Light Microscopy

When a wavelength of light hits a specimen, there are different kinds of interactions that change the initial illuminating light. These interactions may be in the form of absorption, refraction, fluorescence, reflection, or diffraction.

Absorption is a lowering of the amplitude of the wave as it passes through a more or less transparent object. Most cells and tissues show very low absorption with visible light. This results in the transparent nature of these specimens under a light microscope, as well as poor contrast.

Refraction is the change in the traveling velocity of the light wave as it passes from one medium to another. With an increase in refractive index, the light wave will be bent towards the normal; when the refractive index becomes lower at the interface, the light wave will be bent away from the normal. This phenomenon along with concurrent manipulation of the aperture diaphragm of the condenser is important for obtaining contrast in unstained specimens.

Fluorescence is the effect when certain compounds hit by a certain wavelength of light, emit light of another (usually longer) wavelength. The choice of dyes gives rise to contrast of the desired structure being observed.

Reflection of light plays a major role in specimens with low contrast. It occurs usually at any boundary where light passes through different refractive indexes.

Finally, diffraction is a when there is a spreading out of the light beam as it passes an edge of on obstacle. It also plays an important role in image formation.

Conventional Brightfield Microscopy

Brightfield microscopy is best suited for viewing specimens which absorb visible light, such as histological stains. Unstained cells such as ones depicted below offer poor contrast and resolution of structures.

Brightfield microscopy image of cultured epithelial cells using a 20X objective.

Phase-Contrast Microscopy

Phase contrast is used mainly to enhance the contrast of unstained specimens. It is better suited for viewing thinner specimens such as single cells compared to differential interference contrast (DIC or Nomarski). Phase contrast requires clean glass-to-air surfaces of the specimen (i.e. no fingerprints!).

Phase contrast image of cultured epithelial cells using a 20X objective.

Differential Interference Contrast (DIC or Nomarski)

DIC is used if a specimen is too thick for phase-contrast. This happens if the object sections are outside the plane of focus and thus impair the contrast of the image or if the halo, characteristic of phase contrast, interferes with the desired observation of small structures. DIC microscopy produces images that have a shadowed relief, which can be useful for optically transparent specimens, such as cultured cells or thick tissue sections. For best images, the optical field of view should follow Kohler illumination.

Differential Interference Contrast (DIC or Nomarski) image of cultured epithelial cells using a 20X objective.

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