Department of Biomedical Engineering, Yale University, New Haven, CT

1. Multiphoton Microscopy Michael J. Levene Department of Biomedical Engineering, Yale University, New Haven, CT

2. Multiphoton microscopy is a powerful tool True “Molecular Imaging,” with single-molecule sensitivity Wealth of indicators capable of specific targeting -Conventional dyes -GFPs -Intrinsic fluorescence & second harmonic generation Sub-micron resolution Optical sectioning in thick, turbid media Wide variety of biological and clinical applications -Gene expression -Protein interactions -Calcium concentrations -Neural activity -Disease diagnosis -Optical biopsy

3. Two Photon Excited Florescence Two photons can interact simultaneously with a molecule adding their energies to produce an excitation equal to the sum of their individual energies. i.e. 2 red photons can = 1 blue photon 1 photon excitation 2 photon excitation Fluorescence Increasing Wavelength Increasing Energy

4. Two Photon Excitation is Spatially Localized Single photon excitation (488 nm) Two photon excitation (900 nm) Because two photons arriving at the same time are required for excitation the emission depends on the square of the intensity, rather then being linearly proportional. Relative Fluorescence 0 5 10 15 20 25 0 0.1 0.2 0.3 0.4 0.5 Power at focus (mW) At “normal” imaging intensities, excitation is only appreciable at the focal point.

5. Acquisition XY Scanner Pockels Cell Pockels Cell Driver Ti:S Laser External Detectors Pump Laser GaAsP PMT or APD Condenser PMT

6. Advantages of Multi-photon Excitation In addition to limiting photobleaching and photodamage to the image plane, multi-photon excitation has several other advantages: • Near-IR light scatters less than blue light in many biological samples • More efficient light collection • Deeper imaging into scattering tissue • Better looking images; greater effective resolution • Unaffected by chromatic aberrations • Can excite dyes in their UV absorption bands • Can use wide range of useful UV dyes • Good for multicolor imaging

7. Fluorescence lifetime imaging (FLIM) provides additional molecular information Measures the time a fluorophore is in the excited state before emitting a fluorescence photon - Molecular binding - Viscosity - Oxygen concentration - Normalizes changes to quantum efficiency  Corrected concentration changes

8. Epilepsy A disorder characterized by transient but chronic electrical abnormalities in the brain associated with seizures. Affects 0.5% - 1% of population 2.75 million with epilepsy in US 125,000 diagnosed each year Focus on temporal lobe epilepsy (TLE) Complex, partial seizures Hippocampal sclerosis

9. Hypometabolism in Epilepsy PET and MRI studies have show hypometabolism in epileptic focal zones Question remain on the cellular mechanism of hypometabolism How is this related to neuron-astrocyte coupling? Develop imaging tools for assessing metabolic function between neuronal and astrocytic populations Hertz L., J Neurosci Research. 57:417-428 (1999).

10. NADH NADH is fluorescent NAD+ is NOT fluorescent (reduced) (oxidized) Nicotinamide ring Two-photon cross-section of NADH is 1/100 to 1/1000 the magnitude of conventional fluorophores

11. MPM FLIM from Rat Hippocampus

12. MPM FLIM from Human Hippocampus

13. NADH species distribution changes in epilepsy A custom algorithm reveals three distinct species of NADH from 2-component lifetime fits of FLIM data. Tissue from pilocarpine-treated rats displays abnormal NADH concentration changes and redistribution in response to stimulation by bicucilline.

14. Multiphoton microscopy is a powerful tool Wealth of indicators capable of specific targeting -Conventional dyes -GFPs -Intrinsic fluorescence & second harmonic generation Sub-cellular resolution Optical sectioning in thick, turbid media Wide variety of biological and clinical applications -Gene expression -Protein interactions -Calcium concentrations -Neural activity -Disease diagnosis -Optical biopsy

15. Multiphoton microscopy is a powerful tool Can only image < 500 microns below the surface! Wealth of indicators capable of specific targeting -Conventional dyes -GFPs -Intrinsic fluorescence & second harmonic generation Sub-cellular resolution Optical sectioning in thick, turbid media Wide variety of biological and clinical applications -Gene expression -Protein interactions -Calcium concentrations -Neural activity -Disease diagnosis -Optical biopsy

16. 0.25 pitch GRIN lenses Normal lens works by refraction at the surfaces GRIN lens works by refraction throughout length of lens

17. 1 pitch GRIN lenses 0.5 pitch

18. In Situ Imaging of Deep Structures

19. Mouse brain Cell bodies in red (Nissl Stain), Axons in black Thy1-YFP line H mouse http://www.hms.harvard.edu/research/brain/atlas.html Feng et. al., Neuron28 (1)41-51, 2001

20. Mouse brain Cell bodies in red (Nissl Stain), Axons in black Thy1-YFP line H mouse http://www.hms.harvard.edu/research/brain/atlas.html Feng et. al., Neuron28 (1)41-51, 2001

21. Composite GRIN lenses for deep brain imaging 15 mm, NA = 0.1 250 mm ~50 mm 350 mm 657 mm, NA = 0.6 Lenses in collaboration with NSG America High-NA glass is autofluorescent  Use low-NA for regions with internal focus. Resolution determined by NA of end pieces = 0.6 Field of view determined by ratio of NAs = 1/6

22. 20 mm Deep brain imaging, in situ, from Thy1-YFP H mouse Layer V Layer V ~750 mm ~750 mm ~1 mm ~1.5 mm Hippocampus Axon Bundle

23. Conclusions MPM and FLIM are powerful tools, with potential for clinical application Development of GRIN-lens-based systems may Provide platform for the development of new Image-guided surgical techniques.

24. Acknowledgements • Levene Lab • Tom Chia – FLIM and Epilepsy • Joe Zinter – Microscope apparatus • Eben Olson • Veronika Mueller • Amanda Foust • Dr. Rick Torres Yale Neurosurgery Dr. Anne Williamson Dr. Dennis Spencer