Histology: Study of Body Tissues

• Histology involves the study of body tissues, including their arrangement to form organs.
• Histology covers all aspects of tissue biology.
• The focus is on how cells' structure and arrangement optimize functions specific to each organ.
• Histology relies on microscopes and molecular methods due to the small size of cells and matrix components.
• Advances in biochemistry, molecular biology, physiology, immunology, and pathology are essential for understanding tissue biology.
• Understanding the tools and methods of science is crucial for understanding the subject.
• Tissue slices, or "sections," are created for visual examination with transmitted light.
• Thin, translucent sections of tissues and organs are placed on glass slides for microscopic examination of internal structures due to their thickness.
• Ideally, microscopic preparations should preserve the tissue on the slide with the same structural features it had in the body.
• The ideal microscopic preparation is often not feasible, as the preparation process can remove cellular lipid, with slight distortions of cell structure.
• Tissue preparation involves basic steps for light microscopy, as shown in a reference figure.

Steps of Tissue Preparation

• Fixation involves placing tissue pieces in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, thereby preserving cell and tissue structure.
• Dehydration is where tissue are transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, to remove all water.
• Clearing is where alcohol is removed in organic solvents in which both alcohol and paraffin are miscible.
• Infiltration occurs when tissue is placed in melted paraffin until completely infiltrated.
• Embedding is achieved by placing the paraffin-infiltrated tissue in a small mold with melted paraffin and it to harden.
• Trimming involves trimming the paraffin block to expose tissue for sectioning (slicing) on a microtome.
• Preparing tissue for transmission EM involves similar steps, except special fixatives and dehydrating solutions are used with smaller tissue samples.
• Preparing tissue for transmission EM involves epoxy resins which become harder than paraffin to allow very thin sectioning.
• A microtome is used for sectioning paraffin-embedded tissue for light microscopy.
• The trimmed tissue specimen is mounted in the paraffin block holder.
• Each turn of the drive wheel by the histologist advances the holder a controlled distance, generally from 1 to 10 µm.
• After each forward move, the tissue block passes over the steel knife edge and a section is cut at a thickness equal to the distance the block advanced.
• Paraffin sections are placed on glass slides, allowed to adhere, deparaffinized, and stained for light microscope study.
• For TEM, sections less than 1 µm thick are prepared from resin-embedded cells using an ultramicrotome with a glass or diamond knife.
• Fixation preserves tissue structure and prevents degradation by enzymes released from cells or microorganisms.
• Pieces of organs are placed in fixatives (solutions of stabilizing or cross-linking compounds) as soon as possible after removal from the body.
• Tissues are cut into small fragments before fixation for better penetration, as a fixative must fully diffuse through the tissues to preserve all cells.
• To improve cell preservation in large organs, fixatives are often introduced via blood vessels via vascular perfusion.
• Formalin, a buffered isotonic solution of 37% formaldehyde, is a commonly used fixative for light microscopy.
• Formalin and glutaraldehyde, used for electron microscopy, react with amine groups (NH2) of proteins, preventing degradation by common proteases.
• Glutaraldehyde cross-links adjacent proteins, reinforcing cell and ECM structures.
• Electron microscopy provides greater magnification and resolution of very small cellular structures.
• Fixation must be very careful to preserve additional "ultrastructural" detail.
• Glutaraldehyde-treated tissue is immersed in buffered osmium tetroxide, preserving (and staining) cellular lipids as well as proteins in electron microscopy studies.
• Fixed tissue undergoes gradual dehydration by extracting water.
• Dehydration involves transfers through a series of increasing ethanol solutions, ending in 100% ethanol.
• Ethanol is replaced by an organic solvent miscible with both alcohol and the embedding medium.
• Replacing ethanol is called "clearing" because the reagents give the tissue a translucent appearance.
• Fixed tissues are infiltrated and embedded in a firm material for thin sectioning.
• Paraffin, used routinely for light microscopy, and plastic resins, adapted for both light and electron microscopy, are embedding materials.
• Fully cleared tissue is placed in melted paraffin in an oven at 52°-60°C for infiltration with paraffin.
• The paraffin is then allowed to harden in a small container at room temperature.
• Tissues to be embedded with plastic resin are also dehydrated in ethanol, and then infiltrated with plastic solvents that harden when cross-linking polymerizers are added.
• Plastic embedding avoids the higher temperatures needed with paraffin, which helps prevent tissue distortion.
• Hardened blocks with tissue and surrounding embedding medium are trimmed and placed for sectioning in a microtome.
• Paraffin sections are typically cut at 3-10 μm thickness for light microscopy (LM), while electron microscopy (EM) requires sections less than 1 μm thick.