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Introduction to FCS and FRAP University of Edinburgh, November 2013

Introduction to FCS and FRAP University of Edinburgh, November 2013. Paul McCormick Leica Microsystems. FRAP F luorescence R ecovery A fter P hotobleaching. Application. To measure molecular diffusion and active processes in time. Can be Fast or Slow processes – measured in XY:

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Introduction to FCS and FRAP University of Edinburgh, November 2013

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  1. Introduction to FCS and FRAPUniversity of Edinburgh, November 2013 Paul McCormick Leica Microsystems

  2. FRAP Fluorescence Recovery After Photobleaching

  3. Application To measure molecular diffusion and active processes in time. Can be Fast or Slow processes – measured in XY: Movement and localization of macromolecules in living cell (RNA and protein dynamics in the nucleus, mobility of macromolecular drugs). Molecule trafficking in ER and Golgi

  4. beam FRAP Principle Excitation Bleached Area e.g 488 nm fluorescent molecules Movement of fluorescent molecules into the bleached area (recovery) Complete Recovery

  5. FRAP: Mode of operation • Determination of pre-bleach levels. 2. Photobleaching (short excitation pulse) of selected cells / areas. 3. Recovery: diffusion of unbleached molecules into the bleached area and increase of fluorescence intensity. Record the time course of fluorescence recovery at various time intervals, using a light level sufficiently low to prevent further bleaching. 4. Quantification: graph shows the time course of fluorescence recovery (calculated as average percentage recovery of initial fluorescence).

  6. Experimental Set-up Each fluorophore has different photobleaching characteristics. For FRAP experiments it is important to choose a dye which bleaches minimally at low illumination power (to prevent photobleaching during image acquisition) but bleaches fast and irreversibly at high illumination power. If molecules with rapid kinetics are investigated, advanced features can be necessary for FRAP experiments : • higher laser power to bleach faster (to minimize diffusion during bleaching) • time optimized FRAP modules (switching delays between bleach and postbleach image aquisition should be minimized) • small formats and fast acquisition speed

  7. Experimental Set-up One general consideration in FRAP experiments is to minimize the bleaching during acquisition instead of acquiring “nice” images. The data has to be averaged over the selected area anyway to diminish statistical distributed noise. To minimize photobleaching during acquisition these parameters should be adjusted: • decreasing the pixel resolution by zooming out or by lowering the pixel number (e.g. 128x128 instead of 512x512) • decreasing the pixel dwell time using a faster scan speed (this is also preferable to monitor rapid recovery kinetics) • decreasing the laser power during image acquisition to a minimum • using fluorophores which are less susceptible to photobleaching at low laser intensities • frame or line averaging should be avoided to reduce undesired photobleaching in the imaging mode • opening the pinhole leads to a brighter signal with less laser power

  8. FRAP wizard Bleach tools of Leica for FRAP: • ROI-Scan • Fly Mode • Zoom In ROI

  9. FRAP with LAS AF: Guided Steps of Work

  10. FRAP with LAS AF: Guided Steps of Work

  11. FRAP with LAS AF: Guided Steps of Work

  12. FRAP with LAS AF: Guided Steps of Work

  13. ROI 4 ROI 3 ROI 1 FLIP slow ROI 2 FLIP fast FRAP Reference FRAP-wizard - Analysis of data ROI based

  14. FRAP: mobile fraction vs. immobilefraction in ER Fluorescence recovery after photobleaching A) Plot of fluorescence intensity in a region of interest versus time after photobleaching a fluorescent protein. The prebleach (F i ) is compared with the recovery (F ∞) to calculate the mobile and immobile fractions. Information from the recovery curve (from F o to F ∞) can be used to determine the diffusion constant of the fluorescent protein. B) Cells expressing VSVG–GFP were incubated at 40 °C to retain VSVG–GFP in the endoplasmic reticulum (ER) under control conditions (top panel) or in the presence of tunicamycin (bottom panel). Fluorescence recovery after photobleaching (FRAP) revealed that VSVG–GFP was highly mobile in ER membranes at 40 °C but was immobilized in the presence of tunicamycin. Lippincott-Schwartz, et al. - JUNE 2001 VOLUME 2 www.nature.com/reviews/molcellbio

  15. Data Analysis… For qualitative determination of the recovery dynamics, e.g. to compare differences of one molecule at different conditions, a simple exponential equation can be used as a first approximation: • After determination of τ by fitting the above equation to the recovery curve the corresponding halftime of the recovery can be calculated with the following formula: • If the molecule binds to a slow or immobile macromolecular structure it is very likely that the recovery curve does not fit a single exponential equation. To overcome this problem, a biexponential equation can be used.

  16. FRAP wizards – how to gofaster…. Possibilities to minimize delay of time between bleaching and recovery: •Reduce scan format in y 512 ≥ … ≥ 32 : flexible y formats • Use 1400Hz scan speed • Use bidirectional scan • Wizard minimizes automatically in time, additionally different time scales can added for multistep kinetics (postbleach 2&3). • So e.g.1400Hz bidirectional scan with 256 square format results in 118 msec/frame. • Use FlyMode

  17. FLIP Fluorescence Loss In Photobleaching

  18. FLIP • In this photobleaching technique, loss of fluorescence rather than fluorescence recovery is monitored. • Fluorescence in one area of the cell is repeatedly bleached with high laser power while images of the entire cell are collected with low laser power. • Using FLIP you can measure the dynamics of 2D or 3D molecular mobility. • e.g diffusion, transport or any other kind of movement of fluorescently labeled molecules in living cells. The time course of fluorescence loss is monitored here.

  19. FLIP: What’s around an ROI

  20. FLIP: Quantify Kinetics within the ER

  21. Photoactivation – use the FRAP wizard Photoactivation is a photo-induced alteration of the excitation or emission spectrum of a fluorophore (e.g. fluorescent proteins). PA-GFP: Irradiation at ~400nm results in a 100x increase in fluorescence when excited at 488nm (Patterson et al., 2002, Science, 297:1873-77)

  22. Photoactivation – Principle of PA-GFP

  23. FCSFluorescence Correlation Spectroscopy

  24. What is FCS? Fluorescence Correlation Spectroscopy - FCS • fluorescence based measurement method • analyses the movement of single molecules into and out of a small illuminated observation volume (focus of confocal SP5 – about 0.15-0.2 fl). • The movement of the molecules leads to fluctuations of fluorescence intensity that are analyzed by statistical methods. FCS read out parameter • Mean Number of Molecules => Concentration • Diffusion times => Molecule size, Viscosity • Fraction of components => Bound/free ratio => Kinetic parameters of or chemical reactions => Equilibrium parameters • Triplet and other dark states => Inherent properties of molecules => Environmental parameters (pH, …)

  25. I(t) <I> t G() log  FCS data acquisition and analysis • Beam park at position of interest => Particles moving in and out of confocal volume • Registration of intensity fluctuations • Calculation of correlation function • Fit of corresponding biophysical model to correlation function => Get parameters

  26. Calculation of autocorrelation 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 Photons over time (photon mode data = time between photons) Number of photons in time bin (time mode data)

  27. 0 0 0 0 0 0 1 1 1 1 1 1 2 4 2 2 2 4 4 2 2 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 x  (0) = 20

  28. 0 0 0 0 0 0 1 1 1 1 1 2 2 4 2 2 2 4 2 2 2 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (1) = 17

  29. 0 0 0 0 0 0 1 2 1 1 1 2 2 4 2 2 2 2 2 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (2) = 14

  30. 0 0 0 0 0 0 1 2 1 1 1 2 2 2 2 2 2 2 0 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (3) = 9

  31. 0 0 0 0 0 0 1 2 1 1 1 1 2 2 2 2 2 0 0 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (4) = 5

  32. 0 0 0 0 0 0 1 1 1 1 1 1 2 0 2 2 2 0 0 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (5) = 2

  33. 0 0 0 0 0 0 1 1 1 1 1 0 2 0 2 2 2 0 0 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (6) = 1

  34. 0 0 0 0 0 0 1 0 1 1 1 0 2 0 2 2 2 0 0 2 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 x  (7) = 0

  35. Calculation of autocorrelation • Normalization of correlation function • Logarithmic scale

  36. Results from FCS experiments G() 1/N  1/c log  corr  1/D • Amplitude of fluctuations  concentration • Curve shape  diffusion model • Time of half maximal amplitude  Length of fluctuations  diffusion coefficient of fluorescently labeled molecules

  37. Theoretical approach Properties of the optical system Properties of the diffusion process  = I (r) = ... c (r,t) = ... G() Analytical autocorrelation function  concentration, brightness, diffusion properties of up to 3 species log 

  38. Theoretical approach Properties of the optical system I (r) = ... assuming that the product of the illumination PSF and the detection PSF can be approximated as a 3D Gaussian

  39. Theoretical approach Properties of the diffusion process c (r,t) = ... solving the diffusion equation for different cases: 1D, 2D, 3D diffusion; anomalous/obstructed diffusion; directed motion; confined diffusion; diffusion and binding; intramolecular fluctuations

  40. Model application: Difference in diffusion larger complexes generate longer fluctuations... I(t) I(t) t t ... and rapidly decaying correlation functions ... and slowly decaying correlation functions small molecules generate short fluctuations... G() log 

  41. FCCS: Fluorescence cross correlation Extended concept: • labeling of potential binding partners with spectrally different fluorophores • register intensity fluctuations with two spectrally separated channels • looking for correlations (similarities) between the corresponding signals I(t) No correlation t I(t) Good correlation! t

  42. Calculation of crosscorrelation x x 2 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 2 2 0 2 0 2 2 2 2 2 0 2 1 1 1 1 1 1 1 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 1 1 0 1 1 1 0 1 1 1 1 2 0 0 0 2 0 0 2 0 0 0 0 1 0 1 0 0

  43.  Distinguish bound from unbound state kas + kdis G() The higher the cross correlation amplitude in relation to the autocorrelation amplitudes, the higher the degree of binding. log 

  44. Example: In vitro biochemistry Reactants: Atto590-Biotin, Atto488-anti-Biotin-IgG Goals: Estimate bound fraction and Kd from cross-correlation amplitude Conditions: Ex: 488 nm, 594 nm Em1: 500-550 nm Em2: 607-683 nm sampling rate: 1 MHz IgG structure by Gareth White

  45. In vitro biochemistry

  46. Prepare experiment: Choice of dyesfor covalent labeling • Criteria for suitable dyes: – High photostability. – Low triplet transition rate. – Amino- and/or thiol-reactive derivatives should be available. – Fluorescence lifetime within the lower ns-range (small against diffusion time) • Excitation wavelength criterion: availability of laser line. • Emission wavelength criterion: avoid range of autofluorescence. • Avoid non-specific binding of the dye to buffer components (as BSA, detergents, ...), and the interaction partners, especially the unlabelled partner. Hydrophobic dyes (as rhodamine) tend to bind to chamber surfaces, membranes and proteins. • Regard dependence of photochemical properties of some dyes on measurement conditions as pH, light intensity, …(like GFP depends on pH and light intensity).

  47. Prepare experiment: Choice ofdyes List of FCS suitable dyes: Alexa dyes Molecular Probes Cy dyes Amersham Pharmazia Rhodamin Green, 6G, B, Lissamin Sigma, … EvoBlue Evotec DY dyes Dyomics TAMRA ROX TMR Texas Red - GFP - YFP - RFP (from Roger Tsien) - Cross correlation pair: GFP-RFP

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