1. www.confocal-microscopy.com Living up to Life Introduction to FCS and FRAP University of Edinburgh, November 2013 Paul McCormick Leica Microsystems

2. www.confocal-microscopy.com Living up to Life FRAP Fluorescence Recovery After Photobleaching

3. www.confocal-microscopy.com Living up to Life 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. www.confocal-microscopy.com Living up to Life Excitation Bleached Area Movement of fluorescent molecules into the bleached area (recovery) Complete Recovery fluorescent molecules beam e.g 488 nm FRAP Principle

5. www.confocal-microscopy.com Living up to Life FRAP: Mode of operation 1.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. www.confocal-microscopy.com Living up to Life 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. www.confocal-microscopy.com Living up to Life 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 Experimental Set-up

8. www.confocal-microscopy.com Living up to Life FRAP wizard Bleach tools of Leica for FRAP: ROI-Scan Fly Mode Zoom In ROI

9. www.confocal-microscopy.com Living up to Life FRAP with LAS AF: Guided Steps of Work

10. www.confocal-microscopy.com Living up to Life FRAP with LAS AF: Guided Steps of Work

11. www.confocal-microscopy.com Living up to Life FRAP with LAS AF: Guided Steps of Work

12. www.confocal-microscopy.com Living up to Life FRAP with LAS AF: Guided Steps of Work

13. www.confocal-microscopy.com Living up to Life FRAP-wizard - Analysis of data FLIP slow FLIP fast FRAP Reference ROI 2 ROI 1 ROI 3 ROI 4 ROI based

14. www.confocal-microscopy.com Living up to Life FRAP: mobile fraction vs. immobile fraction 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. www.confocal-microscopy.com Living up to Life

16. www.confocal-microscopy.com Living up to Life

17. www.confocal-microscopy.com Living up to Life 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.

18. www.confocal-microscopy.com Living up to Life FRAP wizards – how to go faster…. 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

19. www.confocal-microscopy.com Living up to Life

20. www.confocal-microscopy.com Living up to Life FLIP Fluorescence Loss In Photobleaching

21. www.confocal-microscopy.com Living up to Life 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.

22. www.confocal-microscopy.com Living up to Life FLIP: What’s around an ROI

23. www.confocal-microscopy.com Living up to Life FLIP: Quantify Kinetics within the ER

24. www.confocal-microscopy.com Living up to Life 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) Photoactivation – use the FRAP wizard

25. www.confocal-microscopy.com Living up to Life Photoactivation – Principle of PA-GFP

26. www.confocal-microscopy.com Living up to Life FCS Fluorescence Correlation Spectroscopy

27. www.confocal-microscopy.com Living up to Life Why do we need FCS? Problem in life science: Living systems are highly complex, investigation complicated The approach in current biology research: Identification and precise, numerical characterization of basic processes on the level of individual biomolecules, like proteins, amino acids, lipids, second messengers Therefore a technique is required that works on single molecule level, in natural environment, is non-invasive (physiological measurement conditions) Solution: Single molecule detection such as FCS

28. www.confocal-microscopy.com Living up to Life 28 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, …) What is FCS?

29. www.confocal-microscopy.com Living up to Life 29 Kuschel FCS data acquisition and analysis I(t)I(t) t G()G() log  1.Beam park at position of interest => Particles moving in and out of confocal volume 2.Registration of intensity fluctuations 3.Calculation of correlation function 4.Fit of corresponding biophysical model to correlation function => Get parameters

30. www.confocal-microscopy.com Living up to Life Calculation of autocorrelation 0011222110000011110000 Photons over time (photon mode data = time between photons) Number of photons in time bin (time mode data)

31. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0011444110000011110000  (  0 ) = 20

32. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0012442100000011100000  (  1 ) = 17

33. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0022422000000011000000  (  2 ) = 14

34. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0022220000000010000000  (  3 ) = 9

35. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0021200000000000000000  (  4 ) = 5

36. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0011000000000000000000  (  5 ) = 2

37. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0010000000000000000000  (  6 ) = 1

38. www.confocal-microscopy.com Living up to Life 0011222110000011110000 0011222110000011110000 x 0000000000000000000000  (  7 ) = 0

39. www.confocal-microscopy.com Living up to Life Calculation of autocorrelation  Normalization of correlation function  Logarithmic scale 

40. www.confocal-microscopy.com Living up to Life Results from FCS experiments Amplitude of fluctuations  concentration Curve shape  diffusion model Time of half maximal amplitude  Length of fluctuations  diffusion coefficient of fluorescently labeled molecules G()G() 1/N  1/c  corr  1/D log 

41. www.confocal-microscopy.com Living up to Life Theoretical approach G()G() c (r,t) =... I (r) =...  Properties of the diffusion process Properties of the optical system = Analytical autocorrelation function  concentration, brightness, diffusion properties of up to 3 species log 

42. www.confocal-microscopy.com Living up to Life Theoretical approach I (r) =... Properties of the optical system assuming that the product of the illumination PSF and the detection PSF can be approximated as a 3D Gaussian

43. www.confocal-microscopy.com Living up to Life c (r,t) =... Properties of the diffusion process solving the diffusion equation for different cases: 1D, 2D, 3D diffusion; anomalous/obstructed diffusion; directed motion; confined diffusion; diffusion and binding; intramolecular fluctuations Theoretical approach

44. www.confocal-microscopy.com Living up to Life Model application: Difference in diffusion I(t)I(t) t small molecules generate short fluctuations... G()G() log ... and rapidly decaying correlation functions t I(t)I(t) larger complexes generate longer fluctuations...... and slowly decaying correlation functions

45. www.confocal-microscopy.com Living up to Life 45 FCCS: Fluorescence cross correlation I(t)I(t) t I(t)I(t) t 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 No correlation Good correlation!

46. www.confocal-microscopy.com Living up to Life Calculation of crosscorrelation 0011222110000011110000 0011222110000011110000 x 0011222110000011110000 2000000111100000022211 x

47. www.confocal-microscopy.com Living up to Life 47 Kuschel Distinguish bound from unbound state +   k as k dis G()G() log  The higher the cross correlation amplitude in relation to the autocorrelation amplitudes, the higher the degree of binding.

48. www.confocal-microscopy.com Living up to Life Example: In vitro biochemistry Reactants: Atto590-Biotin, Atto488- anti-Biotin-IgG Goals: Estimate bound fraction and K d from cross-correlation amplitude Conditions: –Ex: 488 nm, 594 nm –Em 1 : 500-550 nm –Em 2 : 607-683 nm –sampling rate: 1 MHz IgG structure by Gareth White

49. www.confocal-microscopy.com Living up to Life In vitro biochemistry

50. www.confocal-microscopy.com Living up to Life Prepare experiment: Choice of dyes for 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).

51. www.confocal-microscopy.com Living up to Life Prepare experiment: Choice of dyes 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

52. www.confocal-microscopy.com Living up to Life Leica FCS: the optics avalanche photodiodes for photon counting TCS SP5 AOBS scanner with X1 extension port FCS adaption for X1 port

53. www.confocal-microscopy.com Living up to Life Leica FCS Setup FCS control unit FCS adaption for X1 port filter block

54. www.confocal-microscopy.com Living up to Life Coverslip Correction Not correctedCorrected HCX PL APO 63x/1.2W Corr CS water immersion lens with correction collar

55. www.confocal-microscopy.com Living up to Life Leica FCS – FCS Wizard Overview

56. www.confocal-microscopy.com Living up to Life Beam Park Calibration

57. www.confocal-microscopy.com Living up to Life Measurement in solution – Determine differences in diffusion time Free dye: Alexa488,  D = 33µs FAM-Labeled DNA, 27 bp,  D =153µs Increase in mass => increase in diffusion time => right shift of the curve

58. www.confocal-microscopy.com Living up to Life Measurement of EYFP in living cells FCS measurement spot HeLa cells expressing pure EYFP which is expected to be freely mobile (Cells courtesy T. A. Knoch, K. Rippe, German Cancer Research Center and KIP, University of Heidelberg)

59. www.confocal-microscopy.com Living up to Life Results: Autocorrelation function of free EYFP in HeLa cell nucleus: ~45 molecules in the focus, concentration of ~60 nM for focal size of 0.15 fl Diffusion correlation time ~500  sec, i.e., diffusion coefficient of ~30  m 2 /sec, i.e., viscosity ~3fold higher than in water Measurement of EYFP in living cells

60. www.confocal-microscopy.com Living up to Life 0.0 sec 0.1 sec 0.2 sec 0.3 sec 0.4 sec Diffusion in the nucleus Molecules start at red spot: Covered area after x seconds Example: Measurement of EYFP in living cells

61. www.confocal-microscopy.com Living up to Life Comparison between confocal imaging and FCS

62. www.confocal-microscopy.com Living up to Life Summary: the method Observation of mobile, fluorescent particles Concentration range: >100 pM, <1 μM Dynamic range: >1 μsec, <1 sec Spatial resolution like the confocal microscope Sensitivity down to single molecule level Specificity Not imaging or scanning method

63. www.confocal-microscopy.com Living up to Life Summary: applications Measurement of absolute concentrations at well-defined positions (e.g. location and rate of expression,...) Binding studies: reaction kinetics, equilibrium constants Transport/diffusion (active/passive, restricted/free, directed/non-directed) Aggregation Conformational changes, environmental sensor (e.g. pH) Biophysical parameters (e.g. membrane phases, viscosity,... ) Mechanics/dynamics of cellular structures

64. www.confocal-microscopy.com Living up to Life Thank you….. Any questions?