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Multiphoton Microscopy
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
Relative Fluorescence
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.
F  I2
FI
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.
Single photon
excitation
(488 nm)
Two photon
excitation
(900 nm)
Acquisition
XY
Scanner
Pump Laser
Ti:S Laser
Pockels Cell
Pockels Cell
Driver
External
Detectors
GaAsP PMT
or APD
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
(reduced)
NAD+ is NOT fluorescent
(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
Concentration Changes of NADH Species
Concentration Increase
250%
Species 1
200%
Species 2
Species 3
150%
Total
100%
50%
0%
Cell Layer
Dendritic Layer
Control
Cell Layer
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.
Dendritic Layer
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
GRIN lenses
Normal lens works by refraction
at the surfaces
GRIN lens works by refraction
throughout length of lens
0.25 pitch
GRIN lenses
0.51pitch
pitch
In Situ Imaging of Deep Structures
Mouse brain
Cell bodies in red (Nissl Stain), Axons in black
http://www.hms.harvard.edu/research/brain/atlas.html
Thy1-YFP line H mouse
Feng et. al., Neuron 28 (1)41-51, 2001
Mouse brain
Cell bodies in red (Nissl Stain), Axons in black
http://www.hms.harvard.edu/research/brain/atlas.html
Thy1-YFP line H mouse
Feng et. al., Neuron 28 (1)41-51, 2001
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
Deep brain imaging, in situ, from Thy1-YFP H mouse
Layer V
20 mm
Axon Bundle
Layer V
~750 mm
~750 mm
~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