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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: [email protected] 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: [email protected]

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/[email protected]

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/[email protected]

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/[email protected]

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/[email protected]

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!
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