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Lucia Gardini LOT/2019 LENS Fluorescence microscopy Single-molecule fluorescence microscopy Information on distributions and time trajectories that would otherwise be hidden Identify and compare sub-populations Probe biological


  1. Lucia Gardini LOT/2019 LENS

  2. Fluorescence microscopy

  3. Single-molecule fluorescence microscopy Information on distributions and time trajectories that would otherwise be hidden Identify and compare sub-populations Probe biological macromolecules and ONE MOLECULE AT A TIME provide informations on their structure and function In vitro and in vivo

  4. PSF and image resolution Abbe formula (x, y)   d Radius of x,y PSF  =   = 0 0 1 . 22 2 S  D 2 n sin 2 NA Abbre forluma (z) Axial amplitude of the PSF 2 / NA 2

  5. Image resolution Rayleigh criterion d d =  /2NA Lateral resolution ~ 250 nm Axial resolution = 2 / NA 2 ~ 500nm

  6. Overcoming the diffraction limit ~ 250 nm

  7. Fluorescence Imaging with One Nanometer Accuracy FIONA y y centre Localized point x x centre Thompson et al. Biophys.J. 2002

  8. Fluorescence Imaging with One Nanometer Accuracy FIONA High quantum yield fluo probes ? High NA aperture N b objectives (1.45) High sensitivity CCD cameras such as EMCCD Thompson et al. Biophys.J. 2002

  9. Yildiz et al. Science 2003 High quantum yield fluo probes High NA aperture objectives (1.45) High sensitivity CCD cameras such as EMCCD

  10. LOCALIZATION ON SURFACES:TIRF MICROSCOPY TOTAL INTERNAL REFLECTION MICROSCOPY b (proximity to the membrane) 100 nm depth in the sample Small volume excited 100 nm High S/N

  11. INCLINED ILLUMINATION LOCALIZATION ON SURFACES:TIRF MICROSCOPY HILO ( Highly inclined and Laminated Optical sheet) b THE ILLUMINATION BEAM ALWAYS PASSES THROUGH THE CENTER OF THE SPECIMEN PLANE ALLOWING OPTICAL SECTIONING 8 FOLD HIGHER SIGNAL/BACKGROUND COMPARED TO TRADITIONAL WIDEFIELD MICROSOCPY

  12. Application in vitro: myosin V walks hand over hand Accuracy: 1.5 nm. Time resolution: 0.5 s 10^4 photons collected Yildiz et al. Science 300:2061, 2003

  13. Singel Molecule FRET (Forster Resonance Energy Transfer) Distanze tipiche tra 30 e 80 Å

  14. R = dist.donor/acc R 0 = dist.caratteristica (50% di en. trasferita) Es.Cy3/Cy5 R 0 = 60 Å (6 nm)

  15. Probing conformational changes and displacements

  16. Lucia Gardini 09/04/2019 LENS

  17. Single molecule localization microscopy: SUPER-RESOLUTION MICROSCOPY NOBEL PRIZE IN CHEMISTRY 2014 Xiaowei Zhuang BREACKTHROUGH PRIZE 2019

  18. The principle of PALM and STORM

  19. The principle of PALM and STORM

  20. The principle of PALM and STORM FIONA

  21. The principle of PALM and STORM

  22. The principle of PALM and STORM

  23. The principle of PALM and STORM

  24. The principle of PALM and STORM

  25. The principle of PALM and STORM

  26. The principle of PALM and STORM

  27. The principle of PALM and STORM

  28. The principle of PALM and STORM

  29. The principle of PALM and STORM

  30. STORM - Photo-switchable Probes Activation laser pulses Imaging laser (657 nm) Cy5 fluorescence Reporter Activator 0 5 10 15 20 Time (s) Cy3 Cy5 More colors 6000 photons Activation Cy3 Cy5 Cy3 Cy5 Activation laser (532 nm)

  31. Direct STORM Dempsey G.T. et al Nat. Methods 2011

  32. 5 μ m B-SC-1 cell, Microtubules stained with anti- β tubulin Cy3 / Alexa 647 secondary antibody

  33. 10 6 molecules, 2-30 min 5 μ m Bates et al, Science 317 , 1749 – 1753 (2007)

  34. 500 nm 5 μ m

  35. █ Cy3 / Alexa 647: Clathrin █ Cy2 / Alexa 647: Microtubule 5 μ m Bates et al, Science 317 , 1749 – 1753 (2007)

  36. 1 μ m

  37. Avg = 172 nm 200 nm

  38. 3D via astigmatic detection

  39. 10 nm lateral 20 nm axial

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