Light Microscopy

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Light Microscopy

• Physical Biochemistry, November 2006
• Dr Ardan Patwardhan, a.patwardhan_at_imperial.ac.uk,
  Faculty of Natural Sciences, Imperial College
  London

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Snells Law
n refractive index of medium nair 1 nwater
1.33 noil 1.5 nglass 1.5
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Thin lens equation
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Special cases
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The compound microscope
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Angular Magnification, M

• Near point shortest distance at which eye can
  accommodate (250mm)
• M Angle (b) subtended by the image when viewed
  through the microscope /angle (a) subtended by
  the object when viewed by the naked eye both at
  near point
• In this case, Mm
• Using a suitable combination of lenses, there is
  no limit to the magnification that can be
  attained!!!

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Factors limiting the smallest details that can be
seen in a microscope

• Optical ResolutionRayleigh resolution
  criterion
• Detector SamplingNyquist criterion

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Resolution

• Rayleigh resolution criterion

Dx
q0
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Rayleigh Resolution Criterion
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Immersion lenses
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Example

• Blue light l 400 nm
• Object in oil n 1.5
• Maximum possible angle sinq 1

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Detectors Sampling
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Less than two samples per cycle results in the
detection of a false signal (aliasing)
Two samples per cycle
Less than two samples per cycle
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Sampling The Nyquist limit

• It is necessary to sample a sine wave at at-least
  twice its frequency in order to be able to
  reproduce it faithfully fsampling gt 2 fsine
• Detector sampling may thus limit the overall
  reproduction of detail rather than optical
  resolution
• Detector element spacing should be less Dx/2 of
  the optical system (as projected onto the
  detector plane)

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Useful magnification

• Design constraints on the magnification of a
  microscopea) The resolution of the optical
  system should match that of the detectorb)
  Field of view that is to be imagedc) Size of
  image
• For visual light microscopy, max useful is 2000X

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Monochromatic lens aberrations
Spherical Aberration
Coma
Field Curvature
Distortion
Astigmatism
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Chromatic Aberration

• Refractive index varies with wavelength !
• Depending on the solvent/embedding medium, can be
  a significant problem

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Specimen contrast

• Staining
• Phase contrast and differential interference
  contrast (DIC)
• Polarization microscopy
• Dark field

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Phase contrast microscopy

• Useful for samples that do not absorb much, such
  as unstained cells in an aqueous solvent
• Due to variations in refractive indices and
  thicknesses of different features, light will be
  phase shifted by varying amounts over the
  specimen
• These phase variations are normally not visible
  but can be made visible if the direct beam is
  phase shifted by 90 relative to the diffracted
  light
• This can be achieved using a piece of glass of
  appropriate thickness

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Example of phase contrast
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Polarization Microscopy

• Linear bifringence when the refractive index
  depends on whether the linear polarization is
  parallel or orthogonal to the optical axis
• Often occurs in ordered specimens such as
  crystals and muscle fibers
• Is exploited in a polarization microscope by
  using polarized light and an analyzer

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Polarisation Contrast

• Muscle filament (A-band bright I-band dark)
• Biophys J. 1990 April 57(4) 815828

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Dark field

• Oblique illumination of specimen
• Scattering from specimen imaged
• Scattering is at a maximum at interfaces between
  different refractive indices

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Conclusions

• Numerical Aperture NA nsin(?)
• Lateral resolution
• Highest lateral resolution 0.1 - 0.2 ?m in
  object plane ? Cellular studies possible but not
  at the molecular level ( at least not without
  specific labelling)