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Winfried Wiegraebe, Stowers Institute for Medical Research Dont 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.orgwiw@stowers-institute.org Slide 2 Winfried Wiegraebe, Stowers Institute for Medical Research 2 Dont Waste Photons 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) Slide 3 Winfried Wiegraebe, Stowers Institute for Medical Research 3 Jablonski Diagram: Energy States of Molecule Slide 4 Winfried Wiegraebe, Stowers Institute for Medical Research 4 Components of a Laser Scanning Microscope Laser Detector (Pinhole) Scanner Objective Beam splitter Sample Slide 5 Winfried Wiegraebe, Stowers Institute for Medical Research 5 1-Photon Focal Region Single Photon Excitation (Confocal Microscope) Objective Pinhole Detector Out of Focus excitation Pinhole provides optical sectioning Slide 6 Winfried Wiegraebe, Stowers Institute for Medical Research 6 1-Photon Focal Region Multiphoton Excitation (Nonlinear Excitation, NLO) Objective Detector 2 photons required for excitation No out-of-focus excitation 2-Photon No pinhole required Pinhole Scattered light is detected Slide 7 Winfried Wiegraebe, Stowers Institute for Medical Research 7 Non-Descanned Detection Descanned Detection Pinhole Scanner Sample Non-Descanned Detection No movement of light on detector Light moves on detector Light moves on sample Slide 8 Winfried Wiegraebe, Stowers Institute for Medical Research 8 Absorption and Emission Spectra Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." Nature Methods 2(12): 910-919. Slide 9 Winfried Wiegraebe, Stowers Institute for Medical Research 9 Spectral Detection 32 channel PMT Special grating as dispersive medium Spectral resolution: 10.7 nm Slide 10 Winfried Wiegraebe, Stowers Institute for Medical Research 10 Photo Conversion of KikGR 561nm: 1.1% 488nm: 3.1% (15x 405nm: 2%) Channel Unmixing Danny.mdb/102705-spec-t channel unmix Slide 11 Winfried Wiegraebe, Stowers Institute for Medical Research 11 Fly Larva expressing ELAV-eGFP Plan-Apochromat 20x/0.75 920nm, 75% 32 channel META detector FlyLarva012706.mdb/Flylarvalambda@920unmixedfilter.lsm Slide 12 Winfried Wiegraebe, Stowers Institute for Medical Research 12 Linear Unmixing = a + b GFPYFP Slide 13 Winfried Wiegraebe, Stowers Institute for Medical Research 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 Slide 14 Winfried Wiegraebe, Stowers Institute for Medical Research 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 Slide 15 Winfried Wiegraebe, Stowers Institute for Medical Research 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 Slide 16 Winfried Wiegraebe, Stowers Institute for Medical Research 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 Slide 17 Winfried Wiegraebe, Stowers Institute for Medical Research 17 Excitation Fingerprinting: Fly Larva expressing ELAV-eGFP FlyLarva012706.mdb/Flylarvaexitationseriesfilter.lsm Plan-Apochromat 20x/0.75 Ch2 BP 480-520IR 850 950 nm Excitation fingerprint Slide 18 Winfried Wiegraebe, Stowers Institute for Medical Research 18 Timescales in Fluorescence Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." Nature Methods 2(12): 910-919. Slide 19 Winfried Wiegraebe, Stowers Institute for Medical Research 19 FLIM: Fluorescence Life Time Imaging Time delay between laser pulse and detected photon Number detected photons t Pulsed Laser Dye Molecule Detector Photon Electron Slide 20 Winfried Wiegraebe, Stowers Institute for Medical Research 20 FLIM: Fluorescence Life Time Imaging Time delay between laser pulse and detected photon Number detected photons t Pulsed Laser Dye Molecule Detector Photon Electron Slide 21 Winfried Wiegraebe, Stowers Institute for Medical Research 21 FLIM: Fluorescence Life Time Imaging Time delay between laser pulse and detected photon Number detected photons t Pulsed Laser Dye Molecule Detector Photon Electron Slide 22 Winfried Wiegraebe, Stowers Institute for Medical Research 22 FLIM: Fluorescence Life Time Imaging Time delay between laser pulse and detected photon Number detected photons t Pulsed Laser Dye Molecule Detector Photon Electron Slide 23 Winfried Wiegraebe, Stowers Institute for Medical Research 23 Fluorescence Lifetime Imaging Slide 24 Winfried Wiegraebe, Stowers Institute for Medical Research 24 2PE Fluorescence vs. Second Harmonic Generation (SHG) mouse ovary http://www.drbio.cornell.edu/Infrastructure/NonlinearMicroscopies_WWW/SHG.htm Slide 25 Winfried Wiegraebe, Stowers Institute for Medical Research 25 Fish Scale (Second Harmonic Generation) Sample by Peter Kestler Slide 26 Winfried Wiegraebe, Stowers Institute for Medical Research 26 FLIM: SHG vs. Fluorescence Second Harmonic Generation in Fish Scale Fluorescence Lifetime of Fluorescine Slide 27 Winfried Wiegraebe, Stowers Institute for Medical Research 27 Excitation: 850nm SHG: 425nm C-Apochromat 40x/1.2 W 850nm, 5% 32 channel META Fish scale SHG101805.mdb/FishscaleMETA800nm.lsm Slide 28 Winfried Wiegraebe, Stowers Institute for Medical Research 28 SHG to Measure Membrane Potential Figure 3 Daniel A. Dombeck et al.: J Neurophysiol (August 10, 2005). Slide 29 Winfried Wiegraebe, Stowers Institute for Medical Research 29 Dont Waste Photons 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 Slide 30 Winfried Wiegraebe, Stowers Institute for Medical Research 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