NEU259

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NEU259
Advanced Light Microscope Techniques
Hiroyuki Hakozaki National Center for Microscopy
and Imaging Research University of California,
San Diego
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Optical Tweezers

• (a) The larger momentum change of the more
  intense rays cause a net force to be applied back
  toward the center of the trap.
• (b) When the bead is laterally centered on the
  beam, the net force points toward the beam waist.

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Optical Tweezers Optics

• IR laser is commonly used for not interfering
  with observation wavelength. CW NdYAG Laser
  (1064nm) is common for this application.
• Expand laser beam to fill back focal of objective
  lens to use entire NA

• Dichroic mirrors to separate observation light
  and laser.
• Position Detector to detect beads displacement .

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Optical Tweezers Example (1)

• RNA polymerase Experiment by Dr. Steven Block,
  Stanford University
• http//www.stanford.edu/group/blocklab/RNAP.html

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Optical Tweezers Expmale (2)

• Dr. Kazuhiko Kinosita at Waseda University
• http//www.k2.phys.waseda.ac.jp/Knotmovies/KnotDNA
  .htm

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Optical Tweezers Summary

• Hold object like tweezers by using laser light.
• Advantage
• Hold and Manipulate object that has different
  refractive index number from medium
• Measure force by using trapping power
• A few pN 100pN. pN 10-12 N
• Can manipurate more than two spot
• Disadvantage
• Cant hold big object
• Cant hold every object in cell because of
  refractive index of object
• References
• Observation of single-beam gradient force optical
  trap for dielectric particles. A. Ashkin et al,
  Optics Letters, Vol. 11, No.5, May 1986 p288-290

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Total Internal Reflection Fluorescence (TIRF)
Microscope

• Total Internal Reflection and Evanescent light

• Optics
• Using edge of NA to get TIR angle
• Move spot at back focal of objective lens to
  control TIR angle and illumination depth

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TIRF MicroscopeImage
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TIRF Microscope Summary

• Using evanescent light coming out from Total
  Internal reflection to illuminate fluorescence
  dye
• Advantage
• Illuminate only 100nm from cover-glass surface.
• Z Resolution is better than confocal microscope
  (500nm)
• Less cell damage because of limited excitation
  area
• Less Background High sensitive imaging.
• Disadvantage
• Imaging area is limited to cover glass surface.
• References
• Cell-substrate contacts illuminated by total
  internal reflection fluorescence. Axelrod D. Cell
  Biol. 1981 Apr89(1)141-5.

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Photoactivated Localization Microscopy (PALM)

• By calculating center of PSF, precision of dye
  position detection can be more than optical
  resolution.

• Activate one dye at a time and measure dye
  position by PSF, you can separate two dyes which
  distance is less than optical resolution.

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PALM Image
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PALM Summary

• Using Photo activated dye to get nano-meter
  spatial resolution. Using TIRF illumination to
  reduce background to increase detection
  efficiency.
• Advantage
• Can get very high spatial resolution (20nm) in
  2D.
• Disadvantage
• Only work at cover glass surface area Not high
  resolution 3D
• Require long time exposure to get image
  (2-12hours)
• Improved to 15-30min exposure time these days by
  using continuous activation.
• Cant use for live sample
• References
• Imaging Intracellular Fluorescent Proteins at
  Nanometer Resolution. Eric Betzig et al. Science
  Vol. 313 15 September 2006 p1642-1645

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4 Pi Microscope

• Point Spread Function
• (a) Confocal Microscope (2Pi)
• (b) 4Pi Microscope (4Pi)
• (c) After deconvolution Process

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4Pi Microscope Image
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4 Pi Microscope Summary(1)

• Using two identical objective lens to double the
  NA. Try to use entire solid angle 4Pi to get
  higher resolution.
• Advantage
• Has better Z resolution than confocal microscope
  because of small PSF.
• XYZ resolution is around 100nm in Z and 150nm in
  XY.
• Disadvantage
• Require special sample preparation
• Use quartz cover glass
• Need to put beads for each cover glass for PSF
  measurement
• Require special alignment to co-align two
  objective lens
• Require deconvolution process
• Expensive - 1M

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4Pi Microscope Summary(2)

• References
• Fundamental improvement of resolution with a
  4Pi-confocal fluorescence microscope using
  two-photon excitation. Stefan Hell et al. Optics
  Communications 93 1992 p277-282
• Properties of a 4Pi confocal fluorescence
  microscope. Stefan Hell et al. J. Opt. Soc. Am. A
  Vol. 19 No.12 p2159-2166
• Measurement of the 4Pi-confocal point spread
  function proves 75nm axial resolution. S. W. Hell
  et al. Appl. Phys. Lett. 64(11), 14 March 1994
  p1335-1337

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Stimulated Emission Depletion (STED) Fluorescence
Microscope

• STED Point spread function
• (a) Excitation Laser PSF (Green)
• (a) Depletion Lasre PSF (Red)
• (b) STED PSF 97nm resolution in Z and 104nm in
  XY
• (c) Confocal PSF 490nm resolution in Z and
  244nm in XY

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STED Microscope Image
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STED Microscope Summary (1)

• Using fluorescence depletion to illuminate small
  spot to increase resolution to 100nm.
• Advantage
• Can get high resolution (100nm) in 3D
• Combining with 4Pi, Z resolution can be 33nm
• 16nm Spatial resolution has been demonstrated
• Disadvantage
• Expensive 1.3M
• Take long time to capture image. Not fast enough
  for live imaging.
• Just published Video Rate STED at 60nm Resolution
• References
• Breaking the diffraction resolution limit by
  stimulated emission stimulated-emission-depletion
  fluorescence microscope. Stefan W. Hell et al.
  Optics Letters Vol.19 No.11 June 1, 1994 p780-782
• Fluorescence Microscopy with diffraction
  resolution barrier broken by stimulated emission.
  Thomas A. Klar et al. PNAS Vol.97 No.15 July 18
  2000 p8206-8210

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STED Microscope Summay(2)

• Focal Spots of Size r/23 Open Up Far-Field
  Fluorescence Microscopy at 33nm Axial Resolution.
  Marcus Dyba et al. Physical Review Letters Vol.88
  No.16 22 April 2002 P163901
• Nanoscale Resolution in the Focal Plane of an
  Optical Microscope. Volker Westphal et al.
  Physical Review Letters April 15 2005 Vol.94
  No.14 p143903
• Video-Rate Far-Field Optical Nanoscopy Dissects
  Synaptic Vesicle Movement. Volker Westphal et al.
  Science Vol320, P246 April 23, 2008