M F B 2010 : W #45 S V F

MFB 2010 : W #45 S V F C S & F C-C    ZEISS LSM 780

Alexei GRICHINE, Mathieu FALLET & Sébastien MAILFERT

October, 2010

S V FCS  FCCS  T S P F C-C S R

S 2 / 21

1 T S General Purpose Fluorescence Correlation Spectroscopy Limitations

2 P Biological samples Calibration : waist evaluation Measurements on living cells

3 F C-C S

4 R

S V FCS  FCCS  T S G P P F C S F C-C S L R S

1 T S General Purpose Fluorescence Correlation Spectroscopy Limitations

2 P Biological samples Calibration : waist evaluation Measurements on living cells

3 F C-C S

4 R

G P 4 / 21

F C S

• Diffusion time measurements • Innovative technique : Spot Variation FCS Diffusion coefficient available Discrimination between free and different models of molecular diffusion (Actin meshwork and Nanodomains)

C FCS 5 / 21

ωz

ωxy

C  A

B Fluorescence fluctuations analysis B Low excitation power B Confocal spot : ωxy from 200 to B Low numbers of molecules (from 1 to 400 nm 100) ◦ B Two main parameters : B Physiological conditions @ 37 C 1 Mean number of molecules : N B Living cells B High spatio-temporal resolution (µs 2 Mean diffusion time : τd to s, 200 to 400 nm)

P 6 / 21

One point confocal measurement Intensity fluctuations computation: Auto-Correlation Function Confocal spot Membrane labeled with fluorescent probe

Nucleus 1.3 n o i Coverstrip t c n u F

n o i t 1.2 a l

e τ

r d r 1/N o C - o t u A

3 1.1 100x10 Intensity fluctuations recording

95 ) z H (

e t a

R 90

t 1.0 n u o

C 0.001 0.01 0.1 1 10 100 1000 85 Time (ms)

80 0 2 4 6 8 10 12 14 16 18 20 Time (s)

< δF(t)δF(t + τ) > G(τ) = < F(t) >2

P 6 / 21

Quite good N evaluation, not too much points for τ evaluation !

O S 7 / 21

S V FCS S

Sample Diaphragm: Water Variable spot Immersion size FCS Objective

Dichroïc Mirror Argon Optical Objective Laser Fiber

Pinhole

Fluorescence Filter Avalanche Photodiode

S V FCS 8 / 21

D  

increasing focal spot size

longer diffusion time

u “FCS diffusion law”

spot area

S V FCS 9 / 21

D :   &  

Experimental Results Simulation Results

Trapping in meshwork GFP (like TfR) Mean Diffusion Time (Td)

Free diffusion

O O O

H H Td (ms) H Dynamic partition TfR-GFP In isolated domains (like Thy1) t0 > 0

GFP t0 = 0 Spot Area

t0 < 0 H O HO Accessible spot size

GFP-Thy1 (GPI Anchor) Lipid nanodomain Fluorescent molecule Spot area (non excited/excited) Actin cytoskeleton Observation volume

L 10 / 21

FCS 

• Higher sensitivity to low probe concentration • Higher sensitivity to fast events (µs to ms) • Diffraction limit 2 • Diffusion coefficients available : 0.1 to 10 µm /s • Difficult to discriminate 2 populations with similar diffusion time

S V FCS  FCCS  T S B  P C :   F C-C S M    R S

1 T S General Purpose Fluorescence Correlation Spectroscopy Limitations

2 P Biological samples Calibration : waist evaluation Measurements on living cells

3 F C-C S

4 R

B  12 / 21

A 

• 10 000 to 20 000 cells per well on 8 wells Labtek (here we use COS7 cells) in DMEM culture medium ◦ • Cells must grow one night to be really adherent @ 37 C, 7% CO2 • Prepare buffer solution for FCS : 500µl of HEPES into 50ml of HBSS (Ca2+)

H     F  ?

• Fab fragment ≈ 50kD • Our tube is at 240ng/µl with ≈ 1.6 dye / Fab • The good FCS dilution is 250ng/ml • We must dilute 1µl of Fab in 1ml of buffer (HBSS (with Ca2+) + HEPES)

F A 

• Remove the cell culture medium from well • Put 200µl of diluted Fab into the well • Incubate 10’ @ RT • Wash 3 times in HBSS (with Ca2+) + HEPES

C    :    13 / 21

1 Laser intensity : 300 µW before objective

2 One drop of Rhodamine 6G solution : Rhodamine drop 2 D = 280µm /s Spot Confocal 3 10 × 20s Intensity fluctuations measurement : for 3 100x10

example 95

90 4 Auto-Correlation computation 85 Countrate (Hz) Countrate 80 5 Mean diffusion time determination : 3D measurement 0 2 4 6 8 10 12 14 16 18 20 Time (s) diffusion with Triplet State fluctuationsIntensity    τ    1.3    −   1   τ    1.2 τ G(τ) = 1 + 1 + nT e T   ! !  d3D    τ s2τ   1 + 1 +  1.1 τd3D τd3D determination Auto-Correlation Function Auto-Correlation 1.0 Mean diffusion time 0.001 0.01 0.1 1 10 100 1000 6 Time (ms) Waist√ evaluation √ wxy = 4Dτd3D = 4 × 280 × τd3D

R 6G  14 / 21

1.0

Abs. Rhodamine 6G Em. Rhodamine 6G (excitation @480nm)

0.8

0.6

0.4 Normalized intensity

0.2

0.0 400 450 500 550 600 650 700 Wavelenght (nm)

• Excitation @488nm : not the max. of absorption • Emission : 525±10nm • Enough signal because high efficiency

M    : EGFR + F A-A488 15 / 21

EGF Receptor

AntiEGFR Antibody

protein Intracellular Green

TransMemb. domain fluorophore 1 Plasma membrane molecule with a labelled anti-EGFR Fab antibody y 30µm 2 XY Scan x XY scan of the sample 20

15 3 Z scan and choice of 1 point (upper membrane 10

for ex.) 5 Position (µm) Position Z Scan 0 4 20 × 5s 20 40 60 80 100x103 Intensity fluctuations measurements : for Countrate (Hz)

example 3 100x10

5 95

Mean diffusion time determination : 1 species 90 τ Slow diffusion ( d1) 85 Countrate (Hz) Countrate Fast diffusion (τd2) but not necessary 80

measurement measurement 0 2 4 6 8 10 12 14 16 18 20   Time (s) 1.20   fluctuationsIntensity 1  1  6 ( ) = +   1.15 G τ 1  τ  N  1 +    1.10 τd1

1.05 determination determination Auto-Correlation Function Function Auto-Correlation

1.00 Mean diffusion time 0.001 0.01 0.1 1 10 100 1000 τd2 τd1 Time (ms) S V FCS  FCCS  T S B  P C :   F C-C S M    R

M    : EGFR + F A-A488 16 / 21

R     

90 EGFR-Alexa488

60 (ms) d τ 50

20 Diffusion Time 10

0 0.0 0.1 0.2 0.3 0.4 Spot Area (µm²)

• T0 ≈ 14.8 ± 3.4 ms 2 • Deff ≈ 0.45 ± 0.03µm /s

M    : EGFR + F A-F 17 / 21

L S S P

Fluoprobes 480XXL (Interchim) : λ exc. : 500nm, λ em. : 630nm Fluoprobes 481XXL (Interchim) : λ exc. : 515nm, λ em. : 650nm

A

Use for FCCS with for example Alexa488 and Fluoprobes 480XXL with only one laser @ 488nm and 2 detectors (’eGFP’ and ’Cy5’channels) No alignment problems but ratio of fluorescence emissions difficult to manage

S V FCS  FCCS  T S P F C-C S R S

1 T S General Purpose Fluorescence Correlation Spectroscopy Limitations

2 P Biological samples Calibration : waist evaluation Measurements on living cells

3 F C-C S

4 R

FCCS 19 / 21

This setup is based on 2 lasers and 2 channels One dye excited @488nm & one dye excited @633nm Cross-correlation is due to interactions between dyes or when dyes are linked

P

Both lasers must excite the same part of the sample with a high presicion Cross-correlation could be due to the crosstalk between channels The main idea is to use large shift between both emission spectra and to play with excitation power

1 T S General Purpose Fluorescence Correlation Spectroscopy Limitations

2 P Biological samples Calibration : waist evaluation Measurements on living cells

3 F C-C S

4 R

R 21 / 21

FCS, FCS  B A

J. Wenger et al.

D. Madge et al. Biophys. J., 92 :913-919, 2007. Phys. Rev. Lett., 29 :705-708, 1972. R. Lasserre et al.

P. Schwille et al. Nat Chem Biol., 4(9) :538-47, 2008. Biophysics Textbook Online, 2004. P.-F. Lenne et al.

L. Wawrezinieck et al. Histochem Cell Biol., 130(5) :795-805, 2008.

Biophys. J., 49 :4029-4042, 2005. K. Chakrabandhu et al. Cell Death Differ., 15(12) :1824-37, 2008. P.-F. Lenne et al. EMBO J., 25 :3245-3256, 2006. C. Eggeling et al. Nature, 457 :1159-1162, 2008. K. Bacia et al. Nat. Methods, 3 :83-89, 2006. A. Rossin et al. Exp Cell Res, 15 ;316(9) :1513-22, 2010.

S V FCS  FCCS 