Page 1

Simultaneous
imaging
of
two
fluorescent
signals
using
a
new
fibered
 fluorescent
confocal
microscopy
system

 Bertrand
Viellerobe
1,
Isabelle
Janssens
2,3,
Karine
Gombert
2,3,
Hedi
Gharbi
1,
François
Lacombe
1
and
Frédéric
Ducongé
2,3
 1)
Mauna
Kea
Technologies,
9,
rue
d’Enghien,
75010
Paris,
France
 2)
CEA,
I²BM,
Service
Hospitalier
Frédéric
Joliot,
4
place
du
général
Leclerc,
91401
Orsay
(France)
 3)
INSERM
U1023,
Université
Paris
Sud,
Laboratoire
d’Imagerie
Moléculaire
Expérimentale,
4
place
du
général
Leclerc,
91401
Orsay
(France)


Introduction Today,
confocal
fluorescence
microscopy
and
mul^photon
 microscopy
 are
 increasingly
 used
 for
 in
 vivo
 studies
 in
 small
 animals.
 Such
 techniques
 allow
 studying
 the
 structure
and
the
physiology
of
living
organism
at
cellular
 scale.
 The
 major
 limita^ons
 of
 such
 imaging
 is
 that
 1‐
 samples
need
to
be
placed
conveniently
on
a
conven^onal
 microscope
 stage
 which
 require
 extensive
 surgical
 prepara^on,
 and
 2‐
 rapid
 image
 collec^on
 is
 required
 to
 minimize
 the
 effects
 of
 movement
 (such
 as
 animal
 breathing).
 To
 solve
 this
 problem,
 novel
 confocal
 approaches
 using
 fiber
 bundle‐based
 systems
 have
 been
 developed
 by
 Mauna
 Kea
 Technologies
 (Paris,
 France).
 Such
 systems,
 named
 Cellvizio®,
 use
 extremely
 small
 bundles
of
fibers,
0.3–2.6
mm
in
diameter
that
can
contain
 upwards
of
30,000
fibers.
Each
fiber
is
used
for
excita^on
 delivery
 and
 recovery
 of
 the
 emission
 back
 through
 the
 fiber
to
a
detector.
Hence,
each
fiber
can
be
compared
as
 an
 independent
 insect
 eye.
 The
 absolute
 advantages
 of
 this
 apparatus
 are
 size,
 flexibility,
 and
 image
 collec^on
 speed
 (up
 to
 of
 12
 frames/s).
 Up
 to
 now,
 two
 Cellvizio®
 systems
were
available
either
with
a
488
nm
or
a
660
nm
 laser
 beam.
 Here,
 we
 describe
 the
 use
 of
 a
 new
 fiber
 bundle‐based
fluorescence
imaging
prototype
(Cellvizio®
 Dual
 Band)
 that
 can
 perform
 simultaneous
 excitaEon
 with
 both
 lasers
 (488
 nm
 and
 660
 nm)
 and
 recovery
 of
 emission
 signal
 with
 
 two
 detectors.
 We
 validate
 the
 system
 comparing
 the
 biodistribu^on
 of
 a
 fluorescent
 RGD‐based
 probe
 (Angiostamp®)
 in
 different
 region
 of
 a
 tumor
xenogran
as
well
as
in
different
organs
of
a
mouse.
 This
fluorescent
probe
is
known
to
bind
the
αvβ3
Integrin,
 a
protein
overexpressed
at
the
surface
of
endothelial
cells
 during
angiogenesis
[1].


Materials and methods

●
Ethics
Statement
 All
 animal
 use
 procedures
 were
 in
 strict
 accordance
 with
 the
 recommenda^ons
 of
 the
 European
 Community
 (86/609/CEE)
 and
 the
French
Na^onal
Commioee
(décret
87/848)
for
the
care
and
use
 of
laboratory
animals.
 ●
Animal
model
 Female
 nude
 mice
 (~23
 g)
 were
 subcutaneously
 injected
 with
 106
 tumor
 cells
 NIH‐MEN2A
 expressing
 the
 oncogen
 RETC634Y.
 Aner
 15
 days,
mice
have
a
tumor
(~30‐50
mm3).

 
●
In
vivo
fluorescence
imaging
using
fDOT/CT

 Angiostamp
 (10
 nmol)
 was
 intravenously
 injected
 into
 the
 tail
 of
 anesthe^zed
animals.
3D
fluorescence
images
were
acquired
3h
or
 7h
post‐injec^on
using
a
prototype
op^cal
imager
(TomoFluo3D).
CT
 imaging
 was
 perform
 using
 the
 SkyScan
 1178
 high‐throughput
 micro‐CT
 (Skyscan,
 Kon^ch,
 Belgium).
 Fusion
 of
 fDOT
 with
 CT
 was
 performed
using
the
Brainvisa
medical
imaging
processing
sonware
 (hop://brainvisa.info/index_f.html)
[2].

 
●
In
vivo
fluorescence
imaging
using
Cellvizio®
prototype
 Aner
 fDOT
 imaging
 ,
 1mg
 of
 FITC‐dextran
 (500
 kDa)
 was
 intravenously
 injected
 
 in
 animals
 before
 surgery.
 
 Then,
 Fluorescence
 imaging
 at
 the
 cellular
 level
 was
 performed
 with
 the
 fibered
confocal
microscope
Cellvizio®
Dual
Band
from
 
Mauna
Kea
 Technologies.
 The
 device
 consists
 in
 a
 flexible
 sub‐millimetric
 microprobe
 containing
 thousands
 of
 op^cal
 fibers
 that
 carry
 light
 from
 two
 con^nuous
 laser
 source
 at
 488
 nm
 and
 660
 nm
 to
 the
 living
 ^ssue.
 The
 fluorescence
 emioed
 aner
 excita^on
 by
 the
 fluorophores
 staining
 the
 ^ssue
 species
 is
 sent
 back
 to
 the
 apparatus,
where
a
dedicated
set
of
algorithms
reconstructs
images
 in
real
^me
at
a
frame
rate
of
12
frames
per
second.
The
probe
that
 was
 used
 is
 a
 UltraMiniO
 probe
 with
 30,000
 op^cal
 fibers,
 a
 240x240
µm
field
of
view,
and
a
1.4
µm
lateral
resolu^on.

Results Macroscopic
imaging
of
Angiostamp®
using
fDOT/CT

 The
 biodistribu^on
 of
 Angiostamp
 was
 first
 evaluated
 using
 fluorescence
 Diffuse
 Op^cal
 Tomography
 (fDOT)
 in
 nude
 mouse
 bearing
 a
 subcutaneous
 xenogran
 tumor
 from
 NIH/MEN2A
 cells.
 This
 imaging
 technique
 has
 been
 considerably
improved
since
past
decade
and
allows
now
 reconstruc^ng
and
quan^fying
fluorescence
signal
in
three
 dimensions
 inside
 small
 animal.
 fDOT
 imaging
 fused
 with
 X‐Ray
 Computed
 Tomography
 (CT)
 demonstrated
 a
 high
 uptake
 of
 the
 tracer
 in
 the
 tumor
 area.
 Interes^ngly,
 the
 uptake
 seems
 heterogeneous
 in
 the
 tumor
 and
 seems
 higher
 in
 the
 booom
 of
 the
 tumor.
 In
 subcutaneous
 xenogran
 models,
 the
 tumour
 cannot
 easily
 grow
 to
 the
 skin
 where
 it
 cannot
 find
 a
 lot
 of
 nutrients,
 but
 it
 preferen^ally
 invades
 the
 ^ssue
 below.
 The
 tracer
 seems
 to
have
a
higher
uptake
in
that
zone
that
should
be
rich
in
 new
blood
vessels.
 However,
although
fDOT
can
now
detect
fluorescence
in
 the
nanomolar
range,
it
has
sEll
a
low
(a
few
mm)
spaEal
 resoluEon
 that
can
 not
permit
to
have
a
 precise
idea
 of
 the
biodistribuEon
of
the
probe
at
the
cellular
scale.


3h post-injection

7h post-injection

Angiostamp®
is
surrounding
the
tumor
blood
vessels
 FITC-dextran

FITC-dextran

AngioStamp ®

AngioStamp ®

Merge

FITC-dextran

Merge

FITC-dextran

Angiostamp®
is
not
surrounding
the
blood
vessels
of
muscle
 FITC-dextran

AngioStamp ®

Angiostamp®
is
surrounding
the
tumor
blood
vessels


AngioStamp ®

AngioStamp ®

FITC-dextran

Merge

FITC-dextran

AngioStamp ®

AngioStamp ®

AngioStamp ®

Merge

Angiostamp®is
slightly
accumulated
in
spleen
 Merge

FITC-dextran

AngioStamp ®

Merge

Fig.
1:
BiodistribuEon
of
Angiostamp®
analyzed
by
fDOT/CT
imaging
 Fluorescence
 signal
 reconstructed
 in
 3D
 (colored)
 was
 fused
 to
 CT 
imaging
of
the
mouse
(gray).



Microscopic
 imaging
 of
 Angiostamp®
 using
 Cellvizio®
 Dual
Band

 Following
fDOT
imaging,
the
mice
were
injected
with
FITC‐ Dextran
 before
 imaging
 with
 the
 fiber
 bundle‐based
 fluorescence
 imaging
 prototype
 (Cellvizio®
 Dual
 Band).
 The
instrument
allowed
to
acquired
in
real‐^me
image
of
 blood
 vessels
 labeled
 with
 FITC‐Dextran
 and
 the
 signal
 from
 Angiostamp®.
 Thanks
 to
 the
 high
 flexibility
 of
 the
 system
different
organs
can
easily
been
analyzed
as
well
as
 different
part
of
the
tumor
xenogran
(scheme
2).



Angiostamp®
is
eliminated
by
glomerulus
of
kidney
 FITC-dextran

AngioStamp ®

Merge

Angiostamp®
is
eliminated
by
glomerulus
of
kidney
 FITC-dextran

AngioStamp ®

Merge

Conclusions Using
the
endoscopic
system,
we
demonstrated
that
we
can
simultaneously
observe
the
biodistribu^on
of
Angiostamp®
with
 blood
 vessels.
 We
 observed
 a
 high
 accumula^on
 of
 Angiostamp®
 surounding
 blood
 vessels
 close
 to
 tumor.
 In
 contrast,
 no
 Angiostamp®
was
localised
close
to
blood
vessels
of
healthy
^ssue
such
as
muscle,
spleen,
liver
or
kidney.
Hence,
the
new
 Cellvizio®
allows
us
to
confirm
that
the
macroscopic
image
obtain
by
fDOT
corresponds
to
tumor
angiogenesis
imaging
and
 maybe
 also
 to
 uptake
 by
 tumor
 associated
 macrophages
 expressing
 the
 αvβ3
 Integrin.
 In
 conclusion,
 the
 simultaneous
 monitoring
 of
 two
 fluorescent
 signals
 by
 endomicroscopy
 can
 be
 useful
 to
 validate
 fluorescent
 probes
 used
 for
 macroscopic
imaging
and
it
opens
a
new
avenue
to
monitor
in
vivo
molecular
events
at
a
microscopic
scale.


Literature cited [1] Garanger, E., Boturyn, D., Jin, Z., Dumy, P., Favrot, M.C. and Coll, J.L. (2005) New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors. Mol Ther, 12, 1168-1175.

Scheme
2:
IllustraEon
of
different
part
of
the
tumor
that
can
be 
imaged
by
the
Cellvizio®
Dual
Band


[2] Garofalakis, A., Dubois, A., Kuhnast, B., Dupont, D.M., Janssens, I., Mackiewicz, N., Dolle, F., Tavitian, B. and Duconge, F. (2010) In vivo validation of free-space fluorescence tomography using nuclear imaging. Opt Lett, 35, 3024-3026.

Acknowledgments

The
authors
would
like
to
thank
Anikitos
Garofalakis
for
for
his
valuable
technical
 assistance
 for
 fDOT/CT
 imaging.
 This
 work
 was
 supported
 by
 grants
 from
 the
 “Agence
Na^onale
pour
la
Recherche”
[projects
ANR‐TechSAN
Do^mager
and
the
 European
 Molecular
 Imaging
 Laboratory
 (EMIL)
 network
 [EU
 contract
 LSH‐2004‐503569].



For further information Please
contact:
francois@maunakeatech.com


 



























or
frederic.duconge@cea.fr



Scheme
1:
Cellvizio®
Dual
Band
system


Merge

Angiostamp®
is
slightly
accumulated
in
liver


Angiostamp®is
not
accumulated
in
spleen
 FITC-dextran

Merge

Angiostamp®
is
not
surrounding
the
blood
vessels
of
muscle


Merge

Angiostamp®
is
not
accumulated
in
liver
 FITC-dextran

Merge

Cellvizio Dual Band Poster WMIC 2011  

Poster WMIC Angiogenesis Cellvizio Dual Band

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