Confocal Microscopy

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Confocal Microscopy

• BMEN 489/689

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Confocal Microscopy

• Utilizes pinholes at the CONjugate FOCAL planes
  in the sample to discriminate against
  out-of-focus light from the sample.
• Marginally improves lateral resolution when
  compared to standard microscope.
• Permits optical sectioning by decreasing depth of
  field.
• Provides a large improvement in contrast.

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Confocal Principle
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Laser Scanning Confocal Microscope
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Realtime Imaging Confocal Microscope
http//www.microscopyu.com/articles/confocal/confo
calintrobasics.html
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Uses of Laser Confocal Microscope

• Flourescence microscopy using contrast agents
• Often fitted with multiple detectors for use with
  multiple contrast agents
• Two photon excitation of contrast agents
• Further improvement in axial contrast through two
  photon absorption process

• Some use in fluorescence microscopy, but LCSM is
  more efficient
• Surface/Structure Characterization

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Lateral Resolution

• Using the FWHM of the Airy disk
• For a conventional microscope
• For a confocal microscope

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Optical Sectioning
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Specs for Olympus MX50-CF

• With a 100x objective (NA 0.8) in air and 488nm
  light
• RFWHM(conv) 0.37µm
• RFWHM(conf) 0.27µm
• ZFWHM(point) 1.02µm
• ZFWHM(plane) 0.55µm

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Lateral versus axial resolution for 200nm
fluorescent microspheres
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Settings for Olympus MX50-CF

• There are two confocal settings on the
  microscope
• CF 1 uses a 45µm pinhole
• CF 2 uses a 25µm pinhole
• Tradeoff between light throughput and resolution.

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Effect of Pinhole
Better axial resolution, but Fewer photons
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Typical microscope image showing diffraction
rings and low contrast
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Contrast

• Gains in resolution are outweighed by the ability
  to discriminate against out-of-focus light
  reaching the detector
• In biological samples, the high turbidity makes
  use of a conventional microscope very difficult

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Optical sectioning is also possible with confocal
microscopy. Here are optical sections of
chromosomes in metaphase and a 3D reconstruction