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Don’t Waste Photons

Winfried Wiegraebe Advanced Instrumentation & Physics Stowers Institute for Medical Research 1000 East 50 th Street, Kansas City, Missouri 64110 USA Phone: (816) 926-4415 Fax: (816) 926-2088 Email: wiw@stowers-institute.org. Don’t Waste Photons.

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Don’t Waste Photons

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  1. Winfried Wiegraebe Advanced Instrumentation & Physics Stowers Institute for Medical Research 1000 East 50th Street, Kansas City, Missouri 64110 USA Phone: (816) 926-4415 Fax: (816) 926-2088 Email: wiw@stowers-institute.org Don’t Waste Photons

  2. Spectral Imaging: Learn more about your flurochrome Linear Unmixing: Separate overlapping emissions Channel Unmixing: if you can not use a spectral detector Excitation Fingerprinting: Optimize NLO imaging FLIM: Fluorescence Lifetime to distinguish between dyes SHG: Second Harmonic Generation to measure membrane potential FCS: Fluorescence Correlation Spectroscopy – Probe fluctuations to measure diffusion, concentration and interaction (next Technology & Methods Seminar) Don’t Waste Photons

  3. Jablonski Diagram: Energy States of Molecule

  4. Components of a Laser Scanning Microscope (Pinhole) Detector Laser Beam splitter Scanner Objective Sample

  5. Single Photon Excitation (Confocal Microscope) 1-Photon Focal Region Objective • Out of Focus excitation • Pinhole provides optical sectioning Pinhole Detector

  6. Multiphoton Excitation (Nonlinear Excitation, NLO) 2-Photon 1-Photon Focal Region • 2 photons required for excitation Objective • No out-of-focus excitation • No pinhole required Pinhole • Scattered light is detected Detector

  7. Non-Descanned Detection Pinhole Descanned Detection • No movement of light on detector Scanner Non-Descanned Detection Sample • Light moves on detector • Light moves on sample

  8. Absorption and Emission Spectra Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." Nature Methods 2(12): 910-919.

  9. Spectral Detection • 32 channel PMT • Special grating as dispersive medium • Spectral resolution: 10.7 nm

  10. Photo Conversion of KikGR 561nm: 1.1% 488nm: 3.1% (15x 405nm: 2%) Channel UnmixingDanny.mdb/102705-spec-t channel unmix

  11. Fly Larva expressing ELAV-eGFP • Plan-Apochromat 20x/0.75 • 920nm, 75% • 32 channel META detector FlyLarva012706.mdb/Flylarvalambda@920unmixedfilter.lsm

  12. = a × + b × Linear Unmixing GFP YFP

  13. Linear Unmixing: Fly Larva expressing ELAV-eGFP • Linear unmixing: • eGFP • Autofluorescence • Plan-Apochromat 20x/0.75 • 920nm, 75% • 32 channel META detector • 3x3 lowpass FlyLarva012706.mdb/Flylarvalambda@920unmixedfilter.lsm

  14. Non-Descanned Detector: Fly Antenna expressing ELAV-eGFP • NDD + Transmission: • DIC • NDD2: BP 575-640 • NDD3: BP 500-550 • Plan-Neofluoar 40x/1.3 Oil • 920nm, 25% • 3x3 Lowpass Fly01306.mdb/2ndAntenna@9202channelHBOoff0.lsm

  15. Channel Unmixing: Fly Antenna expressing ELAV-eGFP • Channel Unmixing: • eGFP • Autofluorescence • Plan-Neofluoar 40x/1.3 Oil • 920nm, 25% • NDD2: BP 575-640 • NDD3: BP 500-550 • 3x3 Lowpass Fly01306.mdb/2ndAntenna@9202channelHBOoff2.lsm

  16. Channel Unmixing: Fly Brain expressing ELAV-eGFP • Channel Unmixing: • eGFP • Autofluorescence • Transmitted • Plan-Apochromat 10x/0.45 • 920nm, 32% • NDD2: BP 575-640 • NDD3: BP 500-550

  17. Excitation Fingerprinting: Fly Larva expressing ELAV-eGFP • Plan-Apochromat 20x/0.75 • Ch2 BP 480-520IR • 850 – 950 nm • Excitation fingerprint FlyLarva012706.mdb/Flylarvaexitationseriesfilter.lsm

  18. Timescales in Fluorescence Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." Nature Methods2(12): 910-919.

  19. FLIM: Fluorescence Life Time Imaging Pulsed Laser Detector Dye Molecule Photon Photon Electron Δ t Number detected photons Time delay between laser pulse and detected photon

  20. FLIM: Fluorescence Life Time Imaging Pulsed Laser Detector Dye Molecule Photon Photon Electron Δ t Number detected photons Time delay between laser pulse and detected photon

  21. FLIM: Fluorescence Life Time Imaging Pulsed Laser Detector Dye Molecule Photon Photon Electron Δ t Number detected photons Time delay between laser pulse and detected photon

  22. FLIM: Fluorescence Life Time Imaging Pulsed Laser Detector Dye Molecule Photon Photon Electron Δ t Number detected photons Time delay between laser pulse and detected photon

  23. Fluorescence Lifetime Imaging

  24. 2PE Fluorescence vs. Second Harmonic Generation (SHG) mouse ovary http://www.drbio.cornell.edu/Infrastructure/NonlinearMicroscopies_WWW/SHG.htm

  25. Fish Scale (Second Harmonic Generation) Sample by Peter Kestler

  26. FLIM: SHG vs. Fluorescence • Second Harmonic Generation in Fish Scale • Fluorescence Lifetime of Fluorescine

  27. Excitation: 850nm → SHG: 425nm • C-Apochromat 40x/1.2 W • 850nm, 5% • 32 channel META • Fish scale SHG101805.mdb/FishscaleMETA800nm.lsm

  28. SHG to Measure Membrane Potential Figure 3 Daniel A. Dombeck et al.: J Neurophysiol (August 10, 2005).

  29. Available: Spectral Imaging: Zeiss LSM 510 META, Leica SP Linear Unmixing: Zeiss LSM 510 META Channel Unmixing: all multi-channel systems Excitation Fingerprinting: Zeiss LSM 510 NLO Special applications: FLIM: Zeiss LSM 510 NLO + B&H FLIM FCS: Zeiss LSM 510 + ConfoCor 3 Future: SHG: Zeiss LSM 510 NLO + special detection optics Don’t Waste Photons

  30. Thanks! • Adv. Instrumentation & Physics • Joseph Huff • Whitney Bartlow • Imaging Facility • Paul Kulesa • Joel Schwartz • Cameron Cooper • Sarah Smith • Danny Stark • Jessica Teddy • Kausik Si • Jeffrey Cotitta, Lisa Sandell, Paul Trainor

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