A window with a view: spying brain function at the two-photon microscope

1. Whatistwophotonmicroscopy? Sensingofbrainstructure and function in vivo Twophotonspectroscopy in vivo: towards the quantitative measureof pH and [Cl]. A window with a view: spying brain function at the two-photon microscope

2. Neuroscience fordummies: whatis and whereis the brain

3. Imaging in deeptissue: confocalmicroscopy Z

4. Twophotons are more thanone t = 0.1 fs (1 10-16 s) ≈ 3 108 m → 3 1014µm/s 0.03 µm

5. 2 photon vs. 1-photon excitation

6. Dependency of total fluorescence as a function of z Fluorescent spheres 0.2 mm (m)

7. A windowwith a view

8. A trip into the brain

9. Spine motility in the juvenilecortex (SSctx, p25) 0 min 30 min 60 min

10. Watching the brain in operation Functional imaging of the brainwith single cellresolution

11. Watching a mouse brain that is watching TV

13. Watching the brain in operation pH and Clhoride imaging in vivo

14. Excitation and inhibition in the brain + - + - + + +

15. The space and timeresolvedmeasureof Cl gradientsis the key tounderstandinhibition in the brain -85 mV +60 mV

16. NerstpotentialforChloride

17. NerstpotentialforChloride

18. NerstpotentialforChloride

19. ClopHensor λecc=543 nm λecc=543 nm λecc=458 nm λecc=488 nm +H+ O- Ka +Cl- Kd OH Static quenching OH Cl- Arosio et al. Nature Meth. 2010.

20. Gradients of intracellular Chloride 042 043 048 049 050 054 053 058

21. A newhope: E2-mKate A newsensorformedby the fusionof E2GFP with the Red proteinmKate

22. ExcitingpropertiesofmKateexcitation

25. How to evaluate the integrity of the bi-molecular sensor? The correct measure of Cl concentration requires that the ratio between red and green fuorescent proteins is equal to 1. If we can demonstrate that the protein remains in the correct conformation, with the green and red proteins attached, the stechiometry is ensured.

28. G/R @458nm exc FCS 0 2

29. G/R @458nm exc FCS 0 2

30. 5 s 60 s 240 s Measuring the shuttlingbetweennucleus and cytoplasm pre-bleach

31. 5 s 60 s 240 s Measuring the shuttlingbetweennucleus and cytoplasm pre-bleach Fun facts about N/C shuttling: proteins with MW<30kD freely diffuse between these two compartments. Larger MW are associated to a very slow turnover

32. 5 s 60 s 240 s We can use the nuclear membrane as a molecularsievetomeasure the sizeof the fluorescentproteins! pre-bleach

33. In vivo FRAP measure in cortical neurons Pre bleach

34. Recovery of fluorescence of YFP

35. Diffusion of CloPhensor is strongly limited

36. Linear spectralcompositionformeasuringcells pH pH 8.0 pH 6.0

37. Houston, wehave a problem…

38. IMG_0823

39. Effects of excitation scattering on the spectra 770 800 830 860 890 920 950 980

40. Unmixing the E2-mKate spectra R(l) = Rrfp(l) + aGsensor(l) G(l) = Gsensor + b Rrfp(l)

41. In vivo mouse cortex P4 P18

42. Road mapto pH and Cl computation Spectralunmixingof R and G channels Useof the pH/Cl invariant R channeltocomputeexcitationscattering Correctionof G channelforexcitationscattering Projectionof the corrected G spectra on the referencespectra: pH computation

43. Looking at the redraw data

44. Comparing the effectsofspectracorrections

45. Comparing the effectsofspectracorrections

46. Computing pH in vivo (p18)

47. Sum ofresiduesallows a statistical test of the data treatment

48. Computing pH in vivo (p18)

49. What about extinction of the emitted light? Cl measure depends on an equally efficient collection of the fluorescence emitted at the green and red channels. Sadly, in a few seconds, I will provide evidences, that that is not the case We can build a model for differential extinction to correct the data. Or…

50. Modeling extinction of emitted fluorescence