lipid Figure 1.48 Tissue stained with heavy metal, under 15,000x PARTS SEEN IN THE IMAGE a) Electron dense Figure 1.50 Toluidine blue staining of oviduct, under 1000x ○ Darker areas in the images are referred to as electron dense PARTS SEEN IN THE IMAGE b) Electron lucent a) Lipid droplets ○ Lighter areas in the images are referred to as b) Nuclei electron lucent c) Cytoplasm c) Nuclear envelope d) Cilia d) Nucleolus PERIODIC ACID-SCHIFF (PAS) TOLUIDINE BLUE PAS is a histochemical stain Toluidine blue is a conventional stain Localizes a unique chemical group which is more A blue-colored basic dye that reacts to the precise than a conventional stain negatively-charged tissue components Reaction of PAS with tissue components produces a Often used as a quick stain for frozen sections as magenta color well as plastic, resin-embedded tissue sections HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 13  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 Figure 1.51 PAS staining of liver showing carbohydrates Figure 1.53 Masson’s trichrome staining of stomach, under (glycogen), under 1000x 200x PARTS SEEN IN THE IMAGE PARTS SEEN IN THE IMAGE a) Glycogen a) Connective tissue b) Nuclei b) Mucus c) Cytoplasm c) Nuclei CRESYL VIOLET-LUXOL FAST BLUE MOVAT’S PENTACHROME This stain is also called the Kluver-Barrera method A complex stain that utilizes 5 dyes to reveal the Commonly used stain for nervous tissue different components of tissue The cresyl violet in the stain binds with the myelin sheath and produces a turquoise color Cresyl violet have the same properties as hematoxylin which stains basophilic structures Figure 1.54 Movat’s pentachrome staining of skin, under 200x PARTS SEEN IN THE IMAGE Figure 1.52 Cresyl violet-luxol fast blue staining of a) Elastic fibers - Stains black brainstem, under 400x b) Collagen fibers - Stains yellow c) Ground substance - Stains blue PARTS SEEN IN THE IMAGE d) Cytoplasm - Stains red a) Nerve cells e) Melanin pigment - Stains brown b) Nuclei c) Myelin ELASTIN (VERHOEFF’S-VAN GIESON) d) Nissl substance Elastin stains are used specifically to reveal elastin, ○ Name given to the unique association of rough a major protein component of elastic fibers and ER and polysomes in neurons sheets ○ They form unique, basophilic complexes only Specificity of the stain is due to unique chemical in neurons properties of elastin, resulting in staining a black or brown color MASSON’S TRICHROME Masson’s trichrome combines 3 dyes Used to reveal the presence of collagen Collagen stains green and cytoplasm stains red or purple The third stain is usually a nuclear counterstain like hematoxylin Figure 1.55 Verhoeff’s-van Gieson staining of elastic artery, under 400x HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 14  EPITHELIAL TISSUES AND HISTOLOGY BASICS TRANS #1 PARTS SEEN IN THE IMAGE a) Elastic sheets (green arrow) b) Smooth muscle cells (red arrow) SILVER Each variation of using silver stain involves precipitation of reduced silver onto tissue elements Figure 1.56 Silver staining of cerebral cortex and lymph node; (a) neurons, (b) reticular fibers, under 1000x PARTS SEEN IN THE IMAGE a) Neuronal cell body b) Dendrites c) Reticular fibers ○ A type of connective tissue fiber that stains with silver due to their unique chemical composition ○ Reticular fiber branch provides the major support framework for lymphoid organs ○ Reticular fibers also surround blood vessels (circular fiber in image) ○ It is widely distributed throughout connective tissue proper REFERENCES Ross, M. H., & Pawlina, W. (2010). Histology: A text and atlas: With correlated cell and molecular biology (9th ed.). Philadelphia: Lip pincott Williams & Wilkins. Mescher, A.L. (2021) Junqueira’s Basic Histology Text & Atlas. 16th Edition, McGraw Hill. Department of Anatomy and Neurobiology and the Office of Faculty Affair (2024)Digital Histology. Virginia Commonwealth University School of Medicine and ALT Lab - VCU. https://digitalhistology.org HG201A TRANSCRIBERS: Plete, Cait, Escote, Gamba, Manila, Padasas, Rodriguez M, Sarmiento, Taha, Toledo, Villas 15