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What is two photon microscopy ? Sensing of brain structure and function in vivo Two photon spectroscopy in vivo: towards the quantitative measure of pH and [Cl]. A window with a view: spying brain function at the two-photon microscope.
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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
Twophotons are more thanone t = 0.1 fs (1 10-16 s) ≈ 3 108 m → 3 1014µm/s 0.03 µm
Dependency of total fluorescence as a function of z Fluorescent spheres 0.2 mm (m)
Spine motility in the juvenilecortex (SSctx, p25) 0 min 30 min 60 min
Watching the brain in operation Functional imaging of the brainwith single cellresolution
Watching the brain in operation pH and Clhoride imaging in vivo
Excitation and inhibition in the brain + - + - + + +
The space and timeresolvedmeasureof Cl gradientsis the key tounderstandinhibition in the brain -85 mV +60 mV
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.
Gradients of intracellular Chloride 042 043 048 049 050 054 053 058
A newhope: E2-mKate A newsensorformedby the fusionof E2GFP with the Red proteinmKate
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.
G/R @458nm exc FCS 0 2
G/R @458nm exc FCS 0 2
5 s 60 s 240 s Measuring the shuttlingbetweennucleus and cytoplasm pre-bleach
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
5 s 60 s 240 s We can use the nuclear membrane as a molecularsievetomeasure the sizeof the fluorescentproteins! pre-bleach
In vivo FRAP measure in cortical neurons Pre bleach
Linear spectralcompositionformeasuringcells pH pH 8.0 pH 6.0
Effects of excitation scattering on the spectra 770 800 830 860 890 920 950 980
Unmixing the E2-mKate spectra R(l) = Rrfp(l) + aGsensor(l) G(l) = Gsensor + b Rrfp(l)
In vivo mouse cortex P4 P18
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
Sum ofresiduesallows a statistical test of the data treatment
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…