Chapter Summary

2.1

  • Detection is the ability to determine the presence of an object.
  • Resolution is the smallest distance by which two objects can be separated and still be distinguished.
  • Magnification means an increase in the apparent size of an image so as to resolve smaller separations between objects.
  • Eukaryotic microbes may be large enough to resolve subcellular structures under a light microscope, although some eukaryotes are as small as bacteria.
  • Bacteria and archaea are usually too small for subcellular resolution. Their shapes include characteristic forms, such as rods and cocci.
  • Different kinds of microscopy are required to resolve cells and subcellular structures of different sizes.

2.2

  • Electromagnetic radiation interacts with an object and acquires information we can use to detect the object.
  • Contrast between object and background makes it possible to detect the object and resolve its component parts.
  • The wavelength of the radiation must be equal to or smaller than the size of the object if we are to resolve the object’s shape.
  • Absorption means that the energy from light (or other electromagnetic radiation) is acquired by the object.
  • Reflection means that the wave front bounces off the surface of a particle at an angle equal to its incident angle.
  • Refraction is the bending of light as it enters a substance that slows its speed.
  • Scattering occurs when a wave front interacts with an object of smaller dimension than the wavelength. Light scattering enables the detection of objects whose detail cannot be resolved.

2.3

  • In bright-field microscopy, resolution depends on
  • The wavelength of light, which limits resolution to about 200 nm.
  • The magnifying power of a lens, which depends on its numerical aperture (nsinθ).
  • The position of the focal plane, the location where the specimen is “in focus” (that is, where the sharpest image is obtained).
  • A compound microscope achieves magnification and resolution through a series of lenses: the condenser, objective, and ocular lenses.
  • A wet mount specimen is the only way to observe living microbes.
  • Fixation and staining of a specimen kills it but improves contrast and resolution.
  • Differential stains distinguish between different kinds of bacteria with different structural features.
  • The Gram stain differentiates between two major bacterial taxa: Proteobacteria (gram-negative) and Firmicutes (gram-positive). Other bacteria and archaea vary in their Gram stain appearance. Eukaryotes stain negative.

2.4

  • Dark-field microscopy uses scattered light to detect objects too small to be resolved by light rays. Advantage: Extremely small microbes and thin extracellular structures can be detected. Limitation: The shape of objects is not resolved. Dust particles easily obscure the image of the specimen.
  • Phase-contrast microscopy superimposes refracted light and transmitted light shifted out of phase so as to reveal differences in refractive index as patterns of light and dark. Advantage: Live cells with transparent cytoplasm, and the organelles of eukaryotes, can be observed with high contrast. Limitation: Phase contrast is less effective for organisms whose cytoplasm has a low refractive index.
  • Interference microscopy superimposes interference bands on an image, accentuating small differences in refractive index. Advantage: The shape of cells can be defined most clearly. Limitations: Interference microscopy requires complex optical adjustment and is less effective for organisms with low refractive index.

2.5

  • Fluorescence microscopy involves detection of specific cells or cell parts based on fluorescence by a fluorophore.
  • Cell parts can be labeled by a fluorophore attached to an antibody stain.
  • Laser confocal microscopy visualizes cells in three dimensions.

2.6

  • Electron microscopy is based on the focusing of electron beams on an object stained with a heavymetal salt that scatters electrons. Much higher resolution is obtained than with light microscopy.
  • Transmission electron microscopy (TEM) involves electron beam penetration of a thin sample.
  • Scanning electron microscopy (SEM) involves scanning of a three-dimensional surface with an electron beam.
  • Cryo–electron microscopy (cryo-EM) involves the observation of samples flash-frozen in water solution. Multiple images may be combined digitally to achieve high resolution.
  • Atomic force microscopy (AFM) uses intermolecular force measurement to observe cells in water solution.

2.7

  • X-ray diffraction analysis, or X-ray crystallography, uses X-ray diffraction (interference patterns) from crystallized macromolecules to determine structure at atomic resolution.
  • Cryocrystallography uses frozen crystals with greatly decreased thermal vibrations and diffusion, enabling the determination of structures of large macromolecular complexes, such as the ribosome.
  • Molecular visualization by crystallography can only model the “appearance” of a molecule at atomic resolution. Different models emphasize different structural features and levels of resolution.