Structured illumination microscopy and reflection confocal

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Structured illumination microscopy and reflection
confocal
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Structured illumination

• Sample image through a grating 3X, moving the
  grating each time by 1/3 pixel
• Only in-focus data will have sharp fringes
• Out-of-focus data will be relatively invariant
• I ((I1-I2)2(I1-I3)2(I2-I3)2)1/2 - throws out
  all the out-of-focus information
• Imperial College Powerpoint

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Structured illumination illuminators

• Zeiss Apotome
• Whole microscope is approx 100K
• Grabs images 1/second, processes in real time
• Thales Optem Optigrid
• Can be added onto existing scopes
• Relative cheap (20K)
• OptiGrid

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Mathematical foundations of structured
illumination

• From a mathematical sense, we can say that we are
  sampling 3-dimensional image space with a
  vertical grid spacing of 1/3 pixel
• This should give us a vertical resolution
  comparable to X and Y
• That is far better than confocal, where Z
  resolution is limited to 0.8 µ

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Extended resolution

• Standing wave fluorescence microscopy
• Principle Perform fluorescence microscopy with a
  laser as illumination source
• Light reflected from coverslip sets up a standing
  wave
• Set intensity threshold such that standing wave
  only excites fluorophores at its peak

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Standing Wave Fluorescence
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Problems

• Works well for very thin specimens
• For, thicker specimens, however, you get
  excitation of fluorophores in other parts of the
  cell by adjacent standing waves
• It is possible to mathematicvally deconvolve, but
  a pain!

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4Pi confocal microscopy

• Instead of using reflection, split laser beam to
  have two interfering beams from opposite sides of
  the specimen
• Significantly, the confocal aperture ensures that
  you are only getting fluorescence from one
  standing wave

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Leica TCS 4Pi
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Actin filaments in fibroblast by 4Pi
Gustafsson, Curr. Opin. Struct. Biol. 9627
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Resolution of techniques
Gustafsson, Curr. Opin. Struct. Biol. 9627
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STED

• Depends on phenomenon of Stimulated Emission
  Depletion
• If we raise a fluorophore to an excited state,
  hitting it with the emitted wavelenth can revert
  it to the ground state
• STED microscopy shines a short ? spot in field
  center, and surrounds it with a doughnut of
  longer ? STED reduces the size of the spot

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STED
Stephan Hell
Resolution can be 2X confocal resolution Hells
group has demonstrated 50 nm resolution Leica is
marketing a STED microscope But Microscope is
very elaborate and expensive Pumps a lot of
energy into the specimen photobleaching and
phototoxicity
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Reflection confocal

• Instead of setting the emission wavelength longer
  than excitation wavelength, set them to the same.
• Gives image contrast based on reflected (or
  really, backscattered) light
• Problem hard to interpret

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Reflection-enhanced backscatter confocal
microscopy

• Cells plated on a specular reflective substrate
  look like they were imaged in DIC when viewed by
  reflection confocal

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Mode of image formation in REBCM
In other words, REBCM is an interferometric
technique that measures height above the coverslip
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Note fringes along filopodia
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REBCM can show individual microtubules
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REBCM offers increased resolution
100 nm fluorescent beads are resolved in REBCM,
but not in fluorescence