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 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)

Jablonski Diagram: Energy States of Molecule

Components of a Laser Scanning Microscope (Pinhole) Detector Laser Beam splitter Scanner Objective Sample

Single Photon Excitation (Confocal Microscope) Region Objective Out of Focus excitation Pinhole provides optical sectioning Pinhole Detector

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

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

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

Photo Conversion of KikGR 561nm: 1.1% 488nm: 3.1% (15x 405nm: 2%) Channel Unmixing Danny.mdb/102705-spec-t channel unmix

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 × GFP YFP

Linear Unmixing: Fly Larva expressing ELAV-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 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 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 Autofluorescence Transmitted Plan-Apochromat 10x/0.45 920nm, 32% NDD2: BP 575-640 NDD3: BP 500-550

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

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

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

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

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

Fluorescence Lifetime Imaging

2PE Fluorescence vs. Second Harmonic Generation (SHG) mouse ovary http://www.drbio.cornell.edu/Infrastructure/NonlinearMicroscopies_WWW/SHG.htm

Fish Scale (Second Harmonic Generation) Sample by Peter Kestler

FLIM: SHG vs. Fluorescence Second Harmonic Generation in Fish Scale Fluorescence Lifetime of Fluorescine

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 Figure 3 Daniel A. Dombeck et al.: J Neurophysiol (August 10, 2005).

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

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