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• UST General Santos presents exercises 1-4, post-laboratory discussion for Human Histology (Laboratory) MT120225, Second Semester A.Y. 2024-2025.

Learning Outcome

• Apply principles of microscopy to identify images using different types of microscopes.
• Describe different types of epithelial tissues based on their structural characteristics.

Microscopy

• Light Microscopy wavelength range for visible light is 0.4 - 0.7 um.
• Tungsten is a common light source.
• Adjustment is possible via light sources or specialized prisms/filters when modifying the condenser.

Light Microscopy Components:

• Light Source
• Condenser
• Stage
• Objective lens
• Ocular lens

Brightfield Microscopy

• The image appears dark against a bright background.
• Stained tissue is examined using ordinary light.
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution is 0.2 um.
• Advantage is ease of use and wide availability.
• Disadvantages include limited resolution, requiring thin sections and difficulty seeing unstained structures.

Dark Field Microscopy

• The image appears bright against a dark background.
• The microscope is utilized for examining urine for crystals (uric acid and oxalate), and demonstrating specific bacteria (spirochetes).
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution is 0.2 um.
• The modification involves utilizes an opaque disc located under the condenser.

Fluorescence Microscopy

• Fluorescent substances appear bright on a dark background.
• Fluorophore is utilized to generate fluorescence in a biological sample.
• Used in studying intercellular junctions, tracing nerve fiber pathways, and detecting growth markers of mineralized tissues.
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution is 0.2 um.
• Modification includes xenon arc lamps, mercury vapor lamps, excitation filters, dichroic mirrors, and emission filters.
• Advantages includes it is highly specific for targeted molecules and can visualize multiple targets simultaneously.
• Disadvantages includes it requires a fluorescent label.

Phase-Contrast Microscopy

• The image formed presents a combination of areas exhibiting varying degrees of light intensity (from dark to light) surrounded by a halo.
• Studying unstained cells and tissues, especially living cells (tissue culture).
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution is 0.2 um.
• Modification utilizes an annular ring positioned in the substage condenser front focal plane
• Advantage includes visualization intracellular parts of unstained living cell and tissues.
• Disadvantage includes a halo effect around structures, and difficulty to interpret complex samples

Differential Interference Contrast Microscopy

• The image formed is 3D shadowed under very oblique illumination.
• Provide contrast in unstained and transparent samples
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Modification uses two coherent beams of light coming from the same light source and prism.

Confocal Microscopy

• Combines components of a light optical microscope with a scanning system.
• Biologic specimen appears in three dimensions.
• Fluorophore is utilized to visualize samples and view stained structures with high resolution and sharp focus.
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution is 0.2 um - 0.5 um.
• Modification features detector aperture (pinhole).
• Avoids stray light that reduces image contrast, greatly improves the resolution of the object, and allows localization of specimen components with greater precision.
• Disadvantage includes high expense and slower image acquisition

Polarizing Microscopy

• Allows recognition of stained or unstained structures made of highly organized subunits (striated muscle, crystalloid inclusions in the testicular interstitial cells).
• Total magnification is objective lens power multiplied by ocular lens power, typically 40x - 1000x.
• Resolution of 0.2 um.
• Modification through addition of polarizer and analyzer.
• Advantage reveals information about the orientation of macromolecules.
• Disadvantage requires specialized filters, only produces images of material having repetitive, periodic macromolecular structure

Electron Microscopy

• Provides more defined ultrastructural intracellular details of individual cells.
• Utilizes electron beams and electromagnetic fields.

Transmission EM

• Produces black and white two dimensional image. Visualizes internal structures of cells and viruses.
• Magnification is 400,000 times.
• Resolution is 3 nm.
• Essential systems include, electron gun and condenser, image-producing objective lens, movable specimen stage, intermediate and projector lenses and image-recording system.
• Advantage includes the ability to achieve atomic-level resolution and detailed internal structures.
• Disadvantage includes expensive tissue preparation is challenging, and materials with high density must be added to the specimen.

Scanning EM

• Produces three-dimensional images of the specimen surface
• Designed for studying surfaces of solid objects
• Magnification is <400,000 times.
• Resolution is <3 nm.
• Essential parts include source of electrons, column with electromagnetic lenses, electron detector, sample chamber, and computer with display.

Epithelial Tissue

• Simple Squamous Epithelium features mesothelium, endothelium and allows rapid diffusion.
• Kidney: The kidney tubules are Simple Cuboidal Epithelium and Bowman's capsule is Simple Squamous Epithelium
• Simple Cuboidal Epithelium lines small excretory ducts in different organs, covers and secretes, and is found in the thyroid or ovary.
• Thyroid Follicles are Simple Cuboidal Epithelium.
• Simple Columnar Epithelium covers digestive organs (stomach, small/large intestine, gallbladder), and offers lubrication, absorption, protection, and secretion.
• Ileum and Jejunum are Simple Columnar Epithelium with microvilli and goblet cells.
• Simple Columnar Epithelium may have apical microvilli or cilia.
  • Non-ciliated: stomach, intestines, gallbladder
  • Ciliated: fallopian tube and uterus
• Pseudostratified Columnar Epithelium lines upper respiratory passages (trachea, bronchi), lumina of epididymis and vas deferens and can be ciliated.
• Olfactory Epithelium and Trachea are Ciliated Pseudostratified columnar epithelium with goblet cells
• Epididymis is Pseudostratified columnar epithelium with stereocilia
• Offers protection, transport, and secretion.
• Transitional Epithelium is exclusively in excretory passages of the urinary system.
• Transitional Epithelium covers the lumina of renal calyces, pelvis, ureters, and bladder.
• Transitional Epithelium changes shape in response to stretching, fluid accumulation, or contraction during voiding of urine.
• Stratified Squamous Epithelium features multiple cell layers, and basal cells are cuboidal to columnar
• Cells toward the surface become squamous and carry out protective functions
  • Types are nonkeratinized or keratinized
• Stratified Squamous Nonkeratinized exhibits live surface cells and covers moist cavities (mouth, pharynx, esophagus, vagina, anal canal).
• Esophagus and Vagina are Non-keratinized stratified squamous epithelium
• Stratified Squamous Keratinized lines external surfaces of the body and contains non-living surface keratinized cells (skin).
• Thick skin, palm, is Keratinized stratified squamous epithelium.
• Stratified Cuboidal Epithelium has limited distribution and lines larger excretory ducts of salivary and sweat glands.
• Stratified Columnar Epithelium has limited distribution and seen in the conjunctiva lining the eyelids.