Microscopy & Histology Techniques

Histology and Tissue Biology

• Histology involves all aspects of tissue biology.
• Focuses on optimizing cell structure and arrangement for organ-specific functions.
• Tissues consist of cells and extracellular matrix (ECM).
• ECM supports cells, transports nutrients, removes wastes, and contains macromolecules like collagen fibrils.
• Cells produce the ECM and are influenced by its molecules.
• Matrix components bind to cell surface receptors that connect to internal structural components.
• During development, cells and matrix become functionally specialized, leading to fundamental tissue types.
• Organs combine tissues in an orderly fashion to enable proper functioning.
• Histology requires microscopes and molecular methods due to the small size of cells and matrix.
• Advances in related fields enhance knowledge of tissue biology.

Preparation of Tissues for Study

• Histologic research commonly involves preparing tissue sections for examination with transmitted light.
• Thin translucent sections from tissues/organs are placed on slides for microscopic study.
• Ideal preparations preserve the original structural features of the tissue.
• The preparation process may cause distortions or remove cellular lipids.

Microscopic Preparation Steps

• Fixation: Preserves cell/tissue structure & inactivates degradative enzymes using cross-linking chemicals.
• Dehydration: Removes water by transferring tissue through increasing concentrations of alcohol solutions.
• Clearing: Removes alcohol using organic solvents miscible with both alcohol and paraffin.
• Infiltration: Tissue is placed in melted paraffin to become fully infiltrated with the substance.
• Embedding: The paraffin-infiltrated tissue is placed in a mold with melted paraffin and allowed to harden.
• Trimming: Paraffin block is trimmed to expose tissue for microtome sectioning.
• TEM tissue preparation follows similar steps but uses special fixatives, dehydrating solutions, & epoxy resins.

Microtome Use

• Microtomes section paraffin-embedded tissues for light microscopy, using controlled advance of the block (1-10 μm).
• After each forward move, the tissue block passes over a steel knife edge and a section is cut.
• Paraffin sections are placed on glass slides, deparaffinized, stained, and then light microscopically studied.
• Sections less than 1 µm thick are prepared from resin-embedded cells for TEM using an ultramicrotome with a glass or diamond knife.

Fixation

• Organs should be placed in fixatives immediately after removal from the body.
• Fixatives must fully diffuse to preserve all cells; tissues are often cut into small fragments to facilitate penetration.
• Vascular perfusion is used to introduce fixatives via blood vessels for rapid fixation in large organs.
• Formalin, a buffered isotonic solution, is often used for light microscopy.
• Formalin and glutaraldehyde react with amine groups to prevent protein degradation.
• Glutaraldehyde cross-links adjacent proteins to reinforce cell and ECM structures and is used for electron microscopy.
• Electron microscopy requires careful fixation to preserve ultrastructural detail.
• Glutaraldehyde-treated tissue is immersed in buffered osmium tetroxide which preserves and stains cellular lipids.

Embedding and Sectioning

• Fixed tissues are embedded for thin sectioning.
• Embedding materials include paraffin for light microscopy and plastic resins for light and electron microscopy.
• Before embedding, fixed tissue is dehydrated in increasing ethanol washes, ending in 100% ethanol to fully remove liquid.
• Ethanol is replaced by a solvent miscible with alcohol and the medium, a step called clearing due to translucence.
• Fully cleared tissue is placed in melted paraffin in an oven which evaporates the clearing solvent and promotes paraffin infiltration.
• The resulting block is cooled and placed on a microtome.
• Plastic resins embedding avoids the higher temperatures of paraffin, further limiting cell distortion.
• Paraffin sections are typically 3-10 µm thick for light microscopy; electron microscopy requires sections under 1 µm.
• Sections are placed and stained on glass slides for light microscopy or on metal grids for electron microscopic staining.
• 1 micrometer (1 µm) is 1/1000 of a millimeter (mm), spatial units also include nanometer and angstrom.

Medical Applications of Tissue Preparation

• Biopsies are removed tissue samples analyzed in a pathology lab.
• Rapid freezing in liquid nitrogen preserves cell structures and hardens tissue for quick sectioning, and is an alternative to fixation.
• A cryostat is used to section blocks with tissue at subfreezing temperature, and frozen sections can then be stained and microscopically examined.
• Freezing is effective in histochemical & lipid studies or with highly sensitive enzymes/small molecules because it doesn't inactivate most enzymes.

Staining to Study Tissue Sections

• Tissue sections must be stained (dyed) to be studied microscopically due to colorless cells/material.
• Staining methods make tissue components conspicuous and distinguishable by use of electrostatic linkages.
• Basophilic components have an affinity for basic dyes and are anionic like nucleic acids.
• Acidophilic components have an affinity for acidic dyes and are cationic like proteins and their ionized groups.
• Basic dyes: toluidine blue, alcian blue, and methylene blue.
• Hematoxylin behaves like a basic dye, staining basophilic tissue components.
• Acid dyes: eosin, orange G, and acid fuchsin.
• They stain acidophilic material such as mitochondria, secretory granules, and collagen.
• The most common staining method is Hematoxylin and Eosin (H&E).
• Hematoxylin stains DNA in the nucleus, RNA-rich parts of the cytoplasm, and cartilage matrix dark blue or purple.
• Eosin stains other cytoplasmic structures and collagen pink, acting as a counterstain that highlights more features.
• Trichrome stains (eg, Masson trichrome) enhance distinctions among extracellular tissue components.
• The periodic acid-Schiff (PAS) reaction stains carbohydrates, polysaccharide rings, and carbohydrate-rich purple.
• The Feulgen reaction is a PAS DNA staining method.
• Enzyme digestion identifies material, for example cytoplasmic basophilia with ribonuclease.
• Lipid-rich structures are revealed by avoiding treatments with heat and fat solvents; and by using lipid-soluble dyes.
• Uncommon staining methods employ metal precipitation/ "impregnation" to visualize ECM fibers and nervous components.

Light Microscopy Types

• Bright-field microscopy
• Fluorescence
• Phase-contrast
• Confocal
• Polarizing
• All based on interactions of light with tissue used to reveal and study tissue features

Bright-Field Microscopy

• Bright-field microscopy allows tissue examination using ordinary light passing through the preparation.
• The microscope is composed of an optical system made by the condenser, objective & eyepiece lenses, and focusing mechanisms.
• Optical components include light focusing condenser, objective enlarging lens and eyepiece.
• Total magnification is objective lens power multiplied by ocular lens power.
• Resolution is the smallest distance permitting two structures to be distinguished as separate and is critical to obtain detailed images.
• Resolving power is dependent on lens quality.
• Maximal resolving power ≈ 0.2 µm with 1000-1500x magnification, where only high resolution helps magnification.
• The eyepiece lens enlarges the image obtained for clarity of the tissue sample.
• Virtual microscopy involves digital conversion to create study images of tissues using common web browsers.

Fluorescence Microscopy

• Certain substances emit light when irradiated by a certain wavelength light, called fluoresence.
• Tissue sections usually exposed to UV light causing visible-light emission; where fluoresence appears bright under dark conditions.
• This microscope requires filters to select rays emitted for visualization.
• Fluorescent stains- Acridine Orange, binds the DNA and RNA.
• DAPI and Hoechst bind DNA and stain nuclei with a blue fluoresence using UV.
• Fluorescein conjugated molecules bind cellular components for identification of structures.
• Labeled antibodies are key to immunohistilogic staining.

Phase-Contrast Microscopy

• Unstained cells/sections studied with modified light microscopes, where cellular details are normally hard to see.
• Phase-contrast microscopy allows the viewing of transparent objects by using a lens system.
• Transparent objects cause the speed of light to change and produce an image where cells and structures are easily visible
• Phase-contrast microscopes are an important tool in cell culture laboratories to examine cells without fixation/staining.
• A version of phase-contrast is differential interference contrast microscopy using Nomarski optics.

Confocal Microscopy

• Regular bright-field microscopes use large light beams that fill the specimen, where stray of excess light reduces contrast within the image.
• Confocal microscopy avoids the problems with light scattering by using small point of high-intensity light and a pinhole plate.
• Point, focal point and aperture are all aligned which improves visualization of cells.
• Mirror system moves point of illumination across specimens automatically, digital images in a field of focus will be recorded.
• Optical sections can be produced for 3D reconstruction.

Polarizing Microscopy

• Polarizing microscopy allows recognition of structures with highly organized subunits that can be stained/unstained.
• Polarizing filter prepares the first one directs the light in a single direction; where tissue structure will rotate the axis of light.
• Birefringence helps in studying.

Electron Microscopy

• Based on interaction of tissue components with electrons in beams.
• Resolution is better than in light microscopy.

Transmission Electron Microscopy

• TEM permits resolution of about 3 nm and magnifies up to 400,000x.
• Thin, resin-embedded tissue sections studied at magnifications up to 120,000x.
• Beam of e- passes through tissue section and creates and image with black white and intermediate shades.
• TEM areas of tissue through which e- passed appear bright and low in material/density.
• Adding compounds with heavy metal ions often improves contrast and resolution.
• Cryofracture and freeze etching techniques are for TEM study of cells without fixation or embedding.

Scanning Electron Microscopy

• SEM gives high res view of cell surfaces, tissues and organs.
• SEM beam does not pass through the specimen, rather scans across it.
• Metal spray coating the surface is done first, reflected electrons captured by a detector.
• Shows only surface views but with 3-D quality.

Autoradiography

• Used to locate newly synthesized molecules in cells/tissue.
• Metabolites provided to cells in the area- after fix, process, section & labeled macromolecules emit radiation.
• Slides coated with photographic emulsion.
• After lightproof exposure, metallic silver under a microscope.

Explore Microscopy: Coverslips, magnification, staining issues (H&E, PAS), fixation artifacts, hematoxylin affinity, and glycogen detection. Enhance your histology skills.