1. Topics Acoustic Optical Modulator Faster scanning methods Laser trapping Fluorescence lifetime imaging

2. Acousto-optic modulator Bragg Diffraction: sound waves diffract light Can achieve ~90% diffraction efficiency into 1 st order spot RF (100 MHz) on Transducer sets up Acoustic wave in Second crystal, Forms grating 0 th order

3. Condition for Constructive interference: Double-slit Experiment a sinθ = nλ n = 0,  1,  2,  3 … After focusing: d = f λ / a

4. 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)

5. AOTF to select laser line and power (drive frequency and RF power, respectively) Laser line selection

6. Acousto-optic beam deflector Scanning in a confocal microscope: very fast Compared to galvo mirrors ~100 fold (paper next week) Sweep beam by Changing deflection (linearized)

7. Faster Imaging than with two galvos: line scanning + one galvo

8. Linear CCD Slit pinholes Detection on line-scanning microscope

9. Scanning via spinning disk

10. Spinning disk microscopy Uses White light: convenient but very poor light budget

11. Microlens focuses on Pinholes, conjugate To specimen plane CCD detection, Much higher quantum efficiency Than PMT Modern Design

12. Light contamination between adjacent pinholes

13. 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.Light path not efficient (need powerful light source) 2.Fixed pixel size 3.Disk needs to match objective 4.Lose spatial control of excitation field 5.Problem with very thick samples

14. Laser Trapping

16. Light Can Be Bent by Air

17. Dielectric material n > n(surroundings) Force range is in pN

18. How to measure the force? Langevin equation Stochastic force

19. Langevin equation power spectrum

21. Position sensing with Quadrant photodiodes x = [(B+D) - (A+C)] / [A+B+C+D] y = [(A+B) - (C+D] / [A+B+C+D]

23. Direct observation of base-pair stepping by RNA polymerase Nature. 2005 Nov 24;438(7067):460-5 Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM

24. www.bact.wisc.edu/landick/research.htm

25. Simple But low resolution Stepping size per base pair = 3.4 Å

26. The Dumbbell Setup

27. The Concept of Force Clamp

28. Summary: 1. Decouple from stage 2. Helium environment 3. Passive force clamp

30. HOT Holographic Optical Tweezers

31. 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 Lifetime Imaging

32. 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

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

34. 2 general approaches: time domain and frequency domain Short pulse lasermodulate CW laser

35. 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

36. 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

37. 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

38. Principles of time-correlated single photon counting TAC or TDC measures time of flight, bins photons

39. B&H addon to Zeiss Laser scanning confocal Electronics all in one PCI board, ~50K addon

40. 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

41. 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

42. Intensity vs fluorescence lifetime image Same dye, different lifetime because of environment

43. FRET Outcomes Donor decreases Acceptor increases LifetimeIntensity

44. 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

45. FLIM as Diagnostic of Joint Disorder H&E staining Widefield fluorescence Widefield FLIM Little info Detail revealed by FLIM Fixed, thin sections

47. Effects of Pinhole Size

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