Presentation is loading. Please wait.

Presentation is loading. Please wait.

Winfried Wiegraebe, Stowers Institute for Medical Research Don’t Waste Photons Winfried Wiegraebe Advanced Instrumentation & Physics Stowers Institute.

Similar presentations


Presentation on theme: "Winfried Wiegraebe, Stowers Institute for Medical Research Don’t Waste Photons Winfried Wiegraebe Advanced Instrumentation & Physics Stowers Institute."— Presentation transcript:

1 Winfried Wiegraebe, Stowers Institute for Medical Research 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.orgwiw@stowers-institute.org

2 Winfried Wiegraebe, Stowers Institute for Medical Research 2 Don’t 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)

3 Winfried Wiegraebe, Stowers Institute for Medical Research 3 Jablonski Diagram: Energy States of Molecule

4 Winfried Wiegraebe, Stowers Institute for Medical Research 4 Components of a Laser Scanning Microscope Laser Detector (Pinhole) Scanner Objective Beam splitter Sample

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

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

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

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.

9 Winfried Wiegraebe, Stowers Institute for Medical Research 9 Spectral Detection  32 channel PMT  Special grating as dispersive medium  Spectral resolution: 10.7 nm

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

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

12 Winfried Wiegraebe, Stowers Institute for Medical Research 12 Linear Unmixing = a × + b × GFPYFP

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

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

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

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

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

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.

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 Δ t Pulsed Laser Dye Molecule Detector Photon Electron

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 Δ t Pulsed Laser Dye Molecule Detector Photon Electron

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 Δ t Pulsed Laser Dye Molecule Detector Photon Electron

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 Δ t Pulsed Laser Dye Molecule Detector Photon Electron

23 Winfried Wiegraebe, Stowers Institute for Medical Research 23 Fluorescence Lifetime Imaging

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

25 Winfried Wiegraebe, Stowers Institute for Medical Research 25 Fish Scale (Second Harmonic Generation) Sample by Peter Kestler

26 Winfried Wiegraebe, Stowers Institute for Medical Research 26 FLIM: SHG vs. Fluorescence  Second Harmonic Generation in Fish Scale  Fluorescence Lifetime of Fluorescine

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

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).

29 Winfried Wiegraebe, Stowers Institute for Medical Research 29 Don’t 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

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


Download ppt "Winfried Wiegraebe, Stowers Institute for Medical Research Don’t Waste Photons Winfried Wiegraebe Advanced Instrumentation & Physics Stowers Institute."

Similar presentations


Ads by Google