Microscopy Techniques and Imaging Methods - Comprehensive Notes

Digital Microscopy

  • Aperio ImageScope [v12.4.6.5003] used for digital pathology viewing and analysis
    • Example file: [07-DVM-512 Pancreas.svs] from ScanScope image
    • Image/file properties observed:
    • Original image resolution: 103584×35856×3103584 \times 35856 \times 3
    • Original file size: 10.4 GB10.4\,\mathrm{GB}
    • File size (e.g., after processing): 802 MB802\,\mathrm{MB}
    • Rendering and loading characteristics: prefetching, trackmap, progressive rendering

Light Microscopy

  • Bright-Field Microscopy
  • Fluorescence Microscopy
  • Phase-Contrast Microscopy
  • Confocal Microscopy
  • Polarizing Microscopy

Light Microscopy: Details

  • Bright-Field Microscopy
    • Condenser collects and focuses a cone of light that illuminates the tissue slide on the stage
    • Objective lenses enlarge and project the illuminated image toward the eyepiece
    • Eyepieces (oculars) magnify the image further and project it to the viewer
  • Fluorescence Microscopy
    • Uses UV light; only fluorescent molecules become visible
    • Enables localization of fluorescent probes
    • Probes can be more specific than routine stains
  • Phase-Contrast Microscopy
    • Enables examination of unstained cells and tissues
    • Particularly useful for living cells
  • Confocal Microscopy
    • Allows visualization of a biologic specimen in three dimensions
    • Two lenses (objective and phototube lens) are aligned to focus light from the focal point of one lens to the focal point of the other
  • Polarizing Microscopy
    • Designed to observe and photograph specimens visible due to optical anisotropy
    • Simple modification of light microscope
    • Polarizer located between light source and specimen; analyzer between objective lens and viewer

Electron Microscopy (EM)

  • Higher resolution than light microscopy
  • Requires very specialized equipment and technical expertise; expensive
  • Fixatives differ from regular histology (e.g., glutaraldehyde)
  • Two main types: Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM)

Electron Microscope Components and Operation (Overview)

  • Light Microscope (LM) path: lamp (light source) → condenser lens → specimen → objective lens → projection lens → ocular lens → eye
  • Transmission Electron Microscope (TEM) path: electron source (cathode) → anode → condenser lens → specimen → projection lens → electron detector/eye
  • Scanning Electron Microscope (SEM) path: electron source → scanning coil → scanning beam → primary interaction with specimen → backscattered/secondary electron detectors → image
  • TEM/SEM share the concept of high-resolution imaging but differ in data collection (transmitted electrons vs scanned surface interactions)
  • Note: EM imagery typically requires a vacuum and heavy metal staining/contrast methods

EM vs Light Microscopy (LM) – Examples

  • Canine chondrocytes from femur growth plate visualized by TEM (high-resolution internal structure)
  • Cartilage LM image examples
  • SEM images of rabbit chondrocytes from femur growth plate
  • Nonmalignant chicken tracheal epithelium illustrated via SEM

Observation in Microscope (General Considerations)

  • Regardless of LM or TEM, critical conditions for any specimen:
    • Well preserved: must retain structure and molecular composition
    • Sufficiently thin to allow light transmission (LM) or appropriate electron interaction (EM)
    • Sufficient contrast to observe details

Immunohistochemistry (IHC)

  • IHC is a technique using an antibody to bind a specific antigen (protein) in a tissue section
  • Visualization is achieved with a fluorophore or colored substrate applied to the antibody–antigen complex

Immunohistochemistry (IHC) – Types of Antibodies

  • Polyclonal antibodies
    • Mixed antibodies produced by different immune cell populations
    • Origin: animals such as rodents and rabbits; goats/horses are also used
  • Monoclonal antibodies
    • Identical copies produced by immortal cell lines (myeloma)
    • Recognize a single epitope

Examples/Applications (IHC-related)

  • Borrelia burgdorferi (agent of Lyme disease) as an example organism detectable by IHC
  • Feline Infectious Peritonitis (FIP) and FCOV as additional contexts

Pancreas: Islets of Langerhans – Stains

  • Left panel shows sections stained with H&E and antibody-based staining
  • Cell types in islets:
    • A cells produce glucagon
    • B cells produce insulin
  • Reference staining: Hematoxylin and Eosin (H&E)

In Situ Hybridization (ISH)

  • In situ hybridization localizes a DNA or RNA sequence in a biological sample
  • Procedure overview:
    • Tissue sections, cells, or chromosomes are mounted on a glass slide
    • A probe (short single-stranded DNA) tagged with a chemical or fluorescent dye is applied
    • The probe finds and binds to its complementary sequence in the sample
    • Localization of bound probe is observed under a microscope

ISH Probes and Targets (Illustrative Examples)

  • Tissue section: Pancreas
  • Control sample
  • Probes used:
    • Digestive enzyme gene
    • Insulin gene

Safety and Work Health Regulations (Lab Guidelines)

  • To comply with work health & safety regulations, please:
    1. Wear a lab coat
    2. Wear full-length pants (or equivalent), socks, and closed-toe/heel shoes
    3. After finishing lab activities, disinfect bench space
    4. Wash hands before leaving the lab
  • Note: No water and food permitted in the lab

Campus Map and Lab Location (Overview)

  • LS 352: LIU POST Campus Map – 720 Northern Boulevard
  • Key landmarks: Broc/Pell Hall/Life Science; College of Veterinary Medicine
  • Various route markers and parking locations shown on the map (East points, Gold Coast Road, Scholar Court, etc.)