In Vivo Imaging SRPT

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In Vivo ImagingSRP-T
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SRP Objectives (1/2)

• To develop a clear vision of current and future
  trends in the field of in vivo nano-imaging
• To identify competent teams, experts and leaders
  in the field (inside or outside N2L / Europe)
• To inventory the work currently done in Europe

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SRP Objectives (2/2)

• To define a technological roadmap for the
  clinical use of in vivo nano imaging in the next
  5 to 10 years
• To help fostering collaborations between European
  teams to initiate new research projects
• To prepare mini consortium for future FP7 calls

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SRP-T Focus

• Nano imaging technologies
• From animal to human (from the research to the
  clinics)
• Multimodal imaging combining different imaging
  technologies

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Description of the Tasks (1)

• Task 1 coordination of the SRP
• Contacts with other related European Networks and
  IP
• Mapping of existing teams, or companies (with
  WP6), inside and outside N2L initiate contacts
• Reporting to WP7 board (every 6 months)
• Task 2 state-of-the-art (cooperation with WP5)
• drafting of the optical in vivo molecular
  imaging SoA section
• mapping of existing technologies / approaches
  research areas
• IP landscape mapping
• participation of N2L experts to international
  conferences (2)

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Description of the Tasks (2)

• Task 3 participation to the drug discovery and
  development workshop
• Task 4 initiation of a thematic working group
  (10 to 12 members max) on probes, contrast
  agents, tracers, nanoparticles, and their
  clinical development use
• identification of experts (survey, personal
  contacts) within or outside N2L
• organization of meetings for the working group
• relationship with other SPRs (T or A)
• Task 5 contribution to the working group on
  nanodiagnostics for the ETP NanoMedicine
  participation in 3 meetings
• Task 6 Tutorial

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Interaction with other SRPs or Networks

• SRPT with SRPT
• E.g. with Nano Assemblies SRPT
• (Prof. Ehud Gazit)
• SRPT with SRPA
• E.g. with Drug Delivery SRPA
• (Prof. Rafi Korenstein)
• Coordination with EMIL ? Invite EMIL
  representative to participate in the working group

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Next Steps

• Build the group get it started by
• Initiating and pursuing contacts (phone,
  visits...)
• Involving more people (from or outside N2L)
• Organizing group meetings
• Refining the questions we want to ask (and
  possibly answer)
• Involving people on the biological clinical side

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IN VIVO OPTICAL IMAGINGAT THE LETI / DTBS
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In vivo fluorescence principle
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.
Dot light source
µsgtgtµa
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Optical Filtering
Filter
Filter
Excitation
Excitation
Detector
Detector
Sample
Sample
Fluorescence Reflectance imaging (FRI)
Transillumination Fluorescence Imaging (TFI)
The excitation signal is much higher than the
fluorescence signal (106 at least). The filter
must be optimized to suppress this excitation
signal.
Organs auto-fluorescence with different optical
filters J. Frangioni, Current Op. in Chem. Bio.,
2003
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Trans-illumination fluorescence

• The measured fluorescence signal depends of
• Fluorophores properties (quantum yield, time of
  life, local concentration)
• Optical properties of the medium at the
  excitation and emission wavelengths (absorption
  and scattering).
• Relative position of the source and the
  detector.

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Trans-illumination fluorescence
Detector
Source
Whatever the excitation source position the
fluorescence is emitted from the same
location The source localization and
characterization is possible only if we use the
transmission signal and the fluorescence signal.
Two acquisitions must be performed one with the
transmitted signal the other with the
fluorescence signal
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Fluorescence tomography set-up
The laser beam is scanned in order to provide a
set of fixed positions under the mouse and the
camera acquires the transmission image and the
fluorescence image.
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Linearity Resolution
Linearity
Spatial resolution

• Depth penetration up to 15mm
• Same linearity / fluorochrome concentration for
  6, 9 and 12mm depth
• Submillimetric spatial resolution in the plan of
  the support
• 4mm spatial resolution in depth (FWHM)

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Small Animal Fluorescence Tomography
a)
c)
d)
In vivo experimentation with TSA tumors in
lungs. Tumors labeled with Transferin / Alexa
750. Acquisition 3h after IV injection
a) superimposition of the reconstructed volume
projection and of the white light image of the
mouse. b) FRI image of the opened mouse. c) slice
through the reconstructed volume. d) volume
rendering on the lung area. In mesh for the low
level signal (lungs), in red for the high level
signal (tumors).
b)
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Perspectives from Small Animal to Human-
Intra-operative device ?- Mammography ?-
Endoscopy ?
Small Animal Fluorescence Tomography
Several mice bearing TSA lung metastasis were
imaged at different stages of tumour development
0 day (i.e. healthy mouse), 9 days and 14 days
after the primary tumour implantation. After in
vivo tomography, lungs and heart are extracted to
perform FRI acquisition.
Healthy lungs
14 days tumour
9 days tumour
12 days tumour
Results of a reconstruction of the lungs over time
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Optical Imaging Probes
Receptor
Targeted cell
biomarker
Probe, Contrast agent

• antibody
• peptides
• saccharides

Organic dyes - Low molecular weight (lt1 kDa)

• Nano-particles
• - quantum dots
• silica or polymeric particles loaded with dyes
• Nanometric sizes
• High molecular weight
• ?modifies the pharmacokinetics of the biomarker
• ?allows a multimeric presentation of the
  biomarker

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Optical Imaging Comparison of Different Dyes
Nanocrystals potentially have several advantages
for optical in vivo imaging ? we want to further
study and exploit their optical properties and
potential in biology
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Concept of multi-functional activable fluorescent
probes
SMART PROBES
Classical or activable optical marker
MRI marker
F
 Drug   gene, anti-sense DNA, Active
principle

• Optical imaging
• Low cost and easy handling image acquisition
  system
• High sensitivity, low quantity of injected
  substances
• 2D and 3D imaging with increased resolution
• Limitation observation of deep tissues

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Activable Probes Principle
cRGD biomarker
Targeted cell

Fluorescent
Targeting
Non fluorescent
Boturyn, Coll, Garanger, Favrot, Dumy, JACS 2004,
126 (18), 5730-5739. Texier, Coll, Dumy, Boturyn,
EN n05 07784 .
Internalization
Fluorescence activation
Activated imaging function
Inactivated imaging function
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ACTIVABLES PROBES
The Cy5-X-Q bridge is cleaved inside the cell
after the molecule reaches its target (tumor) gt
the fluorescent probe can be detected.
Activable probe

• Dramatic increase of the tumor / skin contrast
• Long lasting labeling of the tumors

• Collaborative work with
• P. Dumy, D. Boturyn, J. Razkin (LEDSS, UJF/CNRS)
  ? RAFT vector design and synthesis
• J.L. Coll, Z. Jin, V. Josserand, M. Favrot (IAB,
  INSERM/CHU) ? biological validation

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ACTIVABLE PROBES SUGARS
Synthesis of new fluorescent substrates for
b-galactosidase
O
R
O
R
O
b-galactosidase
C
y
5
R
O
O
X
C
y
5
Q

• Patent pending