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Nanoscopie

Nanoscopie. Rainer Heintzmann , Institute for Photonic Technologies (IPHT), Friedrich Schiller University of Jena. Biophotonics, 2011. Nanoscopy ? What does this mean?. Greek. skopos. To look at dwarfs?. Nanos. Overview:. St imulated E mission D epletion Microscopy 4Pi

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Nanoscopie

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  1. Nanoscopie Rainer Heintzmann, Institute for Photonic Technologies (IPHT), Friedrich Schiller University of Jena Biophotonics, 2011

  2. Nanoscopy ?What does this mean?

  3. Greek skopos To look at dwarfs? Nanos

  4. Overview: • Stimulated EmissionDepletionMicroscopy • 4Pi • Pointillism, PALM and STORM

  5. Overview: • Stimulated EmissionDepletionMicroscopy • 4Pi • Pointillism, PALM and STORM

  6. ps ~Ps0sIex ps Singlet Ps1 StimulatedEmissionDepletion Ps0 STED Microscopy Klar, T. A., S. Jakobs, M. Dyba, A. Egner and S. W. Hell(2000). Proc.Nat. Acad. Sc. U.S.A. 97(15): 8206-8210.

  7. Saturation of the stimulated emission… provides the nonlinearity Klar, T. A., S. Jakobs, M. Dyba, A. Egner and S. W. Hell(2000). Proc.Nat. Acad. Sc. U.S.A. 97(15): 8206-8210.

  8. ps ~Ps1t –1 ~ Iex ~Ps0sIex ps Dispersion Element STED, the details <<1 ns 554nm, 250fs 745-760nm, 13ps Intensity First (excitation) Time Second (STED pulse) Dyes (600-760nm): RH414, Phyridine2 Avoid Reexcitation by STEDding at very long wavelength Problem: ~10 stronger power  bleaching Or Pulsed Laser Diode!

  9. STED, the details Emission Excitation Intensity wavelength 554nm, 250fs STED745-760nm, 13ps Low efficiency  Very strong light Detection Range

  10. ps ~µs S2 ~Ps1t –1 ~ Iex ~Ps0sIex T1 ps T0 Triplet STED, selecting a dye Singlet S1 S0

  11. STED Microscopy STED beam Resulting PSF 1 µm NA 1.3, 10nm pixelsize, no background

  12. STED Microscopy STED beam Resulting PSF 1 µm NA 1.3, 10nm pixelsize, 10% background, STED PSF gets quite dim

  13. STED Images Confocal STED human embryonic kidney labeled with a red-emitting dye (MR 121SE) Microtubules Immunofluorescence Current Opinion in Biotechnology 2005, 16:3–12 From micro to nano: recent advances in high-resolution microscopy; Yuval Garini, Bart J Vermolen and Ian T Young

  14. STED Images Vimentin Hell 2008, Nature Methods, 6,24-32 Willig, K. I., J. Keller, M. Bossi, S. W. Hell (2006): STED microscopy resolves nanoparticle assemblies. New J. Phys. 8: 106.

  15. 3D isoSTED Images 3D-Distribution of Tom20 in a mitochondrion (Vero cell) Spherical nanosized focal spot unravels the interior of cells, Nature Methods 5, 539 - 544 R. Schmidt, C.A. Wurm, S. Jakobs, J. Engelhardt, A. Egner & S. W Hell (2008)

  16. Current STED state of the art - resolution Resolution down to 8 nm (N+Vacancy in Diamond) STED microscopy reveals crystal colour centres with nanometric resolution, E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling and S. W. Hell, Nature Photonics 2009, DOI: 10.1038/NPHOTON.2009.2

  17. Current STED state of the art Field of view 2.5 mm x 1.8 mm ".. vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons" Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement, V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell; Science 320, 246-249

  18. Overview: • Stimulated Emission DepletionMicroscopy • 4Pi Microscopy • Pointillism, PALM and STORM

  19. Bigger NA:4Pi Microscopy

  20. Sample betweenCoverslips Detector Pinhole FluorescenceIntensity z Dichromatic Beamsplitter HighSidelobes z 2 Photon Effect 4 Pi Microscope (Type C) Aperture increase: Stefan W. Hell,Max Planck Inst. bpc, Goettingen, Germany Laser Fluorescence Emisssion

  21. APD2 APD1 Leica 4Pi http://www.leica-microsystems.com

  22. Leica 4Pi http://www.leica-microsystems.com

  23. 4Pi images Deviding Escherichia Coli From: Bahlmann, K., S. Jakobsand S. W. Hell (2001). Ultramicr. 87: 155-164.

  24. Leica 4Pi

  25. Leica 4Pi

  26. 4Pi images Confocal (2-Photon ) 4Pi (2-Photon) Thanks to: Elisabeth Ehler, Reiner Rygiel, Martin Fiala, Tanjef Szellas

  27. Computational spectacles Methods requiring computation

  28. + Object Optics Image Paradigm: Optimize for direct visibility E.g.: Widefield, Confocal, STED Does not necessarily optimize information content!

  29. Data Image Computation +  Paradigm: Optimize for information content  + Object Optics Data

  30. Examples in Medical Imaging SPECT MRI http://www.physics.ubc.ca/research/images/spect.gif http://www.cis.rit.edu/htbooks/mri/images/head.gif PET fMRI CT http://www.cerebromente.org.br/n01/pet/petdep.gif http://www.fmri.wfubmc.edu/other%20pics/lab_brain_logo.JPG http://www.vetmed.lsu.edu/vth&c/Orthopedics/Images/Computed%20Tomography%20(CT)%20Scanner.RV.jpg

  31. Overview: • Stimulated Emission DepletionMicroscopy • 4Pi • Pointillism, PALM and STORM

  32. Overview Pointillism, PALM and STORM

  33. Localization not resolution If positions are know you can paint a picture! Seurat: Tiger If particles can be separated, their relative positions can be measured accurately Douthwaite: Lewis Hamilton

  34. How to separate particles? Spectral precision distance microscopy Problems: Chromatic Aberrations, few dyes Using fluorescence lifetime for separation (FLIM) Problems: Lifetime depends on microenvironm. Use the blinking characteristics P. Edelmann, A. Esa, H. Bornfleth, R..Heintzmann, M. Hausmann, and C. Cremer. Proc. of SPIE , 3568:89-95, 1999 M. Heilemann, D.P. Herten, R.Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum and M. Sauer. Anal. Chem., 74, 3511-3517, 2002. K.A. Lidke, B. Rieger, T.M. Jovin, R. HeintzmannOptics Express13, 7052-7062, 2005.

  35. How to separate particles? Better: Avoid overlap entirely by temporally separating E. Betzig, "Proposed method for molecular optical imaging", Opt. Lett. 20, 237 (1995)

  36. Earth

  37. Identifizieren

  38. ErdebeiNacht Localizing the university buildings: Each Professor has to turn on the light for one minute Resoution Localizing is much moreprecise than resolution

  39. Separation over time

  40. Separation over time Ohne Markieren: Alles Licht Uni Gebäude Pointillistisch: genaue Karte

  41. PALM / STORM Prinzip http://www.youtube.com/watch?v=RE70GuMCzww

  42. Pointillismus siehe auch:STORM, FPALM http://jcs.biologists.org/cgi/reprint/123/3/309.pdf

  43. EM Pointillismus WF PALM E. Betzig et al., Science, DOI: 10.1126/science.1127344, Aug. 2006 POINTILLISMUS MitochondriaCOS-7 Zellen Cryo-Schnitte Cytochrom C Oxidase import Sequenz - dEosFP

  44. Pointillismus Hochauflösende Struktur der Podosomen (Vinculin) Neue Algorithmen, die auch überlappende Emission berücksichtigen können

  45. Pointillismus 400nm Podosomenbildung Susan Cox, Edward Rosten, Marie Walde, James Moneypenny, Gareth Jones

  46. Pointillismus

  47. Konfokale Mikroskopie dSTORM / B3 STED 1 m Methodenvergleich Strukturierte Beleuchtung Weitfeld Fluoreszenz

  48. Stochastic Optical Reconstruction Microscopy Science 319, 810 (2008); Bo Huang, et al. Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy POINTILISM Microtubules – (Cy3-Alexa647)

  49. Stochastic Optical Reconstruction Microscopy POINTILISM Microtubules – (Cy3-Alexa647)

  50. Biplane fPALM Resolution: 3030x75nm over several micrometers 4 micrometer bead POINTILISM Three-dimensional sub–100 nm resolution fluorescencemicroscopy of thick samples, Nature Methods 5, 527-529 M. F Juette, T. J Gould, M. D Lessard, M. J Mlodzianoski, B. S Nagpure, B. T Bennett, S. T Hess & J. Bewersdorf (2008)

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