Human Histology (Laboratory)

• Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs
• Histology involves all components of tissue biology, focusing on how cell structures and arrangement optimize functions specific to each organ
• Histological studies may be conducted using tissue culture, where live animal cells are isolated and maintained in an artificial environment for various researches
• Histological diagnosis is the mainstay or the "gold standard" of clinical practice
• Diagnosis of disease depends 80% on the laboratory data, specifically histologic/histopathologic examination
• The technique of using light microscopes to examine wax-embedded sections is essential
• Proper use or manipulation of microscopes is necessary for histological examination
• Electron Microscopy is preferred in research laboratories for greater magnification and resolution of structures as small as 1nm
• Two main types of electron microscopes are Transmission Electron Microscope and Scanning Electron Microscope
• Parts of a microscope include mechanical parts like the base, pillar, stage, arm, body tube, draw tube, and optical parts like ocular lens and objective lenses
• The mechanical parts of a microscope include the base, pillar, stage, arm, body tube, draw tube, and dust shield
• The optical parts of a microscope include the ocular lens and objective lenses, with varying magnification powers
• The illuminating parts of a microscope include the mirror, substage, iris diaphragm, and Abbe condenser
• The revolving nosepiece holds two or more objective lenses and can be rotated to easily change power
• Tissues can be sectioned in three ways: Longitudinal Section, Transverse (Cross-Section), and Oblique
• Samples of human biological materials can be obtained from different parts of the body using instruments like scalpels, needles, endoscopic tubes, and special flexible cannula
• Histological sections are extremely thin, transparent shavings cut from a little piece of body tissue and laid flat on a glass slide after running through different stages of tissue preparation
• Each feature of the animal's anatomy exists in three space dimensions, and many structural characteristics of the tissue or organ may undergo normal cyclic, intermittent, progressive, or regressive changes with time
• Two important facts in the study of histology sections are that tissues are subject to variations over time and tissue under the microscope is only a two-dimension slice of a three-dimension object
• Two major important considerations with regard to methodology are the kind of microscope used and the preparation of the tissue/organ suitable for viewing with the microscope
• Organs are formed by an orderly combination of cells and extracellular matrix (ECM), with precise arrangement allowing the functioning of each organ and of the organism as a whole
• Preparation of tissue slices/sections is the most common procedure used in histologic research, allowing examination with transmitted light
• Most tissues and organs are too thick for light to pass through, so thin translucent sections are cut and placed on glass slides for microscopic examination
• Ideal microscopic preparation aims to preserve the tissue on the slide with the same structural features it had in the body
• Fixation:

  • Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, preserving cell and tissue structure
  • Formalin is a widely used fixative for light microscopy
  • Glutaraldehyde is a fixative used for electron microscopy
  • Fixative must fully diffuse through tissues to preserve all cells
  • Tissues are usually cut into small fragments before fixation to facilitate penetration
  • Vascular perfusion allows rapid fixation throughout tissues
• Dehydration:

  • Tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water
  • Dehydrated tissues are then infiltrated and embedded in a material that imparts a firm consistency
  • Fixed tissues are dehydrated in ethanol before infiltration
• Clearing:

  • Alcohol is replaced by an organic solvent miscible with both alcohol and the embedding medium to give the tissue a translucent appearance
  • Fully cleared tissue is placed in melted paraffin in an oven at 52°C-60°C to promote infiltration with paraffin
• Embedding:

  • Paraffin is used routinely for light microscopy
  • Plastic resins are adapted for both light and electron microscopy
  • Tissues to be embedded with plastic resin are dehydrated in ethanol and infiltrated with plastic solvents
  • Embedding materials include paraffin and plastic resins
• Sectioning:

  • Microtome is used for sectioning paraffin-embedded tissues for light microscopy
  • Sections for light microscopy are 3-10 μm thick
  • Sections for electron microscopy are less than 1 μm thick
  • Microtome is an instrument used for sectioning tissues
• Staining:

  • Tissue sections must be stained for microscopic study
  • Dyes stain material selectively, forming electrostatic linkages with macromolecules in tissues
  • Hematoxylin stains basophilic tissue components, while acid dyes stain acidophilic components
  • Hematoxylin and eosin (H&E) combination is commonly used for staining
  • Trichrome stains allow greater distinctions among various extracellular tissue components
• Mounting:

  • Final step before microscopic observation
  • Involves placing a glass coverslip on the slide with clear adhesive
• Bright-field Microscopy:

  • Stained tissue is examined with ordinary light passing through the preparation
  • Includes optical components such as condenser, objective lens, and eyepiece
  • Objective lens enlarges and projects the image of the object toward the observer
  • Eyepiece further magnifies the image and projects it onto the viewer's retina or CCD
• Eyepiece:

  • Further magnifies the image and projects it onto the viewer's retina or charge-coupled device (CCD) highly sensitive to low light levels with a camera and a monitor
  • Only enlarges the image obtained by the objective
  • Does not improve resolution
  • Magnifies the image another 10x and projects it to the viewer
• Total magnification:

  • Obtained by multiplying the magnifying power of the objective and ocular lenses
• Resolving Power:

  • Critical power in obtaining a crisp, detailed image with a light microscope
  • Smallest distance between two structures at which they can be seen as separate objects
  • Maximal Resolving Power is 0.2 μm, can permit clear images magnified 1000 to 1500 times
• Virtual Microscopy:

  • Used for the study of bright-field microscopic preparations
  • Involves the conversion of a stained tissue preparation to high-resolution digital images
  • Permits the study of tissues using a computer or other digital device without an actual stained slide or a microscope
• Fluorescence Microscopy:

  • Fluorescence occurs when certain cellular substances emit light with a longer wavelength when irradiated by light of a proper wavelength
  • Tissue sections are usually irradiated with ultraviolet (UV) light
  • Fluorescent substances appear bright on a dark background
  • Instrument has a source of UV or other light and filters to select rays of different wavelengths emitted by the substances to be visualized
• Autoradiography:

  • Method of localizing newly synthesized macromolecules in cells or tissue sections
  • Radioactively labeled metabolites provided to living cells or experimental animals are incorporated into specific macromolecules
  • Slides or TEM grids with radiolabeled cells or tissue sections are coated with photographic emulsion to indicate the locations of radiolabeled macromolecules in the tissue
  • Autoradiographs are tissue preparations in which particles called silver grains indicate the cells or regions of cells in which specific macromolecules were synthesized
• 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 added
  • Some cells can be maintained in vitro for long periods and constitute a permanent cell line
  • Cell culture is widely used to study molecular changes in cancer, analyze infectious viruses, and for genetic or chromosomal analyses
• Enzyme Histochemistry:

  • Method for localizing cellular structures using a specific enzymatic activity present in those structures
  • Examples of enzymes that can be detected histochemically include phosphatases, dehydrogenases, and peroxidase
  • Many enzyme histochemical procedures are used in the medical laboratory for various diagnostic purposes