Don t waste photons
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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

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


Don t waste photons1

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



Components of a laser scanning microscope
Components of a Laser Scanning Microscope

(Pinhole)

Detector

Laser

Beam splitter

Scanner

Objective

Sample


Single photon excitation confocal microscope
Single Photon Excitation (Confocal Microscope)

1-Photon

Focal

Region

Objective

  • Out of Focus excitation

  • Pinhole provides optical sectioning

Pinhole

Detector


Multiphoton excitation nonlinear excitation nlo
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


Non descanned detection
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


Absorption and emission spectra
Absorption and Emission Spectra

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


Spectral detection
Spectral Detection

  • 32 channel PMT

  • Special grating as dispersive medium

  • Spectral resolution: 10.7 nm


Photo conversion of kikgr
Photo Conversion of KikGR

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


Fly larva expressing elav egfp
Fly Larva expressing ELAV-eGFP

  • Plan-Apochromat 20x/0.75

  • 920nm, 75%

  • 32 channel META detector

FlyLarva012706.mdb/Flylarvalambda@920unmixedfilter.lsm


Linear unmixing

= a ×

+ b ×

Linear Unmixing

GFP

YFP


Linear unmixing fly larva expressing elav egfp
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


Non descanned detector fly antenna expressing elav egfp
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


Channel unmixing fly antenna expressing elav egfp
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


Channel unmixing fly brain expressing elav egfp
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


Excitation fingerprinting fly larva expressing elav egfp
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


Timescales in fluorescence
Timescales in Fluorescence

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


Flim fluorescence life time imaging
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


Flim fluorescence life time imaging1
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


Flim fluorescence life time imaging2
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


Flim fluorescence life time imaging3
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



2pe fluorescence vs second harmonic generation shg
2PE Fluorescence vs. Second Harmonic Generation (SHG)

mouse ovary

http://www.drbio.cornell.edu/Infrastructure/NonlinearMicroscopies_WWW/SHG.htm



Flim shg vs fluorescence
FLIM: SHG vs. Fluorescence

  • Second Harmonic Generation in Fish Scale

  • Fluorescence Lifetime of Fluorescine


Excitation 850nm shg 425nm
Excitation: 850nm → SHG: 425nm

  • C-Apochromat 40x/1.2 W

  • 850nm, 5%

  • 32 channel META

  • Fish scale

SHG101805.mdb/FishscaleMETA800nm.lsm


Shg to measure membrane potential
SHG to Measure Membrane Potential

Figure 3

Daniel A. Dombeck et al.: J Neurophysiol (August 10, 2005).


Don t waste photons2

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


Thanks
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