Introduction to Histology

Microscopy Techniques

• Bright Field Microscopy: Visualizes live cells but has limitations in cellular detail due to similar optical densities among components.
• Phase Contrast Microscopy: Enhances sample contrast; uses refractive properties to highlight dense regions (darker) and less dense regions (lighter).
• Differential Interference Contrast (DIC) Microscopy: Modified phase contrast technique ideal for imaging surfaces; poor fixation or staining can lead to sample deterioration.

Staining Methods

• Masson’s Trichrome Stain:
  • Nuclei: black
  • Muscle fibers: red/pink
  • Collagen fibers and cartilage matrix: blue/green
• Mallory Trichrome Stain:
  • Nuclei: red
  • Red blood cells and keratin: orange
  • Collagen fibers, cartilage, and bone matrix: blue
• Periodic Acid-Schiff (PAS): Stains polysaccharides (glycogen) and glycoproteins (mucins), making them bright pink, and highlights basement membranes.
• Nissl Staining: Commonly uses Cresyl violet to visualize nuclei, nucleoli, and ribosomes, particularly in nervous tissues.

Fluorescence Techniques

• Fluorescent Stains: Commonly used stains like DAPI and Hoechst specifically target DNA.
• Fluorophores: Antibodies conjugated with fluorophores provide visualization of proteins in tissues, while fluorescent proteins like GFP label proteins of interest.
• Confocal Microscopy: Enhances image resolution using lasers and pinholes to exclude out-of-focus light, resulting in sharper images.

Autoradiography

• Utilizes radioactivity (e.g., Tritium 3H or Carbon 13) to study molecular interactions in living systems and characterize DNA synthesis.

Immunostaining Techniques

• Direct Immunostaining: Involves a single labeled antibody that detects an antigen directly.
• Indirect Immunostaining: Utilizes two antibodies—primary to detect the target antigen and secondary to amplify the signal.

Enzyme Histochemistry

• Detects enzyme locations based on enzymatic activity for types like phosphatases and peroxidases through reaction products.

Hybridization Techniques

• In Situ Hybridization: Detects specific sequences of DNA or RNA in tissues/cells using radioactive isotopes.
• Fluorescence In Situ Hybridization: Uses fluorescently labeled probes for gene or mRNA detection in cells.

Gold Microscopy

• Immunogold Microscopy: Uses electron-dense gold particles for visualization in Transmission Electron Microscopy (TEM), allowing for specific protein detection.

Staining Artifacts

• Awareness of common artifacts in histological preparations is crucial for accurate interpretation of stains and tissue images.

Small Molecule Fluorophores

• Target and label intracellular organelles like mitochondria and Golgi structures; applicable for tumor imaging and real-time surgical interventions.

Understanding and interpreting images obtained through different types of microscopy techniques, including bright field, phase contrast, and differential interference contrast microscopy. Learn about the advantages and limitations of each technique in visualizing cellular details.