Alexei GRICHINE, Mathieu FALLET & Sébastien MAILFERT. October, 2010

Transcript

1 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

2 SUMMARY 2 / 21 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4

3 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

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)

5 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 37 C Living cells High spatio-temporal resolution (µs to s, 200 to 400 nm)

6 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 Rate (Hz) Count Time (ms) Time (s) G(τ) = < δf(t)δf(t + τ) > < F(t) > 2

7 GENERAL PURPOSE FLUORESCENCE CORRELATION SPECTROSCOPY LIMITATIONS PRINCIPLE 6 / 21 Quite good N evaluation, not too much points for τ evaluation!

8 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

9 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

10 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

11 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

12 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

13 BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS BIOLOGICAL SAMPLES 12 / 21 ADHERENT CELLS to cells per well on 8 wells Labtek (here we use COS7 cells) in DMEM culture medium Cells must grow one night to be really 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 RT Wash 3 times in HBSS (with Ca2+) + HEPES

14 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) = n T e τ 1 T ( 1 + τ ) ( ) 1 + s2 τ τ d3d τ d3d 6 Waist evaluation w xy = 4Dτ d3d = τ d3d Confocal Spot Intensity fluctuations measurement Mean diffusion time determination 100x10 3 Countrate (Hz) Auto-Correlation Function τ d3d Time (s) Rhodamine drop Time (ms)

15 BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS RHODAMINE 6G SPECTRA 14 / Abs. Rhodamine 6G Em. Rhodamine 6G 0.8 Normalized intensity Wavelenght (nm) : not the max. of absorption Emission : 525±10nm Enough signal because high efficiency

16 T S P F C -C S R B C : M M : EGFR + F A -A / 21 2 XY scan of the sample Mean diffusion time determination : 1 species Slow diffusion (τd1 ) Fast diffusion (τd2 ) but not necessary 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 x103 Countrate (Hz) 3 100x10 S V FCS FCCS Countrate (Hz) 5 y 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 Time (s) τ1 d2 10 Time (ms) τ 100 d

17 BIOLOGICAL SAMPLES CALIBRATION : WAIST EVALUATION MEASUREMENTS ON LIVING CELLS MEASUREMENTS ON LIVING CELLS : EGFR + FAB ANTIBODY-ALEXA / 21 RESULTS PREVIOUSLY OBTAINED ON OUR SETUP 90 EGFR-Alexa Diffusion Time τd (ms) Spot Area (µm²) 0.4 T ± 3.4 ms Deff 0.45 ± 0.03µm 2 /s

18 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 488nm and 2 detectors ( egfp and Cy5 channels) No alignment problems but ratio of fluorescence emissions difficult to manage

19 SUMMARY THEORETICAL SURVEY 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4

20 FCCS 19 / 21 This setup is based on 2 lasers and 2 channels One dye & one dye 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

21 SUMMARY THEORETICAL SURVEY 1 THEORETICAL SURVEY General Purpose Fluorescence Correlation Spectroscopy Limitations 2 Biological samples Calibration : waist evaluation Measurements on living cells 3 4

22 21 / 21 FCS, SVFCS AND BIOLOGICAL APPLICATIONS D. Madge et al. Phys. Rev. Lett., 29 : , P. Schwille et al. Biophysics Textbook Online, L. Wawrezinieck et al. Biophys. J., 49 : , P.-F. Lenne et al. EMBO J., 25 : , K. Bacia et al. Nat. Methods, 3 :83-89, J. Wenger et al. Biophys. J., 92 : , R. Lasserre et al. Nat Chem Biol., 4(9) :538-47, P.-F. Lenne et al. Histochem Cell Biol., 130(5) : , K. Chakrabandhu et al. Cell Death Differ., 15(12) : , C. Eggeling et al. Nature, 457 : , A. Rossin et al. Exp Cell Res, 15 ;316(9) : , 2010.