Lecture Notes: Instruments of Microscopy

Overview

• Section covers the parts of a bright field microscope, calculating total magnification, and distinguishing features of various microscopes.

Historical Context

• Anton van Leeuwenhoek: First to discover the microbial world using a simple microscope.
• Joseph Jackson Lister (1830): Created a modern light microscope.
• 20th century: Development of microscopes using non-visible light (e.g., ultraviolet light).

Types of Microscopes

Light Microscopes

1. Bright Field Microscopes

   • Produces bright image; uses stains to increase contrast.
   • Parts include:
     • Eyepiece/Ocular Lens: 10x magnification.
     • Objective Lenses: 4x to 100x magnification.
     • Stage & Stage Knobs: Positions and focuses the specimen.
     • Illuminator & Condenser: Provides and focuses light.
     • Diaphragm/Rheostat: Controls light intensity.
   • Total magnification = Ocular x Objective (e.g., 10x ocular and 4x objective = 40x total).
   • Immersion oil is used with 100x lenses to increase resolution by reducing light scattering.

2. Dark Field Microscopes

   • Modifies bright field microscope with an opaque disc to block direct light.
   • Produces images with bright objects on a dark background.
   • High contrast without staining, suitable for live specimens.

3. Phase Contrast Microscopes

   • Uses refraction and interference to create high contrast images without staining.
   • Ideal for living organisms.
   • Utilizes phase plates and rings to alter light wavelength paths.

4. Differential Interference Contrast (DIC) Microscopes

   • Similar to phase contrast; uses interference patterns for 3D imaging.
   • Suitable for live, unstained specimens.

5. Fluorescence Microscopes

   • Uses fluorochromes to emit visible light upon excitation by UV/blue light.
   • Useful in clinical microbiology for identifying pathogens and specific cellular structures.
   • Immunofluorescence: Uses antibodies to bind and visualize pathogens.
     • Direct (DFA): Primary antibody with fluorochrome.
     • Indirect (IFA): Secondary antibody with fluorochrome.

6. Confocal Microscopes

   • Uses lasers to scan and construct 3D images from 2D slices.
   • Suitable for thick specimens like biofilms.

7. Two-Photon Microscopes

   • Uses long-wavelength light for deep tissue imaging.
   • Suitable for living cells and large specimens.

Electron Microscopes

• Use electron beams for increased magnification and resolution (up to 100,000x).
• Cannot be used on living specimens.

1. Transmission Electron Microscopes (TEM)

   • Electrons pass through thin specimens.
   • Produces detailed 2D images.
   • Max magnification: 500,000x.

2. Scanning Electron Microscopes (SEM)

   • Electrons scanned over specimen surface.
   • Produces detailed 3D images.
   • Specimens are dried and coated with metal.
   • Max magnification: 30,000x.

Scanning Probe Microscopes

1. Scanning Tunneling Microscopes (STM)

   • Maps surface structures at atomic level.

2. Atomic Force Microscopes (AFM)

   • Measures surface variations using a probe.
   • Can detect individual atoms.
   • Magnification up to 100 million times.

Conclusion

• Understanding the parts and uses of different microscopes aids in biological research.
• Bright field, electron, and scanning probe microscopes each have unique applications and capabilities.