1 / 30

Advanced Techniques in Spectral Imaging for Fluorescence Microscopy at Stowers Institute

Explore advanced methodologies in spectral imaging, including Linear Unmixing, Channel Unmixing, and Excitation Fingerprinting, designed to optimize fluorescence microscopy. This seminar at Stowers Institute delves into the intricacies of two-photon excitation, fluorescence lifetime imaging (FLIM), and second harmonic generation (SHG) for enhanced imaging of biological samples. Learn how to efficiently utilize spectral detectors and improve your understanding of the energy states in fluorescent molecules, ensuring you maximize data obtained without wasting photons.

loring
Télécharger la présentation

Advanced Techniques in Spectral Imaging for Fluorescence Microscopy at Stowers Institute

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related