1. The current state of Confocal Scanning Laser Microscopy Hjalmar Brismar Cell Physics, KTH

2. What are we doing in Cell Physics Confocal microscopy –History –Present Applications Areas of development –Excitation –Detection –Scanning

3. Cell Physics Study the biological cell from a physical perspective –Use tools and concepts from physics on biological problems –Develop methods and techniques –Describe biological functions and systems within a physical/mathematical framework We focus on: –Cell volume Osmolyte transport Water transport –Cell mass Measurement techniques Cell cycle/cell mass regulation –Intracellular signalling Frequency modulated Ca2+ signals

4. Instrumentation Microscopy (widefield, confocal, multiphoton) –Fluorescencent probes –Fluorescent labels, antibodies –Genetically engineered, GFP Electrophysiology –Patch clamp –MEA, multi electrode arrays

5. Confocal microscopy Marvin Minsky, 1955 –Laser (1958)1960 –Affordable computers with memory > 64kB –CSLM 1986-87

8. WidefieldConfocal

9. Confocal evolution 1 st generation CSLM (1987) –1 channel fluorescence detection –50 Hz line frequency 2nd generation (commercial systems ca1990) –2-3 channel detection –>=100 Hz 3rd generation (1996) –4 channel detection –500 Hz 4th generation (2001) –32 channels –2.6 kHz –AOM, AOBS control

10. Confocal industry Carl Zeiss (physiology, dynamic measurements) Leica (spectral sensitivity) Biorad (multiphoton) (olympus) (nikon) (EG&G Wallac) …

11. Zeiss 510

12. Spectra Physics Millenia X - Tsunami

13. Leica TCS SP

14. Spectra Physics 2017UV

15. Applications - Techniques GFP –FRAP –FRET Multiphoton excitation

16. GFP- Green Fluorescent Protein Aequoria Victoria

17. GFP Discovered 1962 as companion to aequorin Cloned 1992, expression 1994 238 Aminoacids 27-30 kDa Fluorophore made by 3 aminoacids (65-67) ”protected” in a cylinder

18. Dynamics GFP-Tubulin in Drosophila

19. Protein mobility – bleaching experiments Bleach mobile immobile FRAP – Fluorescence recovery after photbleaching

20. Variants of FP –Blue BFP –Cyan CFP –Green GFP –Yellow YFP –Red DsRed HcRed GFP timer CFP YFPCFP GFP

21. Fluorescence Resonance Energy Transfer FRET Spectral overlap Distance <10 nm DonorAcceptor

22. Interaction - FRET ( Fluorescence Resonance Energy Transfer) ProteinA CFP ProteinB YFP < 5-10 nm Excitation 430-450 nm Emission >570 nm Donor Acceptor

23. FRET: NKA – IP 3 R

24. NKA – IP 3 R Donor GFP-NKA Acceptor Cy3-IP 3 R Before After Donor diff Photobleaching of acceptor removes FRET detected as increased donor signal Distance < 12 nm Ouabain binding to NKA shortens the distance – stronger interaction – increased FRET efficiency 15-25%

25. FRET based Ca 2+ sensor YFP CFP CaM 440 nm 480 nm YFP CFP CaM 535 nm 440 nm + 4 Ca 2+

26. Multiphoton excitation 1-photon 2-photon

27. Builtin confocality 1-photon 2-photon

28. PMT KonfokalMultifoton

29. 0 20 40 60 80 80  m 1-photon2-photon Better penetration (2-400  m) Enables measurements from intact cells in a proper physiological environment. Electrophysiology

30. FRET CFP-YFP multiphoton 2-photon @ 790 nm 1-photon @ 514 nm CFP – YFP separated by a 6 aminoacid linker Fluorochrome distance 5 nm YFP – Calcyon No excitation at 790 nm YFP excited at 880 nm 790 880

31. Development - Excitation Currently used lasers –Ar ion, 458,488,514 nm –HeNe 543, 633 nm –Ar ion 351,364 nm –ArKr 488,568 nm –HeCd 442 nm –Diode 405 nm –HeNe 594 nm –Multiphoton excitation, TiSa 700-1100 We need affordable, low noise, low power consumption lasers 370-700 nm !

32. Development - Detection Spectral separation –Optical filters –Prism or grating Detectors –PMT –Photon counting diodes We need higher sensitivity, QE !

33. Development - Scanning Speed Flexibility

34. Ultrafast 3D spline scan Biological motivation –Ca2+ signals Measurement approach –Intracellular ion measurements –Combined electrophysiology

35. Frequency modulated Ca2+ signals

36. Data from live cell experiments combined with biochemical data is used as input for mathematical modeling-simulations [Ca 2+ ] Ca - wave Models verified by experiments can provide new information and direct the further investigations

37. Approach High resolution 3D recording of Ca2+ High speed recording Combined CSLM - electrophysiology Big cells – hippocampal pyramidal neurons

38. Scan speed

39. Confocal - line scan High time resolution (ms) Scan geometry  cell geometry 2D – cell cultures 2 s.

40. Arbitrary scan – 2D (Patwardhan & Åslund 1994)

41. 2D specimen

42. Tissue – 3D cells

43. 3D arbitrary scan x y z

44. Design criteria Z-axis precision >= optical resolution Bidirectional scan (to gain speed) Focusing distance 20-50+ um >100 Hz Nonharmonic

45. Ideas for ultrafast 3D scan Stage scan –High mass, impossible patch clamp Scan objective –Well defined mass, side effects in specimen ? Scan focusing lens inside objective –Tricky optics ?

46. Piezo focus with specimen protection 40X/0.9NA V/I