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M F B 2010 : W #45 S V F 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 Spot Variation FCS and FCCS studies Theoretical Survey Practice Fluorescence Cross-Correlation Spectroscopy References Summary 2 / 21 1 Theoretical Survey General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Practice Biological samples Calibration : waist evaluation Measurements on living cells 3 Fluorescence Cross-Correlation Spectroscopy 4 References Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Summary 1 Theoretical Survey General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Practice Biological samples Calibration : waist evaluation Measurements on living cells 3 Fluorescence Cross-Correlation Spectroscopy 4 References Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References 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) Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Conventional FCS 5 / 21 ωz ωxy Confocal measurement Advantages B Fluorescence fluctuations analysis B Low excitation power B Confocal spot : !xy 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) Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Principle 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 Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Principle 6 / 21 Quite good N evaluation, not too much points for τ evaluation ! Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Optical Setup 7 / 21 Spot Variation FCS Setup Sample Diaphragm: Water Variable spot Immersion size FCS Objective Dichroïc Mirror Argon Optical Objective Laser Fiber Pinhole Fluorescence Filter Avalanche Photodiode Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Spot Variation FCS 8 / 21 Diffusion law concept increasing focal spot size longer diffusion time d τ e m i t n o i s u “FCS diffusion law” f f i d spot area Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Spot Variation FCS 9 / 21 Diffusion : experimental results & computer simulation Experimental Results Simulation Results Trapping in meshwork GFP (like TfR) Mean Diffusion Time (Td) HO 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 Spot Variation FCS and FCCS studies Theoretical Survey General Purpose Practice Fluorescence Correlation Spectroscopy Fluorescence Cross-Correlation Spectroscopy Limitations References Limitations 10 / 21 FCS limitations • 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 Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References Summary 1 Theoretical Survey General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Practice Biological samples Calibration : waist evaluation Measurements on living cells 3 Fluorescence Cross-Correlation Spectroscopy 4 References Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References 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 Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References Calibration on known solution : spot size evaluation 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 0 1 0 τ 1 B C 1.3 B C B − C B 1 C B τ C B C 1.2 τ G(τ) = 1 + B1 + nT e T C B ! ! C d3D @B AC B τ s2τ C @B 1 + 1 + AC 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) Waistp evaluation p wxy = 4Dτd3D = 4 × 280 × τd3D Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References Rhodamine 6G spectra 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 Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References Measurements on living cells : EGFR + Fab Antibody-Alexa488 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 0 1 Time (s) 1.20 B C fluctuationsIntensity 1 B 1 C 6 ( ) = + B C 1.15 G τ 1 B τ C N B 1 + C @ A 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) Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References Measurements on living cells : EGFR + Fab Antibody-Alexa488 16 / 21 Results previously obtained on our setup 90 EGFR-Alexa488 80 70 60 (ms) d τ 50 40 30 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 Spot Variation FCS and FCCS studies Theoretical Survey Biological samples Practice Calibration : waist evaluation Fluorescence Cross-Correlation Spectroscopy Measurements on living cells References 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
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