1. Don’t Waste Photons Winfried Wiegraebe
Advanced Instrumentation & Physics
Stowers Institute for Medical Research
1000 East 50th Street, Kansas City, Missouri 64110
USA
Phone: (816)
Fax: (816)

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. 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)
Region
Objective
Out of Focus excitation
Pinhole provides optical sectioning
Pinhole
Detector

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

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):

9. Special grating as dispersive medium Spectral resolution: 10.7 nm
A Team Meeting Marketintroduction LSM 5 PASCAL, LSM 510 SW Rel. 2.5, ConfoCor 2 Stand Alone
Spectral Detection
32 channel PMT
Special grating as dispersive medium
Spectral resolution: 10.7 nm 1

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

11. Fly Larva expressing ELAV-eGFP
Plan-Apochromat 20x/0.75
920nm, 75%
32 channel META detector

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

13. Linear Unmixing: Fly Larva expressing ELAV-eGFP
Autofluorescence
Plan-Apochromat 20x/0.75
920nm, 75%
32 channel META detector
3x3 lowpass

14. Non-Descanned Detector: Fly Antenna expressing ELAV-eGFP
NDD + Transmission:
DIC
NDD2: BP
NDD3: BP
Plan-Neofluoar 40x/1.3 Oil
920nm, 25%
3x3 Lowpass

15. Channel Unmixing: Fly Antenna expressing ELAV-eGFP
Autofluorescence
Plan-Neofluoar 40x/1.3 Oil
920nm, 25%
NDD2: BP
NDD3: BP
3x3 Lowpass

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

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

18. Timescales in Fluorescence
Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." Nature Methods 2(12):

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

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

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

22. FLIM: Fluorescence Life Time Imaging
Pulsed Laser
Dye Molecule
Detector
Photon
Electron
Photon
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

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
SHG mdb/FishscaleMETA800nm.lsm

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

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 ConfoCor 3
Future:
SHG: Zeiss LSM 510 NLO + special detection optics

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