1. Critique (everyone) Monitoring dynamic protein interactions with photoquenching FRET. Nature Methods, vol 3, 519-24 (2006) “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumor” Jain et al, Nature Medicine, vol 10 203-7 (2004) Suggested presentation

2. 1.Campagnola, P.J., et al., High resolution non-linear optical microscopy of living cells by second harmonic generation. Biophys. J., 1999. 77: p. 3341-3349. 2.Campagnola, P.J., et al., 3-Dimesional High-Resolution Second Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues. Biophys. J., 2002. 82: p. 493-508. 3.Moreaux, L., O. Sandre, and J. Mertz, Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B, 2000. 17: p. 1685-1694. Assigned Reading for Next Week

3. Outline: 1)Fluorescence Lifetime Imaging (FLIM) 2) Fluorescence Resonance Energy Transfer (FRET) 3) FRET/FLIM 4) FCS

4. Fluorescence Lifetime motivation 1)Sensitive to environment: pH, ions, potential SNARF, Calcium Green, Cameleons Perform in vitro calibrations 1)Results Not sensitive to bleaching artifacts 2)Not sensitive to uneven staining (unless self-quenched) 3)Not sensitive to alignment (intensity artifacts)

5. Fluorescence Quantum Yield φ: important for dyes Ratio of emitted to absorbed photons Measured lifetime is sum of Rates of natural lifetime and non radiative decay paths (k is rate, Inverse of time) Quantum Yield: Natural lifetime Very fast 1-10 ps

6. Einstein A coefficient A 21 =1/ τ Oscillator strength, f, and fluorescence lifetime τ For band centered at 500 nm, Fully allowed transition has lifetime of 4 ns (for one electron) Dyes has several valence electrons, larger f Lifetimes between 1-4 ns Fluorescent lifetime depends on environment: Used in microscopy as contrast υ=light frequency, m=mass of electron, c=speed of light, e= electron charge

7. Unquenched emission: Normal QY, lifetime Quenched emission Decreased QY, lifetime e.g. metals, aggregation Unquenched and Quenched Emission

8. Gold somewhat quenches the fluorescence

9. 2 general approaches: time domain and frequency domain Short pulse lasermodulate CW laser

10. Frequency Domain Methods for Lifetime Measurements: Modulate laser and measure phase change of fluorescence Use cw laser (e.g. argon ion) Modulate at rate near Inverse of emission lifetime 10-100 MHz Measure phase change with Lockin amplifier

11. Modulation Methods in Frequency Domain Modulate laser and ICCD (intensified CCD camera) Better S/N Modulate laser

12. ICCD Detectors for Lifetime measurements: Frequency domain and some time-domain Needs to be gated rapidly Widefield imaging (no sectioning) High quantum yield Very expensive $80K Regular CCDs:10-20K Historically Most common Microchannel plates Amplify signal ~10 fold

13. Time-domain Widefield Lifetime imaging with ICCD Variable delayed gate or many gates is scanned To sample exponential decay: Many frames (for each delay) ICCD has no time intrinsic response: slow readout Gated gain Two-photon has short pulse laser for time-gating

14. French Ti:sapphire Higher viscosity Shorter lifetime Better chance for Non-radiative decay

15. Time domain methods for lifetime measurements With gated electronics and fast detectors (not gain modulated) Best for point detection, PMT on laser scanning Synchronized Gating done by pulsed laser (e.g. ti:sapphire laser) Collect data from multiple gates (windows) At the same time, fit to exponential

16. PMT Detectors for Lifetime measurements ~300 picosecond resolution Better with deconvolution Cost ~$500 ~30 picosecond resolution No dispersion Cost ~$15000 fragile PMTS have low quantum yield (10-20%), MCP worse ~5% Microchannel plate photomultiplier: full of holes, kick off electrons Dispersion in time of flight across 14 dynodes Limits time response

17. 300-500 picosecond resolution Very small area (200 sq microns) Not good for scanning High quantum yield (up to 70% at 700 nm) Low count rate (~10 MHz) $5K Extremely fragile!! Avalanche Photodiode (APD)

18. Time gating measurements of fluorescence decay Temporal Resolution defined by IRF (laser, detector, electronics) IRF=instrument response function, Must be (much) shorter than fluorescence lifetime (delta function) to avoid convolution Measure IRF with reflection or known short lifetime e.g. Rose Bengal (90 ps) Ideal IRF Real IRF Gate away from IRF (laser pulse, PMT response) Lose photons

19. Practical determinations of the Instrument Response Function 1)Laser modern lasers: ti:sapphires 100 femtosecond Lifetimes: nanoseconds Not a factor Was 20-30 years ago before modelocked lasers 2) Detectors APD or PMT response ~200 picoseconds: can be MCP-PMT 30 ps: not typical limitation 3) TCSPC or gating Electronics 20-50 ps (depending on sophistication) Can be convolved with MCP-PMT response

20. Time-correlated single photon counting: most flexibility, most accurate, samples whole decay Best time response Measures time of flight of photons After excitation pulse Bins data at each time interval Rather than gating Collect enough photons to approximate exponential: Slower than gating but Better measurement, Can separate biexponentials: Multiple components

21. Principles of time-correlated single photon counting TAC or TDC measures time of flight, bins photons Been around For decades

22. Time-Correlated Single Photon Counting electronics On laser scanning microscope (recent) TCSPC electronics synchronized with laser scanning electronics: Pixel, line, frame synch Historically very hard: mostly homebuilt (e.g. Gerritsen)

23. Becker &Hickel addon to Zeiss Laser scanning confocal Electronics all in one PCI board, ~50K addon

24. Intensity vs fluorescence lifetime image Same dye, different lifetime because of environment Quenched close to Nucleus due to Higher concentration Lower lifetime

25. Intensity and lifetime measurements CFP-YFP linked by short peptide chain Energy is transferred from CFP to YFP Lifetime reveals info intensity does not

26. Duncan, J. Microscopy 2004 TCSPC FLIM using ECFP 2 distinct lifetimes: meaning?

27. Performance of Frequency and time domain methods TCSPC best for efficiency, S/N But more expensive (ti:sapphire laser) But already have if have 2-photon microscope

28. Long Acquisition Times for TCSPC FLIM: Need enough data to approximate decay Bright stains 10 6 /s Dim stains 10 4 /s May bleach before done imaging Detection with 2-4 gates may be better if Short on photons need 100-100000 Photons/pixel

29. GFP lifetime increases With increasing viscosity Different lifetime for B cells at immuno Junction with natural killer (NK) cell EGFP::MHC

30. Autofluorescence of Rat Ear Contains collagen, elastin : Single exponential not sufficient for multiple components Fits to two discrete components noisy

31. Continuous lifetime distribution Better for multiple components Mean tau For pixels Width, h, of distribution For pixel Unless know components Stretched exp is better Representative of physiology and provides more data

32. FLIM as Diagnostic of Joint Disorder H&E staining Widefield fluorescence Widefield FLIM Little info Detail revealed by FLIM Fixed, thin sections (few microns)

33. FLIM as Cancer Diagnostic Benign Carcinoma H&E staining Widefield FLIM FLIM shows morphology like H&E histology Can optically section and no staining with FLIM With 2-p can do thick tissues (few hundred microns) More contrast Than H&E Probably NADH, FAD

34. Widefield fluorescence Widefield FLIM FLIM Diagnostics of arterial plaque Clear lifetime Difference in Normal and plaque: Not visible by Fluorescence intensity

35. FLIM via endoscope as clinical tool Works like through microscope

36. Lifetime of NADH, FAD changes from normal To cancer White PNAS 2007

38. Donor Excitation Donor Emission Donor Excitation Acceptor Emission Fluorescence Resonance Energy Transfer (FRET) Donor emission overlaps with Acceptor Absorption: Highly distance dependent

39. FRET probes conformational changes Different conformation gives Different FRET signature

40. FRET increases In both cases Inter and Intramolecular Forms of FRET with Proteins CFP-YFP good combo Protein-Protein Interactions In cytoplasm and membranes

41. No FRET for No overlap of donor emission, acceptor absorption When FRET Occurs No FRET for Orthogonal dipole orientation No FRET for molecules more than 10 nm apart R 0 =distance where FRET=0.5

42. Typical Values of R o DonorAcceptorR o (Å) FluoresceinTetramethylrhodamine55 IAEDANSFluorescein46 EDANSDABCYL33 Fluorescein 44 BODIPY FL 57 FluoresceinQSY 7 dye61 Cy3Cy553 CFPYFP50 greenred GFPs and other colored “FPs have transformed FRET microscopy Before had to label proteins, then introduce

43. Number of FRET Publications since 1989

44. Fluorescence Resonance Energy Transfer - Detection of Probe Proximity R 0 typically 40-50 Angstroms 50% transfer

46. Practical Challenges to FRET Quantitation Emission from A contaminates D channel (filters) Emission from D contaminates A channel Unknown labeling levels for D and A Signal variation due to bleaching –Complicates kinetic studies –Bleaching rate of D can actually be slowed by FRET Solutions : Separately labeled D and A controls to define bleedthrough Acceptor destruction by photobleaching to establish Dual wavelength ratio imaging to normalize away variations in label levels and bleaching effects

48. Fluorescent Proteins as D-A Pairs Issue of Spectral Overlap Better overlap,FRET But more bleedthrough Poor Spectral overlap, But less bleedthrough

49. Survey of FRET-Based Assays Protease activity Calcium Ion measurements cAMP Protein tyrosine kinase activity Phospholipase C activity Protein kinase C activity Membrane potential

50. Principle of Operation of Chameleon Calcium Indicators FRET Increases when CaM binds Calcium ions Conformation changes, CFP-YFP closer together

51. Potential Sensor Based on FRET Mechanism and Single Cells Gonzalez and Tsien, Biophys J., 1995 Demonstration on Leech Ganglion Kleinfeld, et al., Neuron, 1999 Improved indicators Gonzalez JE, Tsien RY. 1997. Chemistry and Biology 4:269-277. Donor= Di4-ANEPPS Fast voltage sensor Acceptor=Oxonol Slow voltage sensor FRET pair more sensitive

52. Lifetime and FRET Large change in lifetime for quenched donor upon FRET FRET should have bi-exponential decay, quenched and unquenched: Long and short lifetime components

53. CFP and YFP FRET by Lifetime Imaging Channel changes conformation, distance changes, Donor quenching occurs due to FRET Short lifetime is FRET from Donor For given pixel Ratio of fast to slow decay coefficients is estimate of FRET efficiency

54. Duncan, J. Microscopy 2004 CFP and YFP tethers FRET by Lifetime Imaging Donor Lifetime goes up post acceptor bleaching

55. FRET Outcomes Donor decreases Acceptor increases Donor lifetime decreases Donor fluorescence Anisotropy increases Acceptor decreases

56. FRET pair anisotropy Donor Anisotropy Increases: shorter Lifetime, less likely to Rotate before emission Extent of depol Contains relative orientation Emission dipole usually Parallel to excitation dipole: FRET to other orientation Depolarizes acceptor emission Not constrained by laser

57. Much better dynamic range Than lifetime based changes ~10x Anisotropy measurement more accurate Piston, BJ 2004

59. Fluctuation (fluorescence) Correlation Spectroscopy (FCS) Fluctuations in excitation volume due to Diffusion, reactions

60. Diffusion times for globular proteins 0 100 200 300 400 500 02000004000006000008000001000000 molecular weight (kD) diffusion time (microseconds) Spherical molecule Diffusion coefficient

61. Diffusion Demonstration http://physioweb.med.uvm.edu/diffusion/FrapPages1.htm

62. Normalized Autocorrelation Function Compares probability of detecting photon at time t with some latter time t + τ Autocorrelation the expected value of the product of a random variable or signal realization with a time-shifted version of itself FCS: autocorrelation of the fluorescence photons of one and the same molecule. FCS decay: diffusion, binding Maximum at tau=0 Small tau: large correlation Large tau: tend to 0

63. Form for translational diffusion N=concentration of molecules in focal volume τDτD =diffusion time, R=ω z /ω xy of observation volume Need to measure Point Spread Function To determine observation volume

65. Determination of Point Spread Function of Microscope Abbe` Limit 175 nm Bead Sub-resolution Volume is Ellipsoid Axial ~NA 2

66. PSF and Beam Waist Imaging sub-resolution 100 nm fluorescent beads Use 1/e 2 points to get ω beam waist (87%)  D =  2 /4D

67. 8D for two-photon

68. webb

69. FCS of Rhodamine in Sucrose Solution Higher concentrations Shorter correlation times 1/y=number of molecules In focal volume webb

70. Molecular interactions FCS is a method to study molecular interactions between – fluorescence-tagged ligands and target molecules such as receptors, – unidentified, untagged compounds and tagged ligands in competitive binding – protein-protein interactions – individual events of signal transduction cascades within cells. Concentration range FCS allows molecular interactions to be characterized over a wide dynamic range -concentrations of labeled particles between 200 nM to 200 pM -can be used. Because of the ultra-low measuring volume of 10 -15 l required for FCS measurement, nanoliter to microliter sample volumes are sufficient.

71. Physical state Differences in the physical state of the molecule of interest, such as bound vs. free, cleaved vs. intact, can be discerned by FCS provided that the variant forms differ sufficiently in their size-related diffusion – peptides bound to soluble receptors, – ligands bound to membrane-anchored receptors, – viruses bound to cells, – antibodies bound to cells, – primers bound to target nucleic acids, – regulatory proteins /protein-complexes in interaction with target DNA or RNA – enzymatic products. If the diffusion properties of the reactants are too similar, both reactants have to be labeled with fluorescent dyes with different excitation and emission spectra.

72. Mathematical model for autocorrelation Two component autocorrelation curve

74. Diffusion of structures > 50 nm may be hindered in cytoplasm tau values > 1 ms may not represent free diffusion Binding to immobile species, e.g. microtubules

75. Binding to mobile receptorBinding to immobile receptorMotility along microtubule Concentration Diffusion of receptor Concentration Kd On rate (M -1 sec -1 ) Off rate (sec -1 ) Mobile/immobile Mean squared displacement The slow component in living cells

76. The slow component for hnRNP A2 (tau = 21 msec) may represent granule movement on microtubules

77. tau 2 = off rate

78. Carson, JMB Need to measure Endogenous, added Protein concentrations Determine taus, Mobile fractions By bleaching

79. fluorescent molecules Cross-correlation spectroscopy 3D Gaussian confocal detection volume ~1 femtoliter diffusion trajectories Individual fluorescent molecules are detected as single channel photon count fluctuations. Bound molecules are detected as coincident dual channel fluctuations. Cross-correlation analysis provides a measure of the number and rate of diffusion of bound molecules. Cross-correlation function G rg (t) = 1 1.02 1.04 1.06 1.08 1.1 10100100010000 microseconds Alexa488 RNA Syto61 cross-correlation Dual channel fluctuation 10000 15000 20000 25000 30000 35000 40000 45000 50000 012345678910 seconds Alexa488 RNA Syto61 Count rate

81. Photon Counting Histogram Same raw data as autocorrelation Measure of probability of brightness at given time Allows determination of dimerization or oligerimization By diffusion, assuming hardspheres Dimer would increase τDτD By only 26%. Cannot measure by FCS However brightness would increase by 2x

82. PCH can determine concentrations Gratton, BJ

83. PCH can differentiate fluorophores based on brightness