NEU259

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

2. 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.

3. 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 .

4. Optical Tweezers: Example (1) • RNA polymerase Experiment by Dr. Steven Block, Stanford University • http://www.stanford.edu/group/blocklab/RNAP.html

5. Optical Tweezers: Expmale (2) • Dr. Kazuhiko Kinosita at Waseda University • http://www.k2.phys.waseda.ac.jp/Knotmovies/KnotDNA.htm

6. 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

7. High Resolution: Breaking Diffraction Limit • TIRF Microscope • PALM • 4Pi • STED

8. Confocal Microscope • Can provide diffraction limit resolution in Light Microscope • 200nm XY resolution and 500nm Z resolution. PSF in XZ without Pinhole PSF in XY without Pinhole Confocal Image w/o Pinhole PSF in XZ with Pinhole PSF in XY with Pinhole Confocal Image w/ pinhole

9. 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

10. TIRF Microscope:Image

11. 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) • 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.

12. 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.

13. PALM: Image

14. 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) • 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

15. 4 Pi Microscope • Point Spread Function • (a) Confocal Microscope (2Pi) • (b) 4Pi Microscope (4Pi) • (c) After deconvolution Process

16. 4Pi Microscope: Image

17. 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. • 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 • Expesive

18. 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

19. 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

20. STED Microscope: Image

21. 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 • Take long time to capture image. Not fast enough for live imaging. • 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

22. 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