1. Measure of Chloride and intracellular pH by means of two photon spectroscopy in vivo

2. A window with a view: spying brain function at the two-photon microscope 1)What is two photon microscopy? 2)Sensing of brain structure and function in vivo 3)Two photon spectroscopy in vivo: towards the quantitative measure of pH and [Cl].

3. Neuroscience for dummies: what is and where is the brain

4. Imaging in deep tissue: confocal microscopy Z

5. The ideal tool 1) if photons are emitted ONLY in the focal spot, you need NO rejections of out of focus light because there is NO light emitted away from the focal plane. 2) long wavelength excitation minimize photodamage and scattering of excitation

6. t = 0.1 fs (1 10 -16 s) Two photons are more than one ≈ 3 10 8 m → 3 10 14 µm/s 0.03 µm

7. 2 photon vs. 1-photon excitation

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

9. A window with a view

10. A trip into the brain

11. Spine motility in the juvenile cortex (SSctx, p25) 0 min 30 min 60 min

12. Watching the brain in operation Functional imaging of the brain with single cell resolution

13. Watching a mouse brain that is watching TV

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

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

17. -85 mV+60 mV The space and time resolved measure of Cl gradients is the key to understand inhibition in the brain

18. Nerst potential for Chloride

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

22. Gradients of intracellular Chloride 042043048049050 053 054 058

23. A new hope: E 2 -mKate A new sensor formed by the fusion of E 2 GFP with the Red protein mKate

24. Exciting properties of mKate excitation

27. 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.

30. Proteina cloph 2.0 espressa in cellule (misurata in cellule HEK) L = sqrt (6Dτ) D ≈ 20 um 2 /s Lag time (ms)

31. 0 2 G/R @458nm exc FCS

32. 0 2 FCS

33. Measuring the shuttling between nucleus and cytoplasm pre-bleach 5 s60 s240 s

34. Measuring the shuttling between nucleus and cytoplasm pre-bleach 5 s60 s240 s 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

35. We can use the nuclear membrane as a molecular sieve to measure the size of the fluorescent proteins! pre-bleach 5 s60 s240 s

36. In vivo FRAP measure in cortical neurons Pre bleach

37. Recovery of fluorescence of YFP

38. Diffusion of CloPhensor is strongly limited

39. Linear spectral composition for measuring cells pH pH 6.0 pH 8.0

40. Houston, we have a problem…

41. IMG_0823

42. Effects of excitation scattering on the spectra 770800830860890920950980

43. Unmixing the E 2 -mKate spectra R( ) = R rfp ( ) +  G sensor ( ) G( ) = G sensor +  R rfp ( )

44. P18 P4 In vivo mouse cortex

45. Road map to pH and Cl computation Spectral unmixing of R and G channels Use of the pH/Cl invariant R channel to compute excitation scattering Correction of G channel for excitation scattering Projection of the corrected G spectra on the reference spectra: pH computation

46. Looking at the red raw data

47. Comparing the effects of spectra corrections

49. Computing pH in vivo (p18)

50. Sum of residues allows a statistical test of the data treatment

51. Computing pH in vivo (p18)

52. 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…

53. Modeling extinction of emitted fluorescence

54. Applying the extinction model to the in vivo data

56. The state of the art at the present day (1 wk ago) Intracellular pH Intracellular Chloride

57. rat’s lab S. Sulis Sato, P. Artoni L. Cancedda, J. Szczurkowska S. Luin, A. Idilli, D. Arosio Telethon; FIRB Futuro ricerca; PRIN; Regione Toscana