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

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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 Nd:YAG 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


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Optical Tweezers: Expmale (2)

  • Dr. Kazuhiko Kinosita at Waseda University


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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

    • Can’t hold big object

    • Can’t 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 Microscope:Image

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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 Apr;89(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.

    • Can’t 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

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