Transcript Slide 1
Assigned Reading for Next Week 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. Outline: 1) Fluorescence Lifetime Imaging (FLIM) 2) Fluorescence Resonance Energy Transfer (FRET) 3) FRET/FLIM 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) Fluorescence Quantum Yield φ: important for dyes Ratio of emitted to absorbed photons Quantum Yield: Very fast 1-10 ps kf k f k isc (k is rate, Inverse of time) kf k f k isc k nonrad 0 1 k f Natural lifetime 1 k f kisc Measured lifetime is sum of Rates of natural lifetime and non radiative decay paths Oscillator strength, f, and fluorescence lifetime τ Einstein A coefficient A21=1/τ f 21 m c3 A21 2 2 8o e υ=light frequency, m=mass of electron, c=speed of light, e= electron charge 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 Unquenched and Quenched Emission Unquenched emission: Normal QY, lifetime kf k f k isc Quenched emission Decreased QY, lifetime e.g. metals, aggregation kf k f k isc k nonrad Gold somewhat quenches the fluorescence 2 general approaches: time domain and frequency domain Short pulse laser modulate CW laser 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 (100 to 10 ns) Measure phase change with Lockin amplifier Modulation Methods in Frequency Domain Modulate laser OK for spectroscopy Modulate laser and ICCD (intensified CCD camera) Better S/N for imaging ICCD Detectors for Lifetime measurements: Frequency domain and some time-domain Microchannel plates Amplify signal ~10 fold Needs to be gated rapidly Widefield imaging (no sectioning) High quantum yield Very expensive $20-80K Regular CCDs:10-20K Historically Most common 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. Use laser pulse width much less than fluorescence lifetime Two-photon scope has short pulse laser for time-gating Ti:sapphire Higher viscosity Shorter lifetime Better chance for Non-radiative decay French Optics express Time domain methods for lifetime measurements With gated electronics and fast detectors (not gain modulated) Best for point detection, PMT on laser scanning Collect data from multiple gates (windows) At the same time, fit to exponential Synchronized Gating done by pulsed laser (e.g. ti:sapphire laser) PMT Detectors for Lifetime measurements Dispersion in time of flight across 14 dynodes Limits time response ~300 picosecond resolution Better with deconvolution Cost ~$500 Microchannel plate photomultiplier: full of holes, kick off electrons ~30 picosecond resolution No dispersion Cost ~$15000 fragile PMTS have low quantum yield (10-20%), MCP worse ~5% Avalanche Photodiode (APD) 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!! Time gating measurements of fluorescence decay Temporal Resolution defined by IRF (laser, detector, electronics) Ideal IRF IRF=instrument response function, Must be (much) shorter than fluorescence lifetime (delta function) to avoid convolution Real IRF Gate away from IRF (laser pulse, PMT response) Lose photons Measure IRF with reflection or known short lifetime e.g. Rose Bengal (90 ps) Practical limiting governing 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 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 Principles of time-correlated single photon counting Been around For decades TAC or TDC measures time of flight, bins photons Mark Terasaki Flash animation http://www.terasaki.us/flash/lab/flim01.swf 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) Becker & Hickel addon to Zeiss Laser scanning confocal Electronics all in one PCI board, ~50K addon Intensity vs fluorescence lifetime image Quenched close to Nucleus due to Higher concentration Lower lifetime Same dye, different lifetime because of environment Intensity and lifetime measurements CFP-YFP linked by short peptide chain Energy is transferred from CFP to YFP Lifetime reveals info intensity does not TCSPC FLIM using ECFP 2 distinct lifetimes: meaning? Duncan, J. Microscopy 2004 GFP lifetime increases With increasing viscosity Limits motion, nonrad Different lifetime for B cells at immuno Junction with natural killer (NK) cell EGFP::MHC Performance of Frequency and time domain methods TCSPC best for efficiency, S/N, information content But more expensive (ti:sapphire laser) But already have if have 2-photon microscope Long Acquisition Times for TCSPC FLIM: Need enough data to approximate decay Bright stains 106/s Dim stains 104/s need 100-100000 Photons/pixel May bleach before done imaging Detection with 2-4 gates may be better if Short on photons Autofluorescence of Rat Ear Contains collagen, elastin : Single exponential not sufficient for multiple components Time domain With gated ICCD Fits to two discrete components noisy (large residuals) French, 2001, Biophys J. Continuous lifetime distribution Better for multiple components Mean tau For pixels Unless know components Stretched exp is better Representative of physiology and provides more data Width, h, of distribution For pixel FLIM as Diagnostic of Joint Disorder H&E staining Widefield fluorescence Widefield FLIM Fixed, thin sections (few microns) Little info Detail revealed by FLIM FLIM as Cancer Diagnostic H&E staining Widefield FLIM Benign More contrast Than H&E Carcinoma Probably NADH, FAD 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) FLIM Diagnostics of arterial plaque Widefield auto fluorescence Widefield FLIM Clear lifetime Difference in Normal and plaque: Not visible by Fluorescence intensity FLIM via endoscope as clinical tool Works like through microscope White PNAS 2007 Lifetime of NADH, FAD changes from normal To cancer and high to low-grade Fluorescence Resonance Energy Transfer (FRET) Donor Excitation Donor Excitation Acceptor Emission Donor Emission Donor emission overlaps with Acceptor Absorption: Highly distance dependent FRET probes conformational changes Different conformation gives Different FRET signature Inter and Intramolecular Forms of FRET with Proteins CFP-YFP good combo FRET increases In both cases Protein-Protein Interactions In cytoplasm and membranes When FRET Occurs No FRET for No overlap of donor emission, acceptor absorption No FRET for Orthogonal dipole orientation No FRET for molecules more than 10 nm apart R0=distance where FRET=0.5 Typical Values of Ro green Donor Fluorescein IAEDANS Acceptor Ro (Å) Tetramethylrhodamine 55 Fluorescein 46 EDANS DABCYL Fluorescein Fluorescein BODIPY FL BODIPY FL 33 44 57 Fluorescein Cy3 CFP 61 53 50 QSY 7 dye Cy5 YFP GFPs and other colored “FPs have transformed FRET microscopy Before had to label proteins, then introduce red Number of FRET Publications since 1989 Fluorescence Resonance Energy Transfer Detection of Probe Proximity F FD FA F D FRET Max 0 0 FD FA FA D0 0 D 0 A 0 D R0 typically 40-50 Angstroms 50% transfer 6 0 R FRET 6 R R06 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 0 F • Acceptor destruction by photobleaching to establish D • Dual wavelength ratio imaging to normalize away variations in label levels and bleaching effects Want sharp filters, But throw away photons Fluorescent Proteins as D-A Pairs Issue of Spectral Overlap Better overlap,FRET But more bleedthrough Poor Spectral overlap, But less bleedthrough Survey of FRET-Based Assays • • • • • • • Protease activity Calcium Ion measurements cAMP Protein tyrosine kinase activity Phospholipase C activity Protein kinase C activity Membrane potential Principle of Operation of Chameleon Calcium Indicators FRET Increases when CaM binds Calcium ions Conformation changes, CFP-YFP closer together Potential Sensor Based on FRET Donor= Di4-ANEPPS Fast voltage sensor Acceptor=Oxonol Slow voltage sensor FRET pair more sensitive Mechanism and Single Cells Gonzalez and Tsien, Biophys J., 1995 Improved indicators Gonzalez JE, Tsien RY. 1997. Chemistry and Biology 4:269-277. Demonstration on Leech Ganglion Kleinfeld, et al., Neuron, 1999 Lifetime and FRET Large change in lifetime for quenched donor upon FRET F FD FA F D FRET Max 0 0 0 FD FA FA D 0 D 0 A 0 D FRET should have bi-exponential decay, quenched and unquenched: Short and long lifetime components 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 CFP and YFP tethers FRET by Lifetime Imaging Donor Lifetime goes up post acceptor bleaching Duncan, J. Microscopy 2004 FRET Outcomes Donor decreases Acceptor increases Donor lifetime decreases With FRET: Donor fluorescence Anisotropy increases Acceptor decreases FRET pair anisotropy Donor Anisotropy Increases: shorter Lifetime, less likely to Rotate before emission Emission dipole usually Parallel to excitation dipole: FRET to other orientation Depolarizes acceptor emission Not constrained by laser Extent of depol Contains relative orientation Piston, BJ 2004 Much better dynamic range Than lifetime based changes ~10x Anisotropy measurement more accurate