Biophotonics lecture 7. December 2011

1. Biophotonics lecture7. December 2011

2. Exam date ? Monday, 30 January 2012 or Wednesday, 1 February 2012

3. Last week: • Stimulated emission depletion (STED) microscopy

4. Today: • Imaging deep in tissue: 2-photon microscopy • Enlarging the NA: 4Pi microscopy • Super-resolution: Pointillism, STORM, PALM

5. Imaging deep in tissue:2-photon microscopy

6. Imaging deep in tissue:2-photon microscopy Refractive(andscattering)tissue Imaging singlecells Imaging deep in tissue objectivelens not welldefinedfocalspot welldefinedfocalspot Refractive(andscattering)tissue welldefinedfocalspot welldefinedfocalspot

7. The Problem: Scattering, aberrations, absorption Rayleigh scattering: ~ l-4 Blue: Bad! Red / Infrared: OK!

8. Imaging deep in tissue:2-photon microscopy Solution: imagingusinglongerwavelength objectivelens Refractive(andscattering )tissue not welldefinedfocalspot welldefined, but LARGER focalspot

9. x x z z Focal spot, l=500nm Focal spot, l=1000nm ATF ATF OTF OTF

10. Fluorescence Jablonski diagram Absorption… … and spontaneous emission

11. Fluorescence Jablonski diagram NO absorption…

12. 2-photonfluorescence Jablonski diagram 2-photon absorption… … and spontaneous emission

13. 2-photonfluorescence • 2-photon absorption requires two photons to be present simultaneously • The probability for this growsquadratically with intensity • It will only occur where the local intensity is high

14. x x x z z z Focal spot, l=500nm Focal spot, l=1000nm 2-photon, l=1000nm missingconefilledopticalsectioning

15. 2-photonfluorescence Zipfel, Williams, Webb, Nature Biotechnology21, 1369 - 1377 (2003)

16. DichromaticReflector Wide Area Detector at close destance emissionphotons will still bemultiplyscatteredandcannotbefocussed on a pinhole  Non-descanneddetectionneededtomaximizecapturearea

17. Two Photon Microscopy • Much lessabsorption • Much lessscattering • Feweraberrations • Less out-of-focus bleaching • Inherentopticalsectioning

18. Enlargingthe NA:4Pi Microscopy

19. Sample betweenCoverslips Detector Pinhole FluorescenceIntensity z Dichromatic Beamsplitter HighSidelobes z 2 Photon Effect 4 Pi Microscope (Type C) Stefan W. HellMax Planck Institute of Biophysical Chemistry Göttingen, Germany Aperture increase: Laser Illumination Emission

20. OTF ATF widefield 4Pi

21. widefield, l=500nm 4Pi, l=500nm 4Pi PSFs widefield, l=1000nm 2-photon, l=1000nm 4Pi, l=1000nm 4Pi, l=1000nm, 2-photon

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

23. DevidingEscherichia Coli 4Pi images From: Bahlmann, K., S. Jakob, and S. W. Hell (2001). Ultramicr. 87: 155-164.

24. 4Pi images Confocal (2-Photon ) 4Pi (2-Photon) Thanks to: Elisabeth Ehler, Reiner Rygiel, Martin Fiala, Tanjef Szellas

25. Super-resolution:Pointillism, STORM, PALM

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

27. Localization, not resolution PSF position

28. Localization, not resolution position ?????

29. How to separate particles? Spectral precision distance microscopy Problems: Chromatic Aberrations, few dyes Using fluorescence lifetime for separation (FLIM) Problems: Lifetime depends on microenvironment 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.

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

31. Earth at night Earth

32. Jena at night

33. Jena at night Task: Localizationoftheuniversitybuildings How: EachProfessor hasto turn on the light foroneminute Resoution Localizingismuchmoreprecisethanresolution

34. Separation over time

35. Separation over time Withoutlabelling:everythingisbright Labellingtheuniversitybuildingswidefield: badresolution Pointillistic: accuratemap

36. Pointillism, PALM, STORM PALM Photo-activation and localisation microscopy other techniques:STORM, FPALM http://jcs.biologists.org/cgi/reprint/123/3/309.pdf

37. EM Pointillism, PALM, STORM WF PALM E. Betziget al., Science, DOI: 10.1126/science.1127344, Aug. 2006 MitochondriaCOS-7 Zellen Cryo-Schnitte Cytochrom C Oxidase import Sequenz - dEosFP

38. Pointillism, PALM, STORM Hochauflösende Struktur der Podosomen (Vinculin) New, sophisticated algorithms, which can handle overlappingfluorophores

39. Pointillism, PALM, STORM 400nm Podosomenbildung Susan Cox, Edward Rosten, Marie Walde, James Moneypenny, Gareth Jones

40. Pointillism, PALM, STORM

41. Confocal microscopy dSTORM / B3 STED 1 m Comparing some methods Structured illuminationmicroscopy Widefield fluorescence

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

43. Localisation precision in pointilism:(for Gaussian PSFs) • N photons collected from 1 fluorophore • Positions of these photons are rn=rfluorophore ±with  being the standard deviation defined by the PSF • The fluorophore position is determined as the mean of all photon positionsrfluorophore=rn/ N • This mean position has an error ofrfluorophore with rfluorophore = / • With N photons, the localisation precision is better than the resolution Problem: sparseness of labelling