MIFOBIO 2010 : WORKSHOP #45 SPOT VARIATION FLUORESCENCE CORRELATION SPECTROSCOPY & FLUORESCENCE CROSS-CORRELATION STUDIES ON A ZEISS LSM 780 Alexei GRICHINE, Mathieu FALLET & Sébastien MAILFERT October, 2010
SUMMARY 2 / 21 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4
SUMMARY THEORETICAL SURVEY GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS GENERAL PURPOSE 4 / 21 FLUORESCENCE CORRELATION SPECTROSCOPY Diffusion time measurements Innovative technique : Spot Variation FCS Diffusion coefficient available Discrimination between free and different models of molecular diffusion (Actin meshwork and Nanodomains)
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS CONVENTIONAL FCS 5 / 21 ω z ω xy CONFOCAL MEASUREMENT Fluorescence fluctuations analysis Confocal spot : ω xy from 200 to 400 nm Two main parameters : 1 Mean number of molecules : N 2 Mean diffusion time : τ d ADVANTAGES Low excitation power Low numbers of molecules (from 1 to 100) Physiological conditions @ 37 C Living cells High spatio-temporal resolution (µs to s, 200 to 400 nm)
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS PRINCIPLE 6 / 21 One point confocal measurement Confocal spot Membrane labeled with fluorescent probe Intensity fluctuations computation: Auto-Correlation Function Nucleus 1.3 Coverstrip Auto-Correlation Function 1.2 τ d 1/N 100x10 3 Intensity fluctuations recording 1.1 95 Rate (Hz) Count 90 85 1.0 0.001 0.01 0.1 1 10 100 1000 Time (ms) 80 0 2 4 6 8 10 12 14 16 18 20 Time (s) G(τ) = < δf(t)δf(t + τ) > < F(t) > 2
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS PRINCIPLE 6 / 21 Quite good N evaluation, not too much points for τ evaluation!
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS OPTICAL SETUP 7 / 21 SPOT VARIATION FCS SETUP Diaphragm: Variable spot size FCS Sample Water Immersion Objective Argon Laser Objective Optical Fiber Dichroïc Mirror Pinhole Fluorescence Filter Avalanche Photodiode
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS SPOT VARIATION FCS 8 / 21 DIFFUSION LAW CONCEPT increasing focal spot size longer diffusion time diffusion time τ d FCS diffusion law spot area
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS SPOT VARIATION FCS 9 / 21 DIFFUSION : EXPERIMENTAL RESULTS & COMPUTER SIMULATION Experimental Results Simulation Results GFP Mean Diffusion Time (Td) Trapping in meshwork (like TfR) HO Free diffusion Td (ms) HO TfR-GFP t 0 > 0 Dynamic partition In isolated domains (like Thy1) GFP HO HO t 0 = 0 t 0 < 0 Accessible spot size Spot Area GFP-Thy1 (GPI Anchor) Spot area Lipid nanodomain Actin cytoskeleton Fluorescent molecule (non excited/excited) Observation volume
GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS LIMITATIONS 10 / 21 FCS LIMITATIONS Higher sensitivity to low probe concentration Higher sensitivity to fast events (µs to ms) Diffraction limit Diffusion coefficients available : 0.1 to 10 µm 2 /s Difficult to discriminate 2 populations with similar diffusion time
SUMMARY THEORETICAL SURVEY BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4
BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS BIOLOGICAL SAMPLES 12 / 21 ADHERENT CELLS 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+) HOW TO OBTAIN THE GOOD FAB CONCENTRATION? 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) FAB ANTIBODY LABELING 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
BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS CALIBRATION ON KNOWN SOLUTION : SPOT SIZE EVALUATION 13 / 21 1 Laser intensity : 300 µw before objective 2 One drop of Rhodamine 6G solution : D = 280µm 2 /s 3 Intensity fluctuations measurement : 10 20s for example 4 Auto-Correlation computation 5 Mean diffusion time determination : 3D diffusion with Triplet State τ G(τ) = 1 + 1 + n T e τ 1 T ( 1 + τ ) ( ) 1 + s2 τ τ d3d τ d3d 6 Waist evaluation w xy = 4Dτ d3d = 4 280 τ d3d Confocal Spot Intensity fluctuations measurement Mean diffusion time determination 100x10 3 Countrate (Hz) 95 90 85 80 0 Auto-Correlation Function 2 1.3 1.2 1.1 1.0 4 τ d3d 6 8 10 12 Time (s) Rhodamine drop 14 0.001 0.01 0.1 1 10 100 1000 Time (ms) 16 18 20
BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS RHODAMINE 6G SPECTRA 14 / 21 1.0 Abs. Rhodamine 6G Em. Rhodamine 6G (excitation @480nm) 0.8 Normalized intensity 0.6 0.4 0.2 0.0 400 450 500 550 Wavelenght (nm) 600 650 700 Excitation @488nm : not the max. of absorption Emission : 525±10nm Enough signal because high efficiency
T S P F C -C S R B C : M M : EGFR + F A -A 488 15 / 21 2 XY scan of the sample Mean diffusion time determination : 1 species Slow diffusion (τd1 ) Fast diffusion (τd2 ) but not necessary 6 1 1 G (τ) = 1 + N 1 + τ τd1 Position (µm) Intensity fluctuations measurements : 20 5s for example Z Scan Z scan and choice of 1 point (upper membrane for ex.) x 10 5 0 20 40 60 80 100x103 Countrate (Hz) 3 100x10 S V FCS FCCS Countrate (Hz) 5 y 15 95 90 85 80 0 2 1.20 Auto-Correlation Function 4 Green fluorophore 20 Mean diffusion time Intensity fluctuations determination measurement 3 AntiEGFR Antibody Intracellular domain 30µm Plasma membrane molecule with a labelled anti-egfr Fab antibody XY Scan TransMemb. protein 1 EGF Receptor 4 6 8 10 12 14 16 18 Time (s) 1.15 1.10 1.05 1.00 0.001 0.01 0.1 τ1 d2 10 Time (ms) τ 100 d1 1000 20
BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS MEASUREMENTS ON LIVING CELLS : EGFR + FAB ANTIBODY-ALEXA488 16 / 21 RESULTS PREVIOUSLY OBTAINED ON OUR SETUP 90 EGFR-Alexa488 80 70 Diffusion Time τd (ms) 60 50 40 30 20 10 0 0.0 0.1 0.2 0.3 Spot Area (µm²) 0.4 T0 14.8 ± 3.4 ms Deff 0.45 ± 0.03µm 2 /s
BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS MEASUREMENTS ON LIVING CELLS : EGFR + FAB ANTIBODY-FLUOPROBES 17 / 21 LARGE STOKE SHIFT PROBES Fluoprobes 480XXL (Interchim) : λ exc. : 500nm, λ em. : 630nm Fluoprobes 481XXL (Interchim) : λ exc. : 515nm, λ em. : 650nm AIM 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
SUMMARY THEORETICAL SURVEY 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4
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 PROBLEMS 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
SUMMARY THEORETICAL SURVEY 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4
21 / 21 FCS, SVFCS AND BIOLOGICAL APPLICATIONS D. Madge et al. Phys. Rev. Lett., 29 :705-708, 1972. P. Schwille et al. Biophysics Textbook Online, 2004. L. Wawrezinieck et al. Biophys. J., 49 :4029-4042, 2005. P.-F. Lenne et al. EMBO J., 25 :3245-3256, 2006. K. Bacia et al. Nat. Methods, 3 :83-89, 2006. J. Wenger et al. Biophys. J., 92 :913-919, 2007. R. Lasserre et al. Nat Chem Biol., 4(9) :538-47, 2008. P.-F. Lenne et al. Histochem Cell Biol., 130(5) :795-805, 2008. K. Chakrabandhu et al. Cell Death Differ., 15(12) :1824-37, 2008. C. Eggeling et al. Nature, 457 :1159-1162, 2008. A. Rossin et al. Exp Cell Res, 15 ;316(9) :1513-22, 2010.