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

2. Total Internal Reflection Fluorescence (TIRF) Microscope Total Internal Reflection – Oil to Air : Critical Angle = 41 degree – Oil to Water : Critical Angle = 61 degree – Oil to Glycerol : Critical Angle = 78.5 degree Snell’s Law 90

3. TIRF Microscope Total Internal Reflection and Evanescent light – Refractive Index of Sample need to be smaller than objective lens immersion Optics – Using edge of NA to get TIR angle – Move spot at back focal of objective lens to control TIR angle and illumination depth

4. TIRF Microscope:Image

5. TIRF Microscope: Summary Using evanescent light coming out from Total Internal reflection to illuminate fluorescence dye Product:All major microscope company has TIRFM 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.

6. PALM/STORM PALM : Photoactivated Localization Microscope fPALM : Fluorescence Photoactivation Localization Microscope STORM : Stochastic Optical Reconstruction Microscope dSTORM : Direct STORM Use localization of single fluorophore to break diffraction limit.

7. PALM/STORM Precision of localization of each dye is much better - ~20nm - than optical resolution - ~200nm. Activate one dye at a time and measure dye position by PSF, you can separate two dyes which distance is less than optical resolution. Need to image single molecule fast to increase performance. -Require to use ~200mW laser -Use TIRFM to increase signal detection efficiency

8. Localization Sequence

9. Activation Method : PALM Eos Dronpa

10. Activation Method : STORM Combine two fluorophores to control activation Cy3-Cy5 Combination STORM Imaging Sequence STORM Dye Combinations

11. Activation : dSTORM Dye in Triplet state react with thiolate and form the radical anion of fluorophore F* which has several second life time. F* can be oxidized and recover singlet state to emit fluorescence again. Fluorephore Sate Chart

12. Photo Induced Activation : dSTORM Excite with UV light will bring dark state fluorophores into single state Absorption Spectra of Alexa Fluor 488

13. Two Color Example C, F: PALM Image D: TIRFM Image E: DIC Image

14. PALM, STORM, dSTORM: Summary (1) Using Photo activated dye to get nano-meter spatial resolution. Using TIRF or Near TIRF angle illumination to reduce background to increase detection efficiency. Product: Zeiss, Nikon, Leica Advantage – Can get very high spatial resolution (~10-20nm) in 2D. – Can get high Z resolution (~60nm) – System will be cheaper than other high resolution product – half or less. – dSTORM technique allow to use existing Fluorophores. Disadvantage: – Long Exposure to get image (15-30min) – Computation is required – not direct imaging. – Differernt Analysis software can output different result. – Need to evaluate system performance. – Difficult to image multiple dye compare to confocal microscope – Two Color PALM has been demonstrated – Eos & Dronpa – STORM has advantage in multi color imaging compare to PALM.

15. PALM, STORM: Summary (2) References PALM – Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Eric Betzig et al. Science Vol. 313 15 September 2006 p1642-1645 – Dual-color super resolution imaging of genetically expressed probes within individual adhesion complexes. H. Shroff et al. PNAS Vol.104 No.51 18 December 2007 p20308-20313 – Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure G. Shtengel et al. PNAS Vol.106 March 3 2009 p3125-3130 FPALM – Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. S. T. Hess, Biophys J. Vol 91 (11) Dec. 2006 p4258-72

16. PALM, STORM: Summary (2) References STORM – Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) M. Rust et al. Nature Method Vol.3 No.10 October 2006 p793-795 – Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes M. Bates et al. Science Vol.317September 2007 p1749-1753 – Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy B. Huang et al. Science Vol.319 February 2008 p810-813 dSTORM – Photoswitching microscopy with standard fluorophores. S. van de Linde et al. Appl. Phys. B Vol 93 2008 p725-731 – Direct stochastic optical reconstruction microscopy with standard fluorescent probes. S. van de Linde et al. Nature Protocols Vol.6 No.7 2011 p991-1009 – 4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues. D. Baddeley et al. PLoS One, Vol. 6 (5) 2011 e20645

17. 4 Pi Microscope Point Spread Function – (a) Confocal Microscope (2Pi) – (b) 4Pi Microscope (4Pi) – (c) After deconvolution Process

18. 4Pi Microscope: Image

19. 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. Product:Leica 4Pi Microscope System - $1M Advantage – Has better Z resolution than confocal microscope because of small PSF. – XYZ resolution is around 100nm in Z and 150nm in XY. Disadvantage – Expensive – 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

20. 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

21. Stimulated Emission Depletion (STED) Fluorescence Microscope Making laser spot size – PSF - smaller by using Depletion effect of fluorophore. Depletion

22. STED Concept The Concept of Super Resolution with STED Microscopy

23. STED Resolution STED PSF : 97nm resolution in Z and 104nm in XY Confocal PSF : 490nm resolution in Z and 244nm in XY

24. STED Microscope: Image

25. STED Microscope: Summary (1) Using fluorescence depletion to illuminate small spot to increase resolution to 100nm. Product:Leica STED System $1.3M Advantage – Can get high resolution (100nm) in 3D – Combining with 4Pi, Z resolution can be 33nm – No computation require to construct image Disadvantage – Expensive – Difficult to image multiple dye – Take long time to capture image. Not fast enough for live imaging. – Demonstrated Video Rate STED at 60nm Resolution.

26. STED Microscope: Summay(2) 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 – 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

27. Structured Illumination Microscope Use Moiré pattern effect to detect sub diffraction limit object – Maximum illumination pattern frequency (Fi) is determined by NA Fi max = F 0 – Detectable Moiré pattern frequency (Fd) is also determined by NA Fd max = F 0 – Moiré pattern frequency Fd = Fs – Fi (Fs is sample image frequency) – Fs = Fi + Fd = 2F 0 : Maximum detectable sample frequency is 2F 0 – Structured Illumination improved resolution 2 times ~100nm Moiré pattern a: Sample Image b: Illumination Pattern c: Moiré pattern

28. Saturated Structured Illumination (Non-Linear Illumination) (A) Structured illumination without saturation (B) Fluorescence emission saturation (C) Structured illumination with saturation (A) (B) (C)

29. Structured Illumination Microscope Frequency Map – a) Frequency of LM Original Resolution – b) Structured Illumination Pattern – c) Frequency Components – Black : Original Frequency – Dark Gray : Structured Illumination – Light Gray : Saturated Structured Illumination – d) Frequency of Structured Illumination – e) Rotate illumination to cover frequency in all direction

30. Saturated Structure Illumination Image Resolution Comparison – 50nm Beads sample – b: Conventional Microscope – c: Structured Illumination w/o Saturation – d: Saturated Structure Illumination

31. 3D Structured Illumination 3D Structured Illumination Pattern – 100nm Lateral Resolution – 270nm Axial Resolution – Difficult to use Saturated Technique because of photo stability of dye Creating 3D Structured Illumination 120nm Beads Image

32. 3D Structured Illumination Biological Sample Image

33. Structure Illumination Microscope: Summary(1) Use Moiré pattern to achieve sub diffraction limit resolution Product:Applied Precision Inc OMX, Nikon, Zeiss, Leica Advantage – Can improve lateral and axial resolution 2 times from diffraction limit – 100nm Lateral, 260nm Axial – Can improve lateral resolution 4 times ~50nm by using non-linear excitation – Can image multiple dye easily – Fast acquisition speed <10sec Disadvantage – Low resolution compare to localization microscope technique. – Saturated Illumination require high photo stability and difficult to apply for 3D imaging

34. Structure Illumination Microscope: Summary (2) References – Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. M. G. L. Gustafsson Journal of Microscopy Vol. 198, Pt 2, May 2000, p82-87 – Saturated patterned excitation microscopy—a concept for optical resolution improvement Rainer Heintzmann et al. J. Opt. Soc. Am. A Vol.19 No.8 August 2002 p1599-1609 – Nonlinear structured-illumination microscopy:Wide-field fluorescence imaging with theoretically unlimited resolution. M. G. L. Gustafsson PNAS Vol 102 2005 p13081-13086 – Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination M. G. L. Gustafsson et al. Biophysical Journal Vol.94 June 2008 p4957-4970 – Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy. L. Schermelleh et al. Science Vol.320 2008 p1332-1336

35. 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.

36. 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.

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

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

39. 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