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2007

www.leti.fr

Fluorescence Reflectance Imaging (FRI)

AbstractFluorescence enhanced diffuse optical tomography is an emergent diagnosis tool for the localization and the quantification of fluorescent probes ; this technique comes as a supplement or sometimes replaces the classical ionizing radiation imaging techniques, and in particular if a simple , inexpensive, non invasive and accurate instrumentation is sought. For 5 years now, the CEA-LETI has built a base of knowledge in markers and instrumentation within the framework of small animal imaging. More recently, an instrumentation has been developed, the purpose of which is a specific approach to the examination of underlying structures, deeply embedded within the tissues, and in fine for human being screening.

Cooled CCD camera

lens

Why fluorescence ?

Visualization techniques widely used in vitro Non ionizing technique Cheap

Principle

In vivo Fluorescence imaging limitations

The excitation and emission wavelengths must be in the near infra red: higher than 650 nm and lower than 900 nm.

The scattering coefficient is much higher than the absorption coefficient, therefore the outcoming photons have been highly scattered.Light propagation in biological tissues is modeled as a diffusion process.

µs>>µa

light source

Strong heamoglobin attenuation Strong water attenuation

haemoglobin and water absorption

0,01

0,1

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wavelength (nm)

abso

rpti

on

(cm

-1)

Hb02 cm-1

Hb cm-1

H2O cm-1

Excitation light scattering

~20

cm

CCD Camera Emission filter

Optical fibers

Laser source

Halogen lamp IR filter

IR filtered visible light illumination

•Two optical fibred 690nm laser light 100mW for fluorescence excitation•Scattering of the illumination source with a holographic lens•Field homogeneity better than 30%•Illumination: 2,6 mW/cm²

3D imaging: Trans-illumination Diffuse Optical Tomography

liver

Nodules in the lungs

day 10 day 12 day 14

Course of an experiment

Exemple of in vivo follow up [*]

National funded project Prostafluo

Deep Tissue Screening: endoscopy

The major challenge in deep tissues fluorescence imaging initially consists in bringing the fluorescent marker in sufficient quantity on the target.

The second difficulty in deep tissue screening consists in being freed from the intrinsic fluorescence of biological tissues. To get rid of this autofluorescence, the approach considered consists in using a pulsed signal and time-resolved measurements in order to achieve a temporal discrimination between the autofluorescence signal and the fluorophores emission signal.

This discrimination will be all the more efficient if the fluorescence marker lifetime is significantly different from the autofluorescence lifetime.

[*] A. Koenig, L. Hervé, V. Josserand, M. Berger, J. Boutet, A. Da Silva, J.-M. Dinten, P. Peltié, J.-L. Coll, P. Rizo, “In vivo mice lungs tumors follow-up with fDOT”, to be published in Journal of Biomedical Optics 2008

Edges detection

3D Visualisation

10x10 fluorescence images acquisition -> 5 minutes

Ufl

100 200 300 400 500 600

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600

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2.2x 10

4

Reconstruction -> 5-10 min

IAB

IABIAB

2D fluorescence reflectance image