Introduction to Histology

• Histology involves studying tissues and their arrangement in organs, focusing on how cell structure and arrangement optimize organ-specific functions.
• Tissues comprise cells and the extracellular matrix (ECM).
• The ECM supports cells and facilitates nutrient transport and waste removal.
• Cells produce the ECM, and interactions between matrix components and cell surface receptors create a continuum for coordinated function.
• During development, cells and the ECM specialize, forming fundamental tissue types.
• Organs arise from the combination of these tissues, enabling organ and organismal function.
• Histology relies on microscopy and molecular methods due to the small size of cells and matrix components.
• Advances in various fields enhance the understanding of tissue biology.

Preparation of Tissues for Study

• Tissue preparation involves creating thin sections for light microscopy.
• The goal is to preserve tissue structure, although preparation can cause distortions.

Fixation

• Fixation preserves tissue structure and prevents degradation using stabilizing or cross-linking fixatives.
• Tissues are cut into small fragments to facilitate fixative penetration.
• Vascular perfusion can introduce fixatives to improve preservation in large organs.
• Formalin (buffered isotonic 37% formaldehyde solution) is a common fixative for light microscopy.
• Glutaraldehyde, used for electron microscopy, cross-links proteins and reinforces cell and ECM structures.
• Electron microscopy requires careful fixation to preserve ultrastructural detail, often involving glutaraldehyde followed by osmium tetroxide.

Embedding & Sectioning

• Fixed tissues are embedded in a firm material like paraffin or plastic resins for thin sectioning.
• Dehydration with increasing ethanol solutions is required before infiltration with embedding media.
• Clearing replaces ethanol with an organic solvent miscible with both alcohol and the embedding medium, rendering the tissue translucent.
• Paraffin embedding occurs in an oven at 52°-60°C.
• Plastic embedding avoids high temperatures, minimizing tissue distortion.
• The hardened block is trimmed and sectioned using a microtome.
• Paraffin sections are typically 3-10 μm thick for light microscopy.
• Electron microscopy needs sections less than 1 μm thick.
• Sections are placed on glass slides or metal grids for staining and examination.
• Micrometer (μm), nanometer (nm), and angstrom (Å) are spatial units used in microscopy.

Medical Applications

• Biopsies are tissue samples analyzed in pathology laboratories.
• Rapid freezing in liquid nitrogen is used for quick analysis during medical procedures, employing a cryostat.
• Freezing preserves enzyme activity for histochemical studies and is useful for studying lipids.

Staining

• Staining is essential because most cells and extracellular material are colorless.
• Dyes selectively stain tissue components based on their chemical properties.
• Basophilic components (nucleic acids) are anionic and have an affinity for basic dyes.
• Cationic components (proteins) are acidophilic and stain with acidic dyes.
• Basic dyes include toluidine blue, alcian blue, and methylene blue, while hematoxylin behaves like a basic dye.
• Acid dyes include eosin, orange G, and acid fuchsin.
• Hematoxylin and eosin (H&E) staining is commonly used; hematoxylin stains DNA dark blue or purple, and eosin stains cytoplasm and collagen pink.
• Eosin acts as a counterstain.
• Trichrome stains provide greater distinction among extracellular tissue components.
• The periodic acid–Schiff (PAS) reaction stains carbohydrate-rich structures purple or magenta.
• The Feulgen reaction specifically stains DNA.
• Enzyme digestion can further identify basophilic or PAS-positive material.
• Lipid-rich structures are revealed by avoiding heat and organic solvents and using lipid-soluble dyes like Sudan black.
• Metal impregnation techniques, often using silver salts, visualize certain ECM fibers and cellular elements in nervous tissue.
• Slide preparation takes 12 hours to 2½ days, concluding with mounting a coverslip.

Light Microscopy

• Light microscopy includes bright-field microscopy and specialized applications like fluorescence, phase-contrast, confocal, and polarizing microscopy.
• These are based on the interaction of light with tissue components.

Bright-Field Microscopy

• Bright-field microscopy uses ordinary light to examine stained tissue.
• The microscope includes a condenser, objective lens, and eyepiece.
• Total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses.
• Resolving power is the smallest distance between two structures that can be seen as separate objects.
• The maximum resolving power of a light microscope is approximately 0.2 μm.
• Resolving power, image clarity and richness of detail, depends mainly on the quality of the objective lens.
• Virtual microscopy uses high-resolution digital images of stained tissues.
• It allows tissue study on a computer without a physical slide, scanned with a slide-scanning microscope.

Fluorescence Microscopy

• Fluorescence microscopy uses UV light to irradiate tissue sections, causing fluorescent substances to emit light.
• The fluorescent substances appear bright on a dark background.
• Fluorescent compounds with affinity for specific cell macromolecules can be used as fluorescent stains.
• Acridine orange binds both DNA and RNA.
• DAPI and Hoechst specifically bind DNA and stain cell nuclei blue under UV light.
• Fluorescein can be coupled to molecules that specifically bind to certain cellular components.
• Antibodies labeled with fluorescent compounds are extremely important in immunohistologic staining.

Phase-Contrast Microscopy

• Phase-contrast microscopy allows the study of unstained cells and tissue sections.
• It uses a lens system that produces visible images from transparent objects and can be used with living, cultured cells.
• It is based on the principle that light changes its speed when passing through structures with different refractive indices.
• Differential interference contrast microscopy with Nomarski optics produces a 3D image of living cells.

Confocal Microscopy

• Confocal microscopy uses a point of high-intensity light and a pinhole aperture to achieve high resolution and sharp focus.
• It avoids stray light.
• A computer-driven mirror system moves the point of illumination across the specimen.
• Digital images captured at many individual spots in a very thin plane of focus are reconstructed into a 3D image.

Polarizing Microscopy

• Polarizing microscopy identifies structures made of highly organized subunits.
• Tissue structures containing oriented macromolecules rotate the axis of light and appear as bright structures against a dark background.
• Birefringence is the ability to rotate the direction of vibration of polarized light.
• It is a feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin filaments.
• Digital cameras extend the utility of light microscopic methods.

Electron Microscopy

• Transmission and scanning electron microscopes are based on the interaction of tissue components with beams of electrons.
• The wavelength in an electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution.

Transmission Electron Microscopy

• The transmission electron microscope (TEM) has a resolution around 3 nm.
• It uses electromagnetic "lenses" to focus a beam of electrons through the tissue section.
• Regions of an electron micrograph correspond to tissue areas through which electrons passed readily (appearing brighter or electron-lucent) and areas where electrons were absorbed or deflected (appearing darker or more electron-dense).
• Compounds with heavy metal ions, such as osmium tetroxide, lead citrate, and uranyl compounds, improve contrast and resolution.
• Cryofracture and freeze etching allow TEM study of cells without fixation or embedding, useful in the study of membrane structure.

Scanning Electron Microscopy

• Scanning electron microscopy (SEM) provides a high-resolution view of the surfaces of cells, tissues, and organs.
• The beam of electrons does not pass through the specimen.
• The surface of the specimen is spray-coated with a thin layer of heavy metal that reflects electrons.
• Reflected electrons are captured to produce a black-and-white image.

Autoradiography

• Microscopic autoradiography localizes newly synthesized macromolecules in cells or tissue sections.
• Radioactively labeled metabolites are incorporated into specific macromolecules.
• Slides are coated with photographic emulsion containing silver bromide crystals to detect radiation.
• Silver grains indicate the locations of radiolabeled macromolecules in the tissue under the microscope or TEM.
• The radioactive precursor of DNA (such as tritium-labeled thymidine) can show cells in a tissue replicating DNA and preparing to divide.

Cell & Tissue Culture

• Live cells and tissues can be maintained and studied outside the body in culture (in vitro).
• Cell culture allows the direct observation of cellular behavior under a phase-contrast microscope.
• Cells and tissues are grown in complex solutions of known composition with serum or specific growth factors.
• Cells are dispersed and placed in a dish to which they adhere, usually as a single layer.