Microscopy Overview and Techniques

Microscopy Overview

• Microorganism sizes range from nanometers (viruses) to approximately 200 micrometers (larger protists and bacteria).
• Light microscopes are the fundamental tools for microbiologists, crucial for studying microbial structures.
• Magnification ability is not limited; however, resolution (ability to distinguish two objects) is constrained by physical properties of light, with a limit of 0.2 µm for light microscopes.

Principles of Light Microscopy

• Different light microscopy techniques include:
  • Bright field
  • Dark field
  • Phase contrast
  • Differential interference contrast
  • Fluorescence microscopy
  • Confocal scanning laser microscopy
• Use of lenses is essential; they bend light to create images, with short focal lengths providing higher magnification.
• Resolution is influenced by the wavelength of light and numerical aperture of the lens.

Bright-Field Microscopy

• Examines both stained and unstained specimens, yielding dark images against a bright background.
• Total magnification is the product of the ocular and objective lens magnifications.

Dark-Field Microscopy

• Utilizes scattered light from the specimen, creating a bright image on a dark field.
• Particularly useful for observing live, unstained preparations and certain bacteria (e.g., Treponema pallidum).

Phase Contrast Microscopy

• Invented by Frits Zernike in 1936; enhances contrast by exploiting differences in refractive index.
• Provides dark cells against a light background, effective for observing microbial movement.

Differential Interference Contrast (DIC) Microscopy

• Detects differences in refractive indices and specimen thickness, using polarized light.
• Produces three-dimensional images with bright, colorful appearances for living cells and structures that are invisible in bright-field microscopy.

Fluorescence Microscopy

• Uses specific wavelengths of light to excite fluorochromes, resulting in emitted fluorescence from specimens.
• Practical for identifying pathogens and localizing proteins within cells.

Confocal Scanning Laser Microscopy (CSLM)

• Combines computerized microscopy with laser technology for sharp, three-dimensional images.
• Capable of focusing on single layers of a specimen, achieving a resolution of 0.1 µm.

Sample Preparation and Staining

• Staining enhances specimen visibility, morphological feature accentuation, and specimen preservation.
• Fixation via heat or chemical methods preserves structural integrity; chemical fixation is preferred for delicate organisms.
• Dyes increase contrast, aiding visibility of internal and external structures.

Electron Microscopy Techniques

• Transmission Electron Microscopy (TEM)

  • Provides high resolution (down to 0.2 nm) using electron wavelengths much shorter than light.
  • Requires ultra-thin specimens (20–60 nm) and contrast-enhancing staining methods.

• Scanning Electron Microscopy (SEM)

  • Electron beams scan surfaces to create detailed three-dimensional images.
  • Specimens are coated with heavy metals (e.g., gold) to enhance surface visuals.

• Cryo-Electron Microscopy

  • Achieved a Nobel Prize in Chemistry (2017); visualizes proteins via rapid freezing and multiple imaging angles.
  • Generates high-resolution three-dimensional images of biomolecules.

Takeaways

• Familiarity with various microscopy techniques is essential for selecting the appropriate method for specific research inquiries in microbiology.