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1.06 Light Microscope

Posted26/10/2024
Updated22/12/2024
ByBMF
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Introduction to Cell Biology

  • Cell biology = the study of cells.

Types of Microscopes

  • Light Microscope: Uses light radiation to create cell images.
  • Electron Microscope: Uses electron radiation for higher resolution images (details discussed later).

Plant and animal cells

Figures: Images of actual human, plant cell and bacterial cells.

Figure 1. (a) Nasal sinus cells (viewed with a light microscope), (b) onion cells (viewed with a light microscope), and (c) Vibrio tasmaniensis bacterial cells (seen through a scanning electron microscope) are from very different organisms, yet all share certain basic cell structure characteristics. (credit a: modification of work by Ed Uthman, MD; credit b: modification of work by Umberto Salvagnin; credit c: modification of work by Anthony D’Onofrio, William H. Fowle, Eric J. Stewart, and Kim Lewis of the Lewis Lab at Northeastern University; scale-bar data from Matt Russell)

(a) Human nasal sinus cells (viewed with a light microscope), (b) onion cells (viewed with a light microscope), and (c) Vibrio tasmaniensis bacterial cells (seen through a scanning electron microscope).

Figures: Show generalized structures of animal and plant cells

Compound Light Microscope

  • Compound Light Microscope: Utilizes multiple lenses to magnify specimens up to 1000x. Ideal for viewing stained cells, tissues, and microorganisms.

Parts of a Compound Light Microscope

  1. Eyepiece (Ocular Lens): Typically 10x magnification; the lens you look through.
  2. Objective Lenses: Usually three or four lenses (e.g., 4x, 10x, 40x, 100x). They provide primary magnification.
  3. Nosepiece (Turret): Holds the objective lenses and can be rotated to change magnification.
  4. Stage: Platform where the slide is placed.
  5. Stage Clips: Hold the slide in place.
  6. Condenser: Focuses light onto the specimen.
  7. Diaphragm (Iris or Disc Diaphragm): Controls the amount of light reaching the specimen.
  8. Coarse Focus Knob: Adjusts the focus quickly.
  9. Fine Focus Knob: Makes precise focus adjustments.
  10. Light Source (Illuminator): Provides the light needed to view the specimen.
  11. Base: The bottom support structure of the microscope.

Magnification and Resolution

  • Total Magnification: Product of the magnification of the eyepiece and objective lens.
    Example: 10x eyepiece × 40x objective = 400x total magnification.
  • Resolution: The ability to distinguish two close points as separate entities. Higher resolution allows for clearer and more detailed images. Factors affecting resolution include the wavelength of light and the numerical aperture of the objective lens.

Specimen Preparation

  • Proper specimen preparation is crucial for clear and detailed observation.
  1. Choosing the Specimen:
    • Select thin sections of plant tissues (e.g., lower epidermis of a leaf, onion peel) to allow light to pass through.
  2. Making the Specimen Thin:
    • Thin slices ensure light penetration and prevent overlapping structures, which can obscure details.
  3. Slide Preparation:
    • Materials Needed: Clean microscope slides, cover slips, pipette or dropper, tweezers, scalpel or blade (if necessary), staining reagents.
    • Steps:
      1. Clean the Slide: Ensure the slide is free from dust and debris to avoid image distortion.
      2. Add Water: Use a pipette to place a drop of distilled water in the center of the slide. Water acts as a medium to keep the specimen moist.
      3. Place the Specimen: Carefully place the prepared epidermal tissue into the drop of water.
      4. Flatten the Specimen: Gently press down with a tweezer or the back of a spatula to spread the tissue evenly.
      5. Apply the Cover Slip: Slowly lower a cover slip at an angle to avoid trapping air bubbles, which can interfere with viewing.
      6. Remove Excess Water: Blot the edges with a paper towel if necessary to prevent movement.
  4. Staining (Optional but Recommended):
    • Purpose: Enhances contrast by coloring specific cell structures, making them easier to observe.
    • Common Stains:
      • Iodine Solution: Stains starch granules in cells.
      • Methylene Blue: Stains nuclei and other cell components.
      • Safranin: Stains cell walls and nuclei.
    • Procedure:
      1. Apply a few drops of stain to the specimen on the slide.
      2. Let it sit for a minute or two.
      3. Gently rinse with water to remove excess stain.
      4. Reapply the cover slip if necessary.

Using the Microscope

  1. Initial Setup:
    • Place the slide on the stage and secure it with stage clips.
    • Connect and turn on the microscope’s light source.
  2. Focusing:
    • Start with the Lowest Magnification (e.g., 4x):
      1. Rotate the nosepiece to the lowest objective lens.
      2. Use the coarse focus knob to bring the specimen into general focus.
      3. Use the fine focus knob to sharpen the image.
    • Increasing Magnification:
      1. Once the specimen is clear at low magnification, switch to a higher objective lens (e.g., 10x, then 40x).
      2. Adjust focus with the fine focus knob as necessary.
      3. Avoid using the highest magnification (e.g., 100x) unless necessary, as it may require oil immersion techniques.
  3. Adjusting Light and Contrast:
    • Use the diaphragm to control the amount of light.
    • Adjust the condenser to focus light on the specimen.
    • Alter the light intensity for optimal visibility.
  4. Final Steps:
    • Observe the specimen under different magnifications to examine various details.
    • Take notes or sketches as needed for study purposes.

Safety Considerations

  1. Handling Microscope Components:
    • Always carry the microscope with two hands to prevent drops.
    • Keep the lens and stage clean using lens paper or appropriate cleaning solutions.
  2. Chemical Safety:
    • Handle stains and reagents with care, using gloves and goggles as necessary.
    • Dispose of chemical waste properly according to your institution’s guidelines.
  3. Electrical Safety:
    • Ensure that the microscope’s electrical components are in good condition.
    • Keep cords away from pathways to prevent tripping hazards.

Troubleshooting Common Issues

Confirm that the objective lenses are functioning correctly.

  • Blurry Images:
    • Ensure the specimen is properly focused.
    • Check if the lenses are clean and free from smudges.
    • Verify that the specimen is adequately prepared and thin.
  • Uneven Illumination:
    • Adjust the condenser and diaphragm to ensure even light distribution.
    • Clean the light source and lenses to remove any obstructions.
  • Air Bubbles Under Cover Slip:
    • Carefully lower the cover slip at an angle to minimize trapping air.
    • If bubbles form, remove and reposition the cover slip.
  • Low Magnification Not Showing Details:
    • Increase magnification stepwise, ensuring each step is properly focused.

Ultrastructure and Microscopy

Definition of Ultrastructure

  • Ultrastructure: The detailed structure of a cell visible only with an electron microscope.

Comparison with Light Microscopy

  • Light Microscope:
    • Limited resolution; can visualize larger organelles (e.g., nucleus, mitochondria).
  • Electron Microscope:
    • Higher resolution; reveals fine details of organelles (e.g., ER, ribosomes, cristae in mitochondria).

Practise Questions

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