Darkfield Microscope

Darkfield Microscope

• Used to observe live, light-sensitive, or unstained samples
• Opaque disc in the condenser blocks direct light, allowing only reflected light to enter the objective lens
• Image appears bright with a dark background
• Effective for visualizing living cells that would be distorted by drying or heat, or those that can't be stained with usual methods
• Does not allow for visualization of fine internal details of cells

Phase Contrast Microscope

• Commonly used to observe live samples
• Allows detailed examination of internal structures
• Special objective lenses and ring-shaped diaphragm bring out small differences in refractive indexes of internal structures
• Images appear as various shades of gray to black due to direct and reflected light rays

Fluorescence Microscope

• Uses ultraviolet light (UV)
• Depends on samples that absorb UV light and emit light of longer wavelength
• Some organisms, like Pseudomonas, have natural fluorescence under UV light
• Other samples may be treated with fluorochrome (fluorescent dyes)
• Immunofluorescence: a technique that uses fluorescent antibodies to detect specific antigens
• Useful in rapid detection of specific pathogens
• Images appear as fluorescent objects against a dark background

Light Microscope

• Includes darkfield, phase contrast, and fluorescence microscopes
• Limitations: highest magnification of 2000X, resolving power of 0.2 um
• Viruses and most internal structures of cells cannot be seen clearly

Electron Microscope

• TWO types: Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)
• Developed in 1932
• Uses beams of electrons (100,000 times smaller than beams of light)
• Allows scientists to observe structures that were previously too small to be examined

Scanning Electron Microscope (SEM)

• Provides excellent views of external structures
• Magnification of 10,000X or more, resolving power of 20 nm
• Provides a 3D image of the surface of the sample
• Mechanism: samples are coated with heavy metals (gold or palladium), and a narrow beam of electron is applied to create a secondary electron beam

Transmission Electron Microscope (TEM)

• Allows scientists to observe and study internal structures of samples
• Magnification same as SEM, but resolving power of 2.5 nm or better
• Image is 2D, called electron micrograph
• Disadvantages: can only observe very thin samples, processing is tedious, and staining may be used
• Viewing of samples must be done in vacuum, and distortion of samples is often seen due to sample processing