Lecture Notes on Light Microscopy

Overview

• Focus on light (optical) microscope, commonly used in early microbiologist ventures
• Uses light to visualize specimens

Light Microscope Operation

Basic Principle

• Light passes through a lens to focus on the specimen
• Light then goes through objective lens (magnifies image 4-100 times)
• Travels through microscope tube and eyepiece (further magnifies 10 or 15 times)
• Maximum magnification up to 1,200 times

Components and Functions

• Stage: Platform for specimen
• Stage Clips: To hold specimen in place
• Diaphragm: Controls image contrast by adjusting light exposure
• Slide Adjustment Knobs: Move specimen in field of view
• Objectives: Different magnification levels, held by nosepiece
• Nosepiece: Rotatable holder for objectives
• Tube: Connects nosepiece to eyepiece
• Eyepiece: Further magnifies image; part through which users look
• Coarse Focus Knob: For large focus adjustments
• Fine Focus Knob: For small focus adjustments

Practical Use Steps

1. Plug in and turn on the light source
2. Secure specimen with stage clips
3. Rotate nosepiece to smallest objective
4. Use coarse focus to clear image, then adjust specimen position
   • Note: Image inversion - moving slide right moves image left
5. Use fine focus for image refinement
6. Adjust eyepieces if necessary
   • Focus may differ between eyes
   • Individuals may need personal adjustments
7. Switch objectives as needed, using fine focus as required
8. Note objective magnification (total is 10 times the objective)
   • Special step for 100x objective: use oil immersion
     • Add oil between 40x and 100x, position 100x in oil
     • Avoid scraping lens

Post-Use Steps

• Turn off light, unplug microscope
• Dispose or save specimen
• Clean lenses with lens paper
• Replace microscope cover

Applications

• Studies of bacteria, tissue slices, liquid samples, mitochondria
• Inverted light microscopes:
  • Light source above, objective below stage
  • Ideal for tissue cultures in liquid media

Advantages and Limitations

Advantages

• Easy to use
• Relatively inexpensive
• Common in educational labs
• Suitable for live specimens with minimal damage

Limitations

• Limited resolution and magnification
• Cannot see structures in as much detail as more powerful microscopes

Conclusion

• Overview of the light microscope's operation and applications
• Next, exploration of more powerful microscopy techniques