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

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

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
Fluorescence
Increasing Wavelength
Increasing Energy
2 photon excitation

Two Photon Excitation is Spatially Localized
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.
Single photon excitation
(488 nm)
Two photon excitation
(900 nm)
F

I F

I
2
0 0.1 0.2 0.3 0.4 0.5
0 5 10 15 20 25
Power at focus (mW)
At “normal” imaging intensities, excitation is only appreciable at the focal point.

Pockels Cell
Pockels Cell
Driver
XY
Scanner
External
Detectors
GaAsP PMT or APD
Acquisition
Condenser PMT

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

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

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

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 neuronastrocyte coupling?
Develop imaging tools for assessing metabolic function between neuronal and astrocytic populations
Hertz L., J Neurosci Research . 57:417-428 (1999).

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

MPM FLIM from Rat Hippocampus

MPM FLIM from Human Hippocampus

NADH species distribution changes in epilepsy
200%
150%
100%
50%
0%
250%
Concentration Changes of NADH Species
Species 1
Species 2
Species 3
Total
Cell Layer Dendritic Layer Cell Layer Dendritic Layer
Control Pilocarpine
ROI in CA1 Rat Hippocampus
A custom algorithm reveals three distinct species of NADH from 2component lifetime fits of FLIM data.
Tissue from pilocarpine-treated rats displays abnormal NADH concentration changes and redistribution in response to stimulation by bicucilline.

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

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

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

In Situ Imaging of Deep Structures

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., Neuron 28 (1)41-51, 2001

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., Neuron 28 (1)41-51, 2001

Composite GRIN lenses for deep brain imaging
15 mm, NA = 0.1
~50 m m
350 m m
250 m m
657 m m, 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

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

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.

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