1. Biophotonics lecture 7. 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. Refractive (and scattering) tissue
Imaging deep in tissue: 2-photon microscopy
Imaging single cells
Imaging deep in tissue
objective lens
Refractive (and scattering) tissue
well defined focal spot
well defined focal spot
Refractive (and scattering) tissue
not well defined focal spot
well defined focal spot

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

8. Imaging deep in tissue: 2-photon microscopy
Solution: imaging using longer wavelength
objective lens
Refractive (and scattering ) tissue
well defined, but LARGER focal spot
not well defined focal spot

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

10. … and spontaneous emission
Fluorescence
Jablonski diagram
Absorption…
… and spontaneous emission

11. Fluorescence
Jablonski diagram
NO absorption…

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

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

14. missing cone filled optical sectioning
Focal spot, l=500nm
Focal spot, l=1000nm
2-photon, l=1000nm x z x z x z
missing cone filled optical sectioning

15. missing cone filled optical sectioning
Focal spot, l=500nm
Focal spot, l=1000nm
2-photon, l=1000nm x z x z x z
missing cone filled optical sectioning

16. 2-photon fluorescence
Zipfel, Williams, Webb, Nature Biotechnology  21, (2003)

17. Wide Area Detector
at close destance
Dichromatic Reflector
emission photons will still be multiply scattered and cannot be focussed on a pinhole
 Non-descanned detection needed to maximize capture area

18. Two Photon Microscopy Much less absorption Much less scattering
Fewer aberrations
Less out-of-focus bleaching
Inherent optical sectioning

19. Enlarging the NA: 4Pi Microscopy

20. Sample between Coverslips
Aperture increase:
4 Pi Microscope (Type C)
Sample between Coverslips
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
Detector
Pinhole
Fluorescence Intensity z
Dichromatic
Beamsplitter
2 Photon Effect
High Sidelobes
Laser
Illumination Emission

21. ATF
OTF
widefield
4Pi

22. 4Pi PSFs widefield, l=500nm 4Pi, l=500nm 2-photon, l=1000nm

23. 4Pi PSFs widefield, l=500nm 4Pi, l=500nm 2-photon, l=1000nm

24. Leica 4Pi

25. 4Pi images Deviding Escherichia Coli
From: Bahlmann, K., S. Jakob, and S. W. Hell (2001). Ultramicr. 87:

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

27. Super-resolution: Pointillism, STORM, PALM

28. 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

29. Localization, not resolution
PSF
position

30. Localization, not resolution
position ?????

31. 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, , 2002.
K.A. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann Optics Express 13, , 2005.

32. 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)

33. Earth
at night
Earth

34. Jena at night

35. Jena at night Resoution
Task: Localization of the university buildings
How: Each Professor has to turn on the light for one minute
Resoution
Localizing is much more precise than resolution

36. Separation over time

37. Separation over time Pointillistic: accurate map
Without labelling: everything is bright
Labelling the university buildings widefield: bad resolution
Pointillistic: accurate map

38. Pointillism, PALM, STORM
Photo-activation and localisation microscopy
other techniques: STORM, FPALM
reprint/123/3/309.pdf

39. Pointillism, PALM, STORMWF EM
PALM
E. Betzig et al., Science, DOI: /science , Aug. 2006
Mitochondria COS-7 Zellen
Cryo-Schnitte
Cytochrom C Oxidase import Sequenz - dEosFP

40. Pointillism, PALM, STORM
Hochauflösende Struktur der Podosomen (Vinculin)
New, sophisticated algorithms,
which can handle overlapping fluorophores

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

42. Pointillism, PALM, STORM

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

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

45. 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 positions rfluorophore=rn / N
This mean position has an error of rfluorophore with rfluorophore =  / N
With N photons, the localisation precision is N better than the resolution
Problem: sparseness of labelling