1. Biophotonics lecture 23. November 2011

2. Last week: -Fluorescence microscopy in general, labeling, etc… -How to do optical sectioning and fill the missing cone, the confocal microscope

3. Today: -Structured illumination: an alternative approach to optical sectioning -Super resolution techniques: beyond the Abbe limit -High-resolution structured illumination microscopy

4. Optical sectioning alternatives: structured illumination microscopy (SIM) -Sample is illuminated with a structured illumination pattern, i.e. a line grid. -This can be produced by placing a grid in a conjugate image plane in the illumination pathway. -Three images are taken for three different (lateral) positions of the illumination grid. -These images can be computed into a final, optically sectioned image.

5. For single-shot sectioning

6. In-focus sample slice Out-of-focus sample slice Full-field illumination Structured illumination Acquired wide-field image Acquired structured illumination image x y x z x y x y

7. Acquired structured illumination images I1(r)I1(r)I2(r)I2(r)I3(r)I3(r) I final (r) = max { I 1 (r), I 2 (r), I 3 (r) } - min { I 1 (r), I 2 (r), I 3 (r) } Result: sectioned imageWide-field image

8. Advantages: -Cheaper: no need for laser, scanner, PMTs, AOTFs, etc. -Potentially faster: for a large field of view 3 wide-field images can be acquired faster than a point-wise scan. -Less photo-bleaching: less light is lost as compared to using a pinhole

9. Wide-field Sectioning SIM Frequency support of sectioning SIM -Filled missing cone -Higher axial support -Higher lateral support (1 direction), more on this later But this is NOT an OTF !!

10. Wide-field Confocal Sectioning SIM The image formation in section SIM cannot be written as a convolution of the sample with an intensity PSF.

11. Advantages: -Cheaper: no need for laser, scanner, PMTs, AOTFs, etc. -Potentially faster: for a large field of view 3 wide-field images can be acquired faster than a point-wise scan. -Less photo-bleaching: less light is lost as compared to using a pinhole Disadvantage: -Not a linear imaging modality. Not as useful for quantitative analysis.

12. Imaging beyond the Abbe-limit: the true strength of SIM

13. “Sample“ for simulation Fourier transform of “Sample“ Sample will be “repainted” with a blurry brush rather than a point-like brush. Real space Fourier space Limited resolution in conventional, wide-field imaging

14. Moiré effect high frequency detail high frequency grid low frequency moiré patterns

15. Moiré effect Structured Illumination Microscopy Illumination with periodic light pattern down-modulates high- frequency sample information and makes it accessible for detection. SampleIllumination

16. Laser CCDxz Tube lens Filter Dichromatic reflector Tube lens Objective Sample Diffraction grating, SLM, etc…

17. SampleIllumination Structured Illumination Micropscopy Sample with structured illumination Multiplication of sample and illumination

18. Structured Illumination Micropscopy Multiplication of sample and illumination Real space Fourier space  Convolution of sample and illumination  Convolution of sample and illumination

19. SampleIllumination Structured Illumination Micropscopy

20. Sample

21. Sample Sample & llumination

22. Sample Imaging leads to loss of high frequencies (OTF) Structured Illumination Micropscopy

23. Separating the components… Sample Structured Illumination Micropscopy

24. Separating the components… Shifting the components… Sample Structured Illumination Micropscopy

25. Separating the components… Shifting the components… Recombining the components… Sample Structured Illumination Micropscopy

26. Separating the components… Shifting the components… Recombining the components… using the correct weights. Sample Reconstructed sample Structured Illumination Micropscopy

27. samplewide-field SIM (x only)

28. Missing cone – no optical sectioning Full-field illumination 1 focus in back focal plane

29. Missing cone – no optical sectioning 2-beam structured illumination 2 foci in back focal plane

30. Missing cone filled – optical sectioning 2-beam structured illumination 3 foci in back focal plane better z-resolution

31. 1  m Fourier space (percentile stretch) Liisa Hirvonen, Kai Wicker, Ondrej Mandula, Rainer Heintzmann

32. WF: 252 nm SIM: 105 nm 99 beads averaged wide-field SIM

33. 2 µm excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris Samples Prof. Bastmeyer, Universität Karlsruhe (TH) Axon Actin (Growth Cone)

34. excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris 2 µm Samples Prof. Bastmeyer, Universität Karlsruhe (TH) Axon Actin (Growth Cone)

35. Doublets in Myofibrils Isolated myofibrils from rat skeletal muscle Titin T12 – Oregon green L. Hirvonen, E. Ehler, K. Wicker, O. Mandula, R. Heintzmann, unpublished results 1 µm 124 nm

36. 1  m Molecules in space and time living COS1 cell L. Hirvonen, K. Wicker., O. Mandula and R. Heintzmann, Structured illumination microscopy of a living cell, Europ. Biophys. J. 38, 807-812, 2009

37. Marie Walde, James Monypenny (cooperation G. Jones), King‘s College London f-Actin Vinculin Are podosomes arranged as "sticks and joints"? 5  mPodosomes

38. 0 magnitude spatial frequency Support region of OTF 0 magnitude spatial frequency -K 0 K0K0 -2K 0 -K 0 K0K0 Linear Excitation (low intensity) Non-Linear Excitation (high intensity) -3K 0 Support region of OTF

39. Conventional microscopy Saturated structured illumination 1 µm Linear structured illumination 1 µm Mats Gustafsson, UCSF 50 nm microscpheres nonlinearity: fluorescence saturation, 53J/m 2 3 extra harmonics M.G.L. Gustafsson (2005), PNAS, 37, 13081-13086 Nonlinear Structured Illumination Micropscopy

40. But: Artefacts possible! 0.5 µm

41. But: Artefacts possible!  Sophisticated algorithms are needed!