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

Multiphoton Microscopy

Michael J. Levene

Multiphoton microscopy is a powerful tool

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

True “Molecular Imaging,” with single-molecule sensitivity

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

Increasing Wavelength Increasing Energy

1 photon excitation Fluorescence

2 photon excitation

Two Photon Excited Florescence

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.

At “normal” imaging intensities, excitation is only appreciable at the focal point.

0 5 10 15 20 25

Power at focus (mW)

0 0.1 0.2 0.3 0.4 0.5

Re

lati

ve

Flu

ore

sc

en

ce

F I 2F I

Two Photon Excitation is Spatially Localized

GaAsP PMT or APD

Condenser PMT

External Detectors

Pockels Cell

Pockels Cell Driver

XY Scanner

Ti:

S L

as

er

Pu

mp

La

se

r

Acquisition

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 population2.75 million with epilepsy in US125,000 diagnosed each year

Focus on temporal lobe epilepsy (TLE)Complex, partial seizuresHippocampal 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 neuron-astrocyte 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 ofconventional fluorophores

MPM FLIM from Rat Hippocampus

MPM FLIM from Human Hippocampus

Concentration Changes of NADH Species

0%

50%

100%

150%

200%

250%

Cell Layer Dendritic Layer Cell Layer Dendritic Layer

Control Pilocarpine

ROI in CA1 Rat Hippocampus

Co

ncen

trati

on

In

cre

ase

Species 1

Species 2

Species 3

Total

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.

NADH species distribution changes in epilepsy

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

0.25 pitch

Normal lens works by refractionat the surfaces

GRIN lens works by refractionthroughout length of lens

GRIN lenses

0.5 pitch1 pitch

In Situ Imaging of Deep Structures

Mouse brain

Feng et. al., Neuron 28 (1)41-51, 2001http://www.hms.harvard.edu/research/brain/atlas.html

Cell bodies in red (Nissl Stain), Axons in black Thy1-YFP line H mouse

Mouse brain

Feng et. al., Neuron 28 (1)41-51, 2001http://www.hms.harvard.edu/research/brain/atlas.html

Cell bodies in red (Nissl Stain), Axons in black Thy1-YFP line H mouse

Composite GRIN lenses for deep brain imaging

657 m, NA = 0.6

350 m

~50 m 250 m

15 mm, NA = 0.1

High-NA glass is autofluorescent Use low-NA for regions with internal focus.Resolution determined by NA of end pieces = 0.6Field of view determined by ratio of NAs = 1/6

Lenses in collaborationwith NSG America

Deep brain imaging, in situ, from Thy1-YFP H mouse

Layer V Layer V

Axon Bundle

20 m

Hippocampus

~750 m ~750 m

~1 mm ~1.5 mm

Conclusions

MPM and FLIM are powerful tools, with potentialfor clinical application

Development of GRIN-lens-based systems mayProvide platform for the development of newImage-guided surgical techniques.

Acknowledgements

Levene LabTom Chia – FLIM and EpilepsyJoe Zinter – Microscope apparatusEben OlsonVeronika MuellerAmanda FoustDr. Rick Torres

Yale NeurosurgeryDr. Anne WilliamsonDr. Dennis Spencer