The current state of Confocal Scanning Laser Microscopy

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. Widefield Confocal

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. Zeiss 510 Spectra Physics Millenia X - Tsunami

13. Leica TCS SP

14. Leica TCS SP 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. immobile mobile Bleach Protein mobility – bleaching experiments FRAP – Fluorescence recovery after photbleaching

20. Variants of FP • Blue BFP • Cyan CFP • Green GFP • Yellow YFP • Red DsRedHcRed • GFP timer CFP GFP CFP YFP

21. Fluorescence Resonance Energy Transfer FRET Donor Acceptor • Spectral overlap • Distance <10 nm

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

23. FRET: NKA – IP3R

24. NKA – IP3R After Before Photobleaching ofacceptor removes FRETdetected as increased donor signal Distance < 12 nm Ouabain binding to NKAshortens the distance – stronger interaction –increased FRET efficiency15-25% Donor GFP-NKA Donor diff Acceptor Cy3-IP3R

25. 535 nm 440 nm YFP YFP CFP CaM CaM + 4 Ca2+ 440 nm 480 nm CFP FRET based Ca2+ sensor

26. Multiphoton excitation 2-photon 1-photon

27. Builtin confocality 1-photon 2-photon

28. Konfokal Multifoton PMT PMT

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

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

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. Ca - wave [Ca2+] Data from live cell experiments combined with biochemical data is used as input for mathematical modeling-simulations 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 z y x

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