Darkfield Microscopy

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Contrast Without Staining
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Ways to induce contrast

• Stains
• Dont usually work with living cells
• Close down the condensor aperture
• Results in loss of resolution, diffraction
  artifacts
• Other methods

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Darkfield Microscopy

• Image is made up of highly diffracted light
• Low light, can detect detail

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Demo
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Phase Contrast methods

• Most unstained biological materials do not have
  much contrast so dont absorb very much light
• Most biological materials retard light
  differentially--they have different refractive
  indices
• This leads to differences in phase of the light
  waves passing through the materials
• The human eye cannot see these phase changes

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Air R.I.1.0
Air R.I.1.0
Water R.I.1.33
Glass R.I.1.5
The speed and wavelength of light changes when it
passes through media with different refractive
indices
Phase contrast microscopy makes these phase
difference visible
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Constructive interference
Destructive interference
Tutorial
We can see amplitude (intensity) differences
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Phase Contrast

• Deviated and undeviated light
• Deviated light is retarded about 1/4 wavelength
• Phase contrast methods increase the difference to
  about half a wavelength
• They usually retard the undeviated light by ¼
  wavelength
• Deviated light makes up a greater part of the
  image
• Differences in refractive index result in
  deviation
• Diffraction at edges also important

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Tutorial
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Much light is wasted in the interference
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Polarized Light Microscopy
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Demo
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Isotropic uniform interaction with light
Birefringence
Anisotropic angle of incidence
affects interaction with light
Amorphous no pattern to light interaction
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Interference Contrast Microscopy

• DIC, or Nomarski
• Also utilizes phase shift for contrast
• Light is polarized
• Light traveling through specimen interacts
• Light is split in Wollaston prism, two rays
  vibrate perpendicular to each other
• Path of one beam altered depending on specimen
  characters
• Beams now recombined

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DIC contd

• Beam interference results in color differences
• Also results in contrast
• If there is no interference, gray image

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Demo
Demo2
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Biology dic and electronic processing--can see
microtubules because of diffraction
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Biology Tradescantia, 2.5 hr observation,
Strasburger in 1880
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