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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
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600
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2.2x 10
4
Reconstruction -> 5-10 min
IAB
IABIAB
2D fluorescence reflectance image