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

t = 0.1 fs (1 10-16 s)

Two photons are more than one

≈ 3 108 m → 3 1014 µm/s 0.03 µm

2 photon vs. 1-photon excitation

0 2 4 6 8 101E-5

1E-4

1E-3

0.01

0.1

1

Tota

l flu

ores

cenc

e

Distance from FP

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 min30 min60 min

Watching the brain in operation

Functional imaging of the brain with single cell resolution

Watching a mouse brain that is watching TV

Ap2

p3

p1n1 n2 n3

n4n5

n6

n7

n9

NP

P3

P2

P1

A1

0 45 90 135 180 225 270 315 3600.00

0.05

0.10

0.15

0.20

0.25

Resp

onse

Grid orientation (deg)

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

0 20 40 60 80-140

-120

-100

-80

-60

-40

-20

0Ne

rst P

oten

tial fo

r Chlo

ride

(mV)

Cl (mM)

Nerst potential for Chloride

Nerst potential for Chloride

0 20 40 60 80-140

-120

-100

-80

-60

-40

-20

0Ne

rst P

oten

tial fo

r Chlo

ride

(mV)

Cl (mM)

Nerst potential for Chloride

0 20 40 60 80-140

-120

-100

-80

-60

-40

-20

0Ne

rst P

oten

tial fo

r Chlo

ride

(mV)

Cl (mM)

ClopHensor

OH

Cl-

+Cl- Kd

Ka

+H+

O-

OH

λecc=543 nm λecc=488 nmλecc=458 nm λecc=543 nm

Static quenching

Arosio et al. Nature Meth. 2010.

Gradients of intracellular Chloride

042 043 048 049

050 053 054 058

A new hope: E2-mKate

A new sensor formed by the fusion of E2GFP with the Red protein mKate

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1 emiss mKate2 exc mKate2

emiss

ion flu

ores

cenc

e int

ensit

y (a.

u.)

wavelength (nm)

Exciting properties of mKate excitation

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1 emiss E2GFP emiss mKate2

emiss

ion flu

ores

cenc

e int

ensit

y (a.

u.)

wavelength (nm)

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1 emiss E2GFP emiss mKate2

emiss

ion flu

ores

cenc

e int

ensit

y (a.

u.)

wavelength (nm)

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.

0 2

G/R @458nm exc

FCS

10 1000.0

0.2

0.4

0.6

0.8

1.0

1.2

N gre

en/N

red

pH 7 - exc 458 nm

[Cl-] (mM)

kCld = 5823 mM

0 2

G/R @458nm exc

FCS

10 1000.0

0.2

0.4

0.6

0.8

1.0

1.2

N gre

en/N

red

pH 7 - exc 458 nm

[Cl-] (mM) G

reen

/Red

nor

m. t

o [C

l- ]=

0

kCld = 5823 mM

kCld = 472 mM

Measuring the shuttling between nucleus and cytoplasm

pre-bleach 5 s 60 s 240 s

Measuring the shuttling between nucleus and cytoplasm

pre-bleach 5 s 60 s 240 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 s 60 s 240 s

In vivo FRAP measure in cortical neurons

Pre bleach

0 1000 2000 3000 4000 5000 6000 70000,5

0,6

0,7

0,8

0,9

1,0

YFP H lineFrecovered= 97 % = 217 3 s

(I n(t)/

I all(t)

) / (I

npre /I all

pre )

time (s)

Recovery of fluorescence of YFP

Diffusion of CloPhensor is strongly limited

0 1000 2000 3000 4000 5000 6000 70000,5

0,6

0,7

0,8

0,9

1,0

YFP H E2GFP-lssmKate2

Frecovered= 97 % = 217 3 s

(I n(t)/

I all(t)

) / (I

npre /I all

pre )

time (s)

y = A exp(-t/) + FrecoveredFrecovered= 84 % = 856 20 s

800 850 900 950 1000

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Inte

nsity

(ADU

)

Wavelength (nm)

0,0 0,2 0,4 0,6 0,8 1,00,0

0,2

0,4

0,6

0,8

1,0 No correction of R>G bleed through Corrected for R>G bleed through

pH 6.8

pH 6.4

pH 7.0

pH 7.2

Com

pone

nt p

rojec

ted

on p

H 8.

0

Component projected on pH 6.0

pH 7.6

Linear spectral composition for measuring cells pH

pH 6.0

pH 8.0

6,0 6,4 6,8 7,2 7,6 8,00

15

30

45

60

75

90 No R>G Bleed through R>G corrected

Angle

pH

Houston, we have a problem…

IMG_0823

Effects of excitation scattering on the spectra

750 800 850 900 950 10000

1000

2000

3000

4000

Fluo

resc

ence

(AU)

Wavelenght (nm)

770 800 830 860 890 920 950 980

0.20.40.60.81.0

Fluo

resc

ence

750 800 850 900 950 10000.5

1.5

2.5

Corre

ction

Wavelength (nm)

750 800 850 900 950 10000.2

0.4

0.6

0.8

1.0

Norm

alise

d flu

ores

cenc

e

Wavelength (nm)

0.50

0.75

1.00

1.25

1.50

1.75

2.00

inten

sity (

norm

. on

R910

) pH 6 pH 6.4 pH 6.8 pH 7 pH 7.2 pH 7.6 pH 8

800 820 840 860 880 900 920 940 960 980 10000.4

0.6

0.8

1.0

excitation wavelength (nm)

Unmixing the E2-mKate spectra

R(l) = Rrfp(l) + a Gsensor (l)G(l) = Gsensor + bRrfp (l)

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

800 850 900 950 10000,25

0,50

0,75

1,00

1,25

1,50

1,75

Wavelength (nm)

Comparing the effects of spectra corrections

800 850 900 950 10000,25

0,50

0,75

1,00

1,25

1,50

1,75

Wavelength (nm)

pH 7.37R=3.63

Comparing the effects of spectra corrections

800 850 900 950 10000,25

0,50

0,75

1,00

1,25

1,50

1,75

pH 7.14R=0.82

Wavelength (nm)

pH 7.37R=3.63

Computing pH in vivo (p18)

0

2

4

6

8

10

12

14

MinimumResidue

IndividualRed

Corrrectionon mean Red

Unmix onlyRaw Data

Sum

of r

esidu

es

0

2

4

6

8

10

12

14

16

IndividualRed

Mean RedUnmixing

Resid

ue o

f pH

com

puta

tion

Raw Data

Sum of residues allows a statistical test of the data treatment

Computing pH in vivo (p18)

7,0

7,2

7,4

7,6

7,8

MinimumResidue

IndividualRed

Corrrectionon mean Red

Unmix onlyRaw Data

pH

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 0 50 100 150 200 250 3000,95

1,00

1,05

1,10

1,15

1,20

1,25

1,30re

d/gr

een

depth (um)

Equation y = A + B*xAdj. R-Square 0,97895

Value Standard ErrorMean Mean 0,97355 0,00139Mean Mean 8,17063E-4 7,57754E-6

Differential extinction of YFP fluorescence

Modeling extinction of emitted fluorescence

140 160 180 200 220 240 260 280-20

0

20

40

60

80

100

120 Clprime % (2)

Clpr

ime

Depth (micron)

Applying the extinction model to the in vivo data

Applying the extinction model to the in vivo data

140 160 180 200 220 240 260 280-20

0

20

40

60

80

100

120 Clprime ClGamma

Clpr

ime

Depth (micron)

The state of the art at the present day (1 wk ago)

Intracellular pH Intracellular Chloride

6,6 6,7 6,8 6,9 7,0 7,1 7,2 7,3 7,4 7,50,0

0,2

0,4

0,6

0,8

1,0

1 10 1000,0

0,2

0,4

0,6

0,8

1,0

P8P22

P18

P5

Perc

enta

ge

Intracellular pH

P4

P5A P8A P4A

P18 P18A P22aCl P32Ag P29Ag

Perc

enta

geChloride (nM)

rat’s lab

S. Sulis Sato, P. ArtoniL. Cancedda, J. Szczurkowska S. Luin, A. Idilli, D. Arosio

Telethon; FIRB Futuro ricerca; PRIN; Regione Toscana