Extracellular Matrix & Tissue Preparation

Histology and Tissue Organization

• Histology examines tissues and how their structure optimizes organ-specific functions.
• Tissues consist of cells and the extracellular matrix (ECM).
• ECM supports cells, transports nutrients, and removes waste.
• Cells produce ECM and are influenced by it.
• ECM components bind to cell surface receptors, connecting to intracellular components for coordinated function.
• Specialized cells and ECM form fundamental tissue types.
• Organs consist of orderly arranged tissues, enabling organ and organism function.
• Microscopy and molecular methods are essential due to the small size of cells and matrix components.
• Advances in various fields enhance tissue biology understanding.
• Knowledge of scientific methods is crucial for understanding tissue biology.
• This section reviews common methods for studying cells and tissues, with emphasis on microscopic approaches.

Preparation of Tissues for Study

• Histologic research often involves tissue sections for transmitted light examination.
• Tissues are sectioned thinly to allow light to pass through for internal structure observation
• Ideal preparations preserve in vivo structural features.
• Preparation can remove cellular lipids and distort structure
• Key preparation steps for light microscopy include fixation, embedding, and staining.

Fixation

• Organs are placed in fixatives immediately after removal from the body
• This process stabilizes cell structure and cross-links compounds that may degrade cells
• Small tissue fragments are used to facilitate fixative penetration.
• Vascular perfusion is used to introduce fixatives via blood vessels for rapid, widespread fixation in large organs.
• Formalin is a common fixative for light microscopy.
• Glutaraldehyde is a fixative used for electron microscopy.
• Glutaraldehyde and Formalin react with amine groups of proteins, preventing protease degradation.
• Glutaraldehyde cross-links proteins and reinforces ECM structures.
• Electron microscopy requires careful fixation to preserve ultrastructural detail.
• Glutaraldehyde-treated tissue is immersed in osmium tetroxide to preserve and stain lipids

Embedding & Sectioning

• Fixed tissues require infiltration and embedding to facilitate thin sectioning and provide support.
• Paraffin is used for light microscopy while plastic resins are used for light and electron microscopy
• Dehydration is achieved by using a series of increasing concentrated ethanol solutions, until 100% ethanol is achieved in order to remove water
• Ethanol is replaced by a solvent miscible with alcohol and the embedding medium
• Infiltration with the reagents used here gives the tissue a translucent appearance
• Tissue is embedded in melted paraffin in an oven, which evaporates the solvent and promotes paraffin infiltration
• Plastic resin embedding avoids high temperatures, which can distort tissue.
• A microtome section hardened blocks containing tissue.
• Paraffin sections have a thickness of 3-10µm for light microscopy.
• Electron microscopy requires sections of less than 1µm thick.
• Micrometer (µm) is 1/1000 of a millimeter(mm).
• Nanometer (nm) is 0.001 µm, 10-6 mm, or 10-9 m.
• Angstrom (Å) equals 0.1 nm or 10-4 µm.
• Sections are placed on slides for light microscopy or on metal grids for electron microscopy.

Staining

• Most cells and ECM are colorless and must be stained for microscopic study.
• Staining methods make tissue components conspicuous and distinguishable.
• Dyes stain material selectively, acting like acidic or basic compounds and forming electrostatic linkages with macromolecules.
• Basophilic components have an affinity for basic dyes.
• Acidophilic components stain readily with acidic dyes.
• Toluidine blue, alcian blue, and methylene blue are examples of basic dyes.
• Hematoxylin behaves like a basic dye, and stains basophilic tissue components.
• Acid dyes like eosin, orange G, and acid fuchsin stain acidophilic components.
• H&E stains DNA, cytoplasm, and cartilage a dark blue or purple.
• Eosin stains other cytoplasmic structures and collagen pink.
• Eosin is considered a counterstain when it is applied separately to distinguish additional tissue features
• Trichrome stains enhance distinctions among extracellular tissue components.
• Periodic acid-Schiff (PAS) reaction stains carbohydrate-rich structures purple or magenta.
• Cell nuclei DNA can be stained using a modification of the PAS procedure called the Feulgen reaction.
• Enzyme digestion helps identify basophilic or PAS-positive material.
• Pretreatment with ribonuclease reduces cytoplasmic basophilia.
• Lipid-rich structures are revealed by dyes such as Sudan black.

Light Microscopy

• Bright-field, fluorescence, phase-contrast, confocal, and polarizing microscopy rely on light interactions with tissue components.
• These methods reveal and study tissue features.

Bright-Field Microscopy

• Bright-field microscopy examines stained tissue with ordinary light.
• Microscopes include an optical system and mechanisms for specimen movement and focus.
• The condenser focuses light on an object
• The objective lens enlarges an image and projects it to the observer.
• The eyepiece further magnifies image and projects it onto the viewer's retina.
• Total magnification is objective lens power multiplied by ocular lens power.
• Resolution is the smallest distance between structures that allows them to be seen as separate objects
• The maximum resolution is approximately 0.2µm permitting clear images at 1000-1500x magnification.
• Objective lenses with higher magnification are designed to also have higher resolving power
• Eye piece lens only enlarges the image, and does not improve resolution

Fluorescence Microscopy

• Certain substances emit light of a longer wavelength when irradiated appropriately, a phenomenon called fluorescence.
• Fluorescence microscopy irradiates sections with UV light, with emission in the visible spectrum.
• Fluorescent substances appear bright on a dark background.
• It uses a light source and filters to select rays of different wavelengths.
• Fluorescent compounds with cellular macromolecule affinity can be used as fluorescent stains
• Acridine orange binds both DNA and RNA
• DAPI and Hoechst specifically bind DNA and stain cell nuclei.
• Fluorescein-labeled compounds identify cellular components.
• Antibodies labeled with fluorescent compounds are used in immunohistologic staining.

Phase-Contrast Microscopy

• Used to study unstained cells and tissue sections.
• Phase-contrast microscopy uses a lens system to create visible images from transparent objects.
• This can be used with living culture cells.
• Phase-contrast uses the changes in light speed when passing through structures to allow structures to appear lighter or darker in relation to each other
• Prominent in cell culture laboratories for examining cells without fixation or staining.
• Differential interference contrast microscopy uses Nomarski optics for a 3D aspect of living cells.

Confocal Microscopy

• Uses a small, high-intensity light and a pinhole aperture to improve resolution and focus.
• Greatly improves localization of specimen components.
• Computer-driven mirror system moves the point of illumination across the specimen rapidly.
• Digital images can be reconstructed to create a 3D image

Polarizing Microscopy

• Allows for recognition of the subunits of highly organized structures that consist of highly organized subunits.
• Polarizing filter causes light to vibrate in one direction
• A second filter perpendicular to the first will cause no light to pass through
• Tissue that contains oriented macromolecules, can rotate the axis of polarized light
• The tissue appears as bright structures against a dark background
• Birefringence refers to the rotation of the vibration direction of polarized light and is useful to identify crystalline substances
• Use in substances that are composed of cellulose, collagen microtubules, such as actin filaments

Electron Microscopy

• Transmission and scanning electron microscopes relies on the interaction between tissue components and beams of electrons.
• Wavelength allows a 1000 fold increase in resolution.

Autoradiography

• Localizes newly synthesized macromolecules in cells or tissue sections.
• Metabolites are radioactively labeled and incorporated into macromolecules.
• Slides with radiolabeled sections are coated with photographic emulsion.
• Silver bromide crystals act as microdetectors of radiation.
• This allows for cellular components to be easily viewed using light and electron microscopes.

Cell & Tissue Culture

• Live cells and tissues can be maintained and studied outside of body in culture, also known as in vitro.
• Achieved with complex solutions of known composition with addition of serum or specific growth factors
• Cells from tissues are dispersed mechanically or enzymatically and placed in clear dishes to which they adhere.
• Explants are called primary cell cultures.
• Improvements in culture technology and growth factors now allow most cell types to be maintained in vitro.

Enzyme Histochemistry

• Localizes cellular structures using a specific enzymatic activity present in those structures.
• Enzymes that can be detected histochemically include the following:
• Phosphatases remove phosphate groups
• Dehydrogenases transfer hydrogen ions
• Peroxidase promotes transfer of hydrogen ions to hydrogen peroxide

Visualizing Specific Molecules

• A macromolecule present in a tissue can be identified by using tagged compounds that bind the molecule of interest.
• These compounds need to be visible with either light or electron microscopy.
• Most commonly labels consist of fluorescent compounds, radioactive atoms that can be detected with autoradiography, metal particles, or molecules of peroxidase
• These methods can be used to detect and localize sugars, proteins, and nucleic acids

Immunohistochemistry

• Uses labeled antibodies to identify and locate proteins.
• Every immunological technique requires an antibody of the protein or antigen that is being detected
• Antibodies can be polyclonal or monoclonal
• Can be directly or indirectly expressed

Hybridization Techniques

• Nucleic acids can be labeled to easily identify gene presence
• Also useful for localizing specific mRNA
• In hybridization, the two strands of nucleic acid bind, which allows for the detection of a specific DNA sequence

Interpretation of Structures in Tissue Sections

• Tissue sections can be distorted during sampling because structures within the cell degrade during histological tissue preparation
• Microscopic preparations can display minor artifacts such as shrinkage, cracks from the fixation or removal of substances.
• No single stain can stain all tissue components together, requiring that the tissue be stained through different methods
• Examining the sections helps in determining whole composition and structure
• Additionally, when 3-dimensional materials are cut during sectioning, they appear as though they have only length and width

This covers the role of the extracellular matrix (ECM) in tissues and the essentials of tissue preparation for microscopic examination. It also highlights the interaction between cells and the ECM, and the importance of tools and methods in scientific study.