Measure of Chloride and intracellular pH by means of two photon spectroscopy in vivo
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].
Neuroscience for dummies: what is and where is the brain
Imaging in deep tissue: confocal microscopy Z
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
t = 0.1 fs ( s) Two photons are more than one ≈ m → µm/s 0.03 µm
2 photon vs. 1-photon excitation
Dependency of total fluorescence as a function of z Fluorescent spheres 0.2 mm ( m)
A window with a view
A trip into the brain
Spine motility in the juvenile cortex (SSctx, p25) 0 min 30 min 60 min
Watching the brain in operation Functional imaging of the brain with single cell resolution
Watching a mouse brain that is watching TV
Watching the brain in operation pH and Clhoride imaging in vivo
Excitation and inhibition in the brain
-85 mV+60 mV The space and time resolved measure of Cl gradients is the key to understand inhibition in the brain
Nerst potential for Chloride
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
Gradients of intracellular Chloride
A new hope: E 2 -mKate A new sensor formed by the fusion of E 2 GFP with the Red protein mKate
Exciting properties of mKate excitation
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.
Proteina cloph 2.0 espressa in cellule (misurata in cellule HEK) L = sqrt (6Dτ) D ≈ 20 um 2 /s Lag time (ms)
0 2 exc FCS
0 2 FCS
Measuring the shuttling between nucleus and cytoplasm pre-bleach 5 s60 s240 s
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
We can use the nuclear membrane as a molecular sieve to measure the size of the fluorescent proteins! pre-bleach 5 s60 s240 s
In vivo FRAP measure in cortical neurons Pre bleach
Recovery of fluorescence of YFP
Diffusion of CloPhensor is strongly limited
Linear spectral composition for measuring cells pH pH 6.0 pH 8.0
Houston, we have a problem…
IMG_0823
Effects of excitation scattering on the spectra
Unmixing the E 2 -mKate spectra R( ) = R rfp ( ) + G sensor ( ) G( ) = G sensor + R rfp ( )
P18 P4 In vivo mouse cortex
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
Looking at the red raw data
Comparing the effects of spectra corrections
Computing pH in vivo (p18)
Sum of residues allows a statistical test of the data treatment
Computing pH in vivo (p18)
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…
Modeling extinction of emitted fluorescence
Applying the extinction model to the in vivo data
The state of the art at the present day (1 wk ago) Intracellular pH Intracellular Chloride
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