Histology: Tissues, Cells, and ECM

• Histology defined: study of tissues' arrangement and components within the body.
• The structural arrangement optimizes tissue function.
• Histology examines tissue architecture (structure, color, shape) to understand its physiology.
• Organization: Cell --> Tissue --> Organ.

Tissue Components

• Cells
• Extracellular matrix (ECM)

General Tissue Makeup: Cell

• Cells are variety of types.
• Functions: protection, absorption, secretion, storage.
• "Ingredients" needed for byproduct release.

ECM

• Fibers - for connection and structural support.
• Types of fibers: collagen (swirl), elastic (hair-like), and reticular.
• Ground Substance - small molecules supplying raw materials or nutrients to cells.
• Components include GAG, proteoglycan, and glycoprotein.
• Fluids akin to plasma, mostly water, fill spaces and flow across tissues.
• Tissue appearance and function vary due to cell type, ECM component proportions, and tissue arrangement into organs.
• Organ differentiation happens at the tissue level.

Histotechniques: Tissue Acquisition (Specimen Collection)

• Cytological swab: cheek scraping for cell examination.
• Biopsies: diagnosis of cancer cells via small incision.
• Surgical resection: tumor removal via open surgery.

Histotechniques: Tissue Processing

• Fixation: halts biological activity, acting as preservative.
• Prevents decomposition via apoptosis and bacterial activity.
• Protects cells/tissues from harsh chemicals.
• Common fixative: 10% buffered formalin.
• Dehydration: removes water using ascending alcohol concentrations.
• Clearing: xylene makes tissue translucent/transparent.
• Infiltration: paraffin wax infiltrates open spaces.
• Embedding: paraffin wax encases tissue in paraffin block for cutting support.
• Trimming: slicing tissue block near the tissue.
• Sectioning: microtome cuts 3-10 um tissue layers, producing tissue ribbons.
• Staining: colors tissue.
• Mounting: cover slip addition for 10-year preservation using Canada Balsam glue.
• Labelling

Histology Staining: Dyes

• H&E is a common dye combination.
• H = Hematoxylin (OH+) / Primary Stain
• Positively charged (basic dyes) - stains negatively acidic structures (DNA & RAN in nucleus and ribosomes)
• Has a blue hue.
• Structures staining well are "basophilic."
• E = Eosin (H-) / Counter Stain
• Negatively charged (acidic dye) - dyes positively charged basic structures (proteins in cytoplasm).
• Has pink hue.
• Well-stained structures called "acidophilic."
• H&E follows the principle of pH.

Histochemistry

• Utilizes natural chemical interactions between tissues and chemicals.
• H&E is a type of histochemistry.
• Other histochemical stains include:
• Trichrome: visualizes collagen using dye combinations.
• Periodic Acid Schiff: magenta color reveals carbohydrate-rich regions and identifies mucopolysaccharide metabolic issues.

Immunohistochemistry

• Employs antigen-antibody interactions to recognize proteins or foreign materials.
• Apply primary antibody to recognize protein.
• Tumor markers (CA-19).
• Apply the secondary antibody with chromogen to recognize the primary antibody.
• Apply chemicals react with the chromogen to mark brown region of tissue.
• Direct immunohistochemistry uses one antibody.
• Indirect immunohistochemistry uses two or antibodies.

Immunofluorescence

• Antigen-antibody interactions are used similar to immunohistochemistry.
• Primary antibody identifies target protein.
• A secondary antibody offers a fluorescent marker.
• Fluorophore emits frequency in color spectrum when exposed excitation by UV light or laser.
• Multiple proteins are able to be visualized.
• Acridine orange is used for staining.

Microscopy - Observing Tissue Slides

• Microscopes magnify prepared tissue slides for observation at various levels.

Light Microscopy

• Natural light illuminates tissues for inspection.
• Applied to tissues dyed using histochemistry & immunohistochemistry, plus immunofluorescence.
• Most common microscope used.
• Types
• Brightfield Microscope - Tissue image appears like a 'shadow'.
• Dark Field Microscope - tissue emits light against black background.
• Phase Contrast Microscope
• Polarizing Microscope
• Fluorescence Microscope
• Confocal Microscope
• Based on light interactions with tissue components.
• Light microscopes utilize photons.
• Bright field can be customized to dark field.
• All light microscopes are not brightfield, but all brightfield ones are light.

Electron Microscopy

• Transmission Electron Microscopy
• Scanning Electron Microscopy
• Electron molecule interaction with tissue structures.
• Electron Microscopy employs electrons.
• Tissues are heavy metal-soaked (gold).

Microscope System

• Mechanical Part
• Function: Supports equipment and improves resolution.
• Parts: arm, base, head, stage, stage clip, stage control, nosepiece.
• Illuminating Part: Fine/coarse adjustment knob, diopter adjustments.
• Lens (Magnifying) Part
• Illumination, Light Switch, Brightness adjustments, Condenser, Eyepiece, and Objective lens [Scanner 4x, Low power objective 10x, High power objective 40x, Oil Emersion Objective].
• Eyepiece + Objective lens = Total magnification of 40x, 100x, 400x, 1000x.
• Resolving power distinguishes two structures at smallest distance.
• Parfocal theory minimizes focusing.

Phase Contrast Microscope

• Cells or tissues need not be stained for observation of live cells and tissues
• Bends light and leverages different refractory properties.

Confocal Microscopy

• (1) A small point of high-intensity light, often from a laser, and (2) a plate with a pinhole aperture in front of the image detector.
• Creating such optical sections at a series of focal planes through the specimen allows them to be digitally reconstructed into a 3D image
• Polarizing microscope - they perform birefringence (crystals).
• Virtual Microscopy
• Software or app simulating the use of microscope on a digital screen
• Allows navigation of the virtual tissue slide similar to Google Map
• Transmission Electron Microscopy
• Instead of photons, electrons are used as a source of "illumination"
• Small size of electrons allow greater magnification and resolution of tissues and cells
• Electrons pass through the tissue, casting different shades of "shadows"
• Darker areas on TEM (electron dense areas): more cellular or tissue materials are concentrated (nucleus)
• Lighter areas on TEM (electron light areas): less cellular or tissue materials are present & allowed electrons to pass (cytoplasm and cellular membrane)
• Up to 400000x magnification
• Scanning Electron Microscopy
• Instead of electrons passing through the tissue, they are scattered by the tissue
• Able to capture three-dimensional surface contours of the tissue
• More powerful because it gives 3d image
• Interpretation of Histology slides
• Artifacts (foreign objects/aggregate chemicals) are natural part of histology (artifacts in tissue is not natural/normal)
• Many steps in histotechnology and staining, each step an opportunity to introduce artifacts Tissue tearing, shrinkage, folding, fading stains, foreign particles Maybe advantageous in tissue ID Presentation of Histology slides Mind the presentation One, 7-micron slice of a large organ does not represent the entire organ
• Representation of Tissue slides
• Typical tissue images are 2D representation of 3D structures
• Levels of section
• Planes of section

4 Basic Tissue Types

• Epithelial tissue - closely aggregated cells, functions: always in covering and lining of the body look at the very top of the tissue cell rich but ECM poor Connective tissue - always seen under the epithelial tissue cell poor but ECM rich rich in oxygen lots of collagen in appearance rich in reticular Muscle tissue - for protection cell rich and ECM poor has striation for contraction and constriction
• Nervous tissue - usually found in the brain and spinal cord branching appearance, long ending used for transmitting and recieving signals

Epithelial Tissues

• Composed of closely aggregated polyhedral cells adhering strongly to one another and to a thin layer of ECM, forming cellular sheets that line the cavities of organs and cover the body surface
• Line all external and internal surfaces of the body and all substances that enter or leave an organ must cross this type of tissue it is important that they are aggregated for retrieval of substance external - they serve as a protection for the substances that must maintain inside the body internal - e.g. small intestine - it is important because they have the first gives (absorb) on the nutrients

Principal Functions of Epithelial Tissues

Covering (for protection), lining (absorption), and protecting surfaces (e.g. epidermis) - lining epithelial Absorption (e.g. the intestinal lining) - lining epithelial Secretion (e.g. parenchymal cells of glands) - glandular epithelium usually seen in connective tissue because glands function to produce hormone Exocrine - secretes by product on external environments and secretes through lumen Endocrine - secretes by product inside the organ and secretes through connective tissue itself such as capillaries Myoepithelial cells Specialized sensory cells

Characteristic features of Epithelial Cells

Shapes and dimensions of epithelial cells are quite variable, ranging from tall columnar to cuboidal to low squamous cells Squamous - for covering, e.g. skin

• flat like a fried egg Cuboidal - for secretion, e.g. glands cubes, perfect square Columnar - for absorption, e.g. digestive tract based on the shape of nucleus Epithelial cell nuclei vary in shape and may be elliptic (oval), spherical, or flattened, with nuclear shape corresponding roughly to cell shape Most epithelia are adjacent to connective tissue containing blood vessels Avascular, High Mitotic Index avascular - no vain or vesicle or blood high mitotic index - can generate new cells in short period of time

Epithelial cells generally show polarity

Basepole Basement Apical pole most upper part Lateral surfaces lower part cell to cell junction

###E pithelial cell junctions, basal lamina, and basement membranes Tight Junctions (Zonula Occludens) Term "zonula" indicates that the junction forms a band completely encircling each cell Most apical of the junctions Band of branching strands in the membrane around each cell's apical end. The seal between the two cell membranes is due to tight interactions between the transmembrane proteins claudin and occludin Serve as fences restricting movements of membrane lipids and proteins at the apical cell surface into the lateral and basal surfaces, and vice versa. It serves as protection and filtration of large molecules it uses claudin and occludin, funtion: serves as fences of the filtration of large molecule looks like 'zip loc' to sealed the substances and prevent leak

• Abberant Claudin and Occludin Expression Paracellular movement of molecules Adherent Junction (Zonula adherens) Also encircles the epithelial cell, usually immediately below the tight junction Firmly anchoring a cell to its neighbors Mediated by cadherins, catenins "terminal web" function: anchors cell to cell ,it for small molecules Desmosome (Macula Adherens) Resembles a single "spot-weld” and does not form a belt around the cell Disc-shaped structures Desmosomes contain larger members of the cadherin family called desmogleins and desmocollins Provide firm cellular adhesion and strength throughout the epitheliu reason why cells are aggregated has microfilaments weak desmosomes - vertigus bulgaris Gap Junction (Nexus) Mediate intercellular communication The transmembrane gap junction proteins, connexins, form hexameric complexes called connexons, each of which has a central hydrophilic pore about 1.5 nm in diameter Permit intercellular exchange of molecules with small diameters Allowing cells in many tissues to act in a coordinated manner its job is to not aggregate cells but to communicate to cells in a coordinated mannee Hemidesmosome On the basal epithelial surface, cells attach to the basal lamina by anchoring junctions Structures resemble a half-desmosome ultrastructurally Integrins Bind primarily to laminin molecules in the basal lamina

Basement Membrane

Basal surface of all epithelia rests on a thin extracellular, felt-like sheet of macromolecules A semipermeable filter for substances reaching epithelial cells from below 2 parts on TEM: Basal lamina- lamina lucida and densa half of it is epithelial tissue lamina are usually seen in connective tissue Reticular lamina Functions of Basement Membrane: Filter Structural support Organize proteins Maintaining cell polarity Cell-to-cell interactions Scaffold that allows rapid epithelial and regeneration.

Specializations of the Apical Cell Surface

Microvilli: Seen on apical cell surfaces of epithelia specialized for absorption Increase total surface area by 20- or 30-fold E.g. small intestine Covered with glycocalyx (PAS+) Each microvillus contains bundled actin filaments function: absorption O non-motile always seen in columnar has regular pattern Cilia Long, highly motile apical structures; containing internal arrays of microtubules Primary cilium

Not motile for detection of light, odors, motion, and flow of liquid Motile cilia/typical cilia: abundant on cuboidal or columnar cells Axoneme: 9 doublet + 2 singlet assembly microtubules Basal bodies: similar structure to centrioles Exhibit rapid beating patterns Respiratory epithelium, reproductive system has irregular pattern

O has prominent hair-like structure function: geneate current motile

Stereocilia

Less common type of apical process Absorptive epithelial cells lining the male reproductive system (e.g. epididymis Increase the cells' surface area, facilitating absorption More specialized stereocilia with a motion-detecting function are important components of inner ear sensory cells Contain arrays of microfilaments and actin-binding proteins Typically much longer and less motile than microvilli, and may show branching distally function: for absorption usually seen in male reproductive system that stores and give nutrients has a flame pattern keratinization Unique to stratified epithelia of the skin and certain parts of oral cavity Dead layer of the epithelial cells (keratin layer) hang onto the living layer underneath → extra protection against frequent contact and frictions E.g. skins serves as additional protection. far from cell nutrients but retain metabolic function

Types of Epithelia - Covering/Lining Epithelia

Organization of cells that cover surface According to the number of cell layers: Simple - cell continuous layer Stratified - 2 or more layers Pseudostratified - pseudo means fake, looks like stratified Shape of cell (apical surface): Squamous (keratinized vs non-keratinized) - protection Cuboidal - secretion Columnar (ciliated vs non-ciliated) - absorption Transnational epithelium (bladder)- shape Nucleus is from smaller to bigger Secretory Epithelia & Glands Function of gland - for secrete Classification Manner Exocrine : Remains connected with the surface epithelium - form Endocrine Losethee connecito w originak epithelium NUMBER OF CELL LAYERS + CELL SHAPE + SURFACE SPECIALIZATION +EPITHELIUM SIMPLE STRATIFIED PSEUDOSTRATIFIED SQUAMOUS CUBOIDAL COLUMNAR

Transitional epithelium (bladder)- shape Stratified squamous - keratin, non-keratin CILIATED NON CILIATED KERATIN NON KERATIN

Pseudostratified columnar - ciliated, non-ciliated Organs specific names for certain covering epithelia Mesothelium: lining of body Urothelium: lining of major urinary tract

Single Squamous Epithelium

• Allows rapid exchange for exchange of materials such as (H2O, CO2, O2
• Lung, Kidneys, endothelium, mesothelium

- Stratified squamous

protection for laceration and friction Simple cuboidal cells Kidneys, Structure, support. Absorption

Simple Columnar Epithelium

• Absorption secretion, structural support
• Intestinal lining (brush border/ microvilli), glands, and ducts
• Simple Columnar Ciliated Epithelium
• Absorption, secretio, generating currents
• Lining of uterine tubes Squamous ( cells, fat nucleus) Cuboidal cells