Transcript Slide 1
Topics • • • • Acoustic Optical Modulator Faster scanning methods Laser trapping Fluorescence lifetime imaging Acousto-optic modulator 0th order RF (100 MHz) on Transducer sets up Acoustic wave in Second crystal, Forms grating Bragg Diffraction: sound waves diffract light Can achieve ~90% diffraction efficiency into 1st order spot Double-slit Experiment Condition for Constructive interference: a sinθ = nλ n = 0, 1, 2, 3 … After focusing: d=fλ/a Applications of Acousto-optic Modulators in microscopes 1) Select Wavelength (tunable filter AOTF): vary drive frequency: Achieve same angle of deflection (wavelength dependent, spacing of grating) 2) Control Laser Power: vary RF power to change diffraction fraction 3) Control Beam angle for fast scanning: vary frequency for same , fixed power (achieves different deflected angle) Laser line selection AOTF to select laser line and power (drive frequency and RF power, respectively) Acousto-optic beam deflector Sweep beam by Changing deflection (linearized) Scanning in a confocal microscope: very fast Compared to galvo mirrors ~100 fold (paper next week) Faster Imaging than with two galvos: line scanning + one galvo Detection on line-scanning microscope Slit pinholes Linear CCD Scanning via spinning disk Spinning disk microscopy Uses White light: convenient but very poor light budget Modern Design Microlens focuses on Pinholes, conjugate To specimen plane CCD detection, Much higher quantum efficiency Than PMT Light contamination between adjacent pinholes Spinning disk microscopy Advantages: 1. Can image very rapidly ( image collection not limited by scanning mirrors 2. Use of cooled CCD camera yields lower noise than PMT (un-cooled) higher quantum yield Disadvantages: 1. 2. 3. 4. 5. Light path not efficient (need powerful light source) Fixed pixel size Disk needs to match objective Lose spatial control of excitation field Problem with very thick samples Laser Trapping Light Can Be Bent by Air • Dielectric material • n > n(surroundings) • Force range is in pN How to measure the force? Stochastic force Langevin equation F ( x) x x Langevin equation F ( x) x x k BT power spectrum S ( f ) 2 2 ( f f c2 ) Position sensing with Quadrant photodiodes x = [(B+D) - (A+C)] / [A+B+C+D] y = [(A+B) - (C+D] / [A+B+C+D] Direct observation of base-pair stepping by RNA polymerase Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM Nature. 2005 Nov 24;438(7067):460-5 www.bact.wisc.edu/landick/research.htm Simple But low resolution Stepping size per base pair = 3.4 Å The Dumbbell Setup The Concept of Force Clamp Summary: 1. Decouple from stage 2. Helium environment 3. Passive force clamp HOT Holographic Optical Tweezers Fluorescence Lifetime Imaging Sensitive to environment: pH, ions, potential SNARF, Calcium Green, Cameleons Perform in vitro calibrations • Results Not sensitive to bleaching artifacts • Not sensitive to uneven staining (unless self-quenched) • Not sensitive to alignment (intensity artifacts) Fluorescence Quantum Yield φ: important for dyes Ratio of emitted to absorbed photons Quantum Yield: 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 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 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 Measure phase change with Lockin amplifier Time-domain Widefield Lifetime imaging with ICCD Variable delayed gate is scanned To sample exponential decay: Many frames ICCD has no time intrinsic response: slow readout Gated gain Two-photon has short pulse laser for time-gating 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 TAC or TDC measures time of flight, bins photons B&H addon to Zeiss Laser scanning confocal Electronics all in one PCI board, ~50K addon 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) 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% Intensity vs fluorescence lifetime image Same dye, different lifetime because of environment FRET Outcomes Donor decreases Acceptor increases Intensity Lifetime 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 FLIM as Diagnostic of Joint Disorder H&E staining Fixed, thin sections Widefield fluorescence Little info Widefield FLIM Detail revealed by FLIM Effects of Pinhole Size Intensity and lifetime measurements CFP-YFP linked by short peptide chain Energy is transferred from CFP to YFP Lifetime reveals info intensity does not