Microscopy

Overview

  1. Light microscopy is the foundation of cell biology.

  2. Key Historical Milestones:

    • 1655: Robert Hooke observed "cells" in cork.

    • 1674: Anthony Van Leeuwenhoek reported protozoa; saw bacteria 9 years later.

    • 1838: Schleiden & Schwann proposed cell theory:

      • All organisms are made of cells.

      • Cells are independent but work together for life.

      • The smallest unit of life is the cell (now questioned due to viruses).

      • Cells arise from other cells.

  3. Neuron Discoveries:

    • Reticular theory: Neurons are interconnected like a vascular system.

    • Neuronal theory (correct): Neurons are individual cells.

    • Camillo Golgi developed the "Golgi stain," proving neuronal theory.

Resolution

  1. Limited by wavelength of illuminating source.

    • Sizes visible with light microscopes:

      • Plant cells: ~100 μm

      • Animal cells: ~10 μm (smaller for cancer cells)

      • Bacteria/mitochondria: ~1 μm

    • Electron microscopes: Resolve down to nanometer (nm) or Angstrom scale.

  2. Key Factors:

    • Diffraction limits resolution for most microscopes.

    • Abbe’s equation defines theoretical resolution limits:

      • d=0.612λnsin⁡θd = \frac{0.612 \lambda}{n \sin \theta}d=nsinθ0.612λ​

      • Shorter wavelengths = better resolution.

Contrast Solutions in Light Microscopy

  1. Dyes:

    • Colorimetric dyes (e.g., hematoxylin) absorb specific light wavelengths.

    • Fluorochromes: Absorb light and fluoresce, providing better visualization.

  2. Light Manipulation:

    • Phase contrast and Nomarski (DIC) optics enhance contrast in living cells.

    • Computer image enhancement improves resolution.

Types of Microscopes

  1. Bright-field Microscope:

    • Used for fixed tissues (e.g., in pathology).

    • Protocol includes fixation, dehydration, embedding, sectioning, and staining.

  2. Phase Contrast Microscope:

    • Highlights refractive index differences in living cells.

    • Used for studying cellular structures without staining.

  3. Differential Interference Contrast (DIC) Microscope:

    • Produces 3D effects; useful for live cell imaging.

    • Essential for neurobiologists (e.g., patch-clamping neurons).

  4. Darkfield Microscope:

    • Highlights small structures (e.g., mitochondria) with high contrast.

  5. Polarizing Light Microscope:

    • Detects ordered structures like collagen in scar tissue or microtubules.

Takeaways

  1. Light microscopes are diffraction-limited; super-resolution microscopy surpasses this.

  2. Selection of microscope and specimen preparation depend on research goals.

  3. Contrast methods (dyes, light manipulation) enhance visualization.

Fluorescent Immunocytochemistry (FIC)

Definition:
The use of antibodies tagged with fluorochromes to locate specific proteins within or on cells.

Key Concepts:

  1. Fluorochromes: Fluorescent dyes used in labeling.

  2. Antigens and Antibodies:

    • Antigen: Molecule eliciting an immune response.

    • Antigenic Determinant/Epitope: Specific part of the antigen recognized by the antibody.

    • Antibody: Molecule with variable regions (specific to epitopes) and Fc regions (common, where fluorochromes bind).

  3. Techniques:

    • Direct: Fluorochrome-tagged primary antibody binds directly to the antigen.

    • Indirect: Primary antibody binds to the antigen, and a secondary fluorochrome-tagged antibody labels the primary, preserving specificity and affinity.

Antibody Production

  1. Polyclonal Antibodies:

    • Derived from multiple B-cell clones.

    • Broad recognition of antigens but less specific and prone to cross-reactivity.

  2. Monoclonal Antibodies:

    • Produced by hybridomas (fused myeloma and B cells).

    • Highly specific and can be cultured indefinitely.

    • Applications include disease treatment, e.g., cancer therapies.

Advanced Fluorescence Techniques

  1. ELISA:

    • Detects antigen concentration in samples.

    • Variants include direct, indirect, and sandwich ELISA, using enzymatic reactions to indicate antigen presence.

  2. Apoptosis Detection:

    • Annexin V: Binds phosphatidylserine exposed on apoptotic cells.

    • Propidium Iodide: Stains nuclei of necrotic cells with ruptured membranes.

  3. GFP (Green Fluorescent Protein):

    • Acts as a reporter molecule to track gene expression in live cells.

    • Enables tracking of labeled cells and overlaying with other microscopy techniques.

Emerging Applications and Ethical Considerations

  1. Bispecific Antibodies:

    • Engineered to bind two different antigens.

    • Potential for targeted cancer therapies by linking immune and tumor cells.

  2. Human-Pig Chimeras:

    • GFP-labeled human cells injected into pig embryos for organ growth research.

    • Raises ethical debates on the limits of genetic engineering.