poster emim 2013
DESCRIPTION
Description of Cellvizio Dual Band, on a technical and applicative point of view.TRANSCRIPT
UltraMiniO MicroprobeResolution 1,4 µmWorking Distance 60 µm30000 fiber-optics bundle
PRINCIPLE & ARCHITECTURE
Perfect imaging modality doesn’t exist to date if we consider spatial resolution, sensitivity, ease of use or penetration capabilities. Conventional microscopy is widely used for in vitro and invasive studies whereas whole body imagers can’t reach cellular details in vivo.
• Conventional microscopy • Requires substantial numbers of animals: high costs and ethics issues • Allows for In vitro imaging of phenomena at a given time: no dynamic imaging • Provides high resolution
• Whole body imagers MRI, CT, Optical fluorescence • Well suited for in vivo imaging of biodistribution of molecular biomarkers • Non invasive: compatible with longitudinal studies • Cellular information unreachable due to low spatial resolution:
INTRODUCTION
CELLVIZIO® DUAL BAND is a multicolor probe-based Confocal Laser Endomicroscope • Fills the gap between conventional microscopy and whole body imagers (Fig 1 on the right) • From bench to bedside: Cellvizio® is FDA approved and CE cleared for clinical indications
• Delivers dynamic in vivo fluorescence imaging of molecular events with cellular resolution (1,4 µm) • Can access any tissue with minimal invasiveness including deep brain, abdominal cavity, GI tract, etc... • Longitudinal studies made possible: evaluation of drug candidates actions on the same animal over time • Turn to Optical Biopsy and real-time diagnostics (Fig 2 below) • Point Of Care molecular imaging station
Multicolor Probe-based Confocal Laser Endomicroscopy In Vivo Molecular Imaging with Cellular Resolution
EMIM 2013 | TORINO | ITALY
IN VIVO OUTCOMES
Cellvizio ® Dual Band is a confocal microscope which makes use of a 488 or 660 nm excitation which is injected one by one in tens of thousands of tiny fibers optics grouped in a flexible fiber bundle. Excitation is conducted by the fibers down to the tissue to be examined where it is focused by some distal optics which defines the field of view, the lateral and the axial resolution of the system. Endogenous or exogenous fluorescence is then produced, which is collected by the very same individual fiber and redirected towards a single detector, an avalanche photodiode (APD). Scanning the laser onto the proximal end of the fiber bundle is performed by a combination of a two fast oscillating mirrors, providing an overall frame rate of 9 to 50 frames per second (fps), which compensates for motion artifacts. Dedicated image processing then operates in real time to first compensate for fiber-to-fiber differences in transmission and background, but also to remove the well-known fiber honeycomb pattern and reconstruct a smooth and readable image.
Cellvizio® Dual Band works with a large variety of fiber-optic probes that have been designed to fit with various applications constraints, with diameters as small as a needle tip (300 µm) or with resolution that can reach 1,4 µm. The system is able to simultaneously track two different molecular signatures in vivo and in situ, allowing therefore colocalization studies to be conducted on the go in the living animal. The system’s wavelengths (488 and 660 nm) cover a large spectrum of in vivo compatible fluorescent dyes, proteins, biosensors, antibodies or genetically engineered animal models used routinely in translational research. The tremendous advances in biomarker discovery is putting in vivo diagnostics to a whole new precision level. Cellvizio® Dual Band sets the stage to a better understanding of molecular pathways that are leading to cancer, inflammation, infection or neurodegenerative diseases.
Simultaneousimagingoftwofluorescentsignalsusinganewfibered
fluorescentconfocalmicroscopysystem
BertrandViellerobe1,IsabelleJanssens2,3,KarineGombert2,3,HediGharbi1,FrançoisLacombe1andFrédéricDucongé2,31)MaunaKeaTechnologies,9,rued’Enghien,75010Paris,France
2)CEA,I²BM,ServiceHospitalierFrédéricJoliot,4placedugénéralLeclerc,91401Orsay(France)
3)INSERMU1023,UniversitéParisSud,Laboratoired’ImagerieMoléculaireExpérimentale,4placedugénéralLeclerc,91401Orsay(France)
Acknowledgments TheauthorswouldliketothankAnikitosGarofalakisforforhisvaluabletechnical
assistance for fDOT/CT imaging. This work was supported by grants from the
“AgenceNa^onalepourlaRecherche”[projectsANR‐TechSANDo^magerandthe
European Molecular Imaging Laboratory (EMIL) network [EU contract
LSH‐2004‐503569].
Introduction Today,confocalfluorescencemicroscopyandmul^photon
microscopy are increasingly used for in vivo studies in
small animals. Such techniques allow studying the
structureandthephysiologyoflivingorganismatcellular
scale. The major limita^ons of such imaging is that 1‐
samplesneedtobeplacedconvenientlyonaconven^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
bundlesoffibers,0.3–2.6mmindiameterthatcancontain
upwardsof30,000fibers.Eachfiberisusedforexcita^on
delivery and recovery of the emission back through the
fibertoadetector.Hence,eachfibercanbecomparedas
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®
systemswereavailableeitherwitha488nmora660nm
laser beam. Here, we describe the use of a new fiber
bundle‐basedfluorescenceimagingprototype(Cellvizio®
Dual Band) that can perform simultaneous excitaEon
with both lasers (488 nmand 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
tumorxenogranaswellasindifferentorgansofamouse.
Thisfluorescentprobeisknowntobindtheαvβ3Integrin,
aproteinoverexpressedatthesurfaceofendothelialcells
duringangiogenesis[1].
Materials and methods
●EthicsStatement
All animal use procedures were in strict accordance with the
recommenda^ons of the European Community (86/609/CEE) and
theFrenchNa^onalCommioee(décret87/848)forthecareanduse
oflaboratoryanimals.
●Animalmodel
Female nudemice (~23 g) were subcutaneously injectedwith 106
tumor cellsNIH‐MEN2A expressing the oncogen RETC634Y. Aner 15
days,micehaveatumor(~30‐50mm3).
●InvivofluorescenceimagingusingfDOT/CT
Angiostamp (10 nmol) was intravenously injected into the tail of
anesthe^zedanimals.3Dfluorescence imageswereacquired3hor
7hpost‐injec^onusingaprototypeop^calimager(TomoFluo3D).CT
imaging was perform using the SkyScan 1178 high‐throughput
micro‐CT (Skyscan, Kon^ch, Belgium). Fusion of fDOTwith CTwas
performedusingtheBrainvisamedicalimagingprocessingsonware
(hop://brainvisa.info/index_f.html)[2].
●InvivofluorescenceimagingusingCellvizio®prototype
Aner fDOT imaging , 1mg of FITC‐dextran (500 kDa) was
intravenously injected in animals before surgery. Then,
Fluorescence imagingat the cellular levelwasperformedwith the
fiberedconfocalmicroscopeCellvizio®DualBandfrom MaunaKea
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,whereadedicatedsetofalgorithmsreconstructsimages
inreal^meataframerateof12framespersecond.Theprobethat
was used is a UltraMiniO probe with 30,000 op^cal fibers, a
240x240µmfieldofview,anda1.4µmlateralresolu^on.
Results
MacroscopicimagingofAngiostamp®usingfDOT/CT
The biodistribu^on of Angiostamp was first evaluated
using fluorescence Diffuse Op^cal Tomography (fDOT) in
nude mouse bearing a subcutaneous xenogran tumor
fromNIH/MEN2A cells. This imaging techniquehas been
considerably improvedsincepastdecadeandallowsnow
reconstruc^ngandquan^fyingfluorescencesignalinthree
dimensions insidesmallanimal. fDOT imaging fusedwith
X‐Ray Computed Tomography (CT) demonstrated a high
uptakeof 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
xenogranmodels, the tumour cannot easily grow to the
skin where it cannot find a lot of nutrients, but it
preferen^ally invades the^ssuebelow.The tracer seems
tohaveahigheruptakeinthatzonethatshouldberichin
newbloodvessels.
However,althoughfDOTcannowdetectfluorescence in
thenanomolarrange,ithassEllalow(afewmm)spaEal
resoluEonthatcannotpermit tohaveaprecise ideaof
thebiodistribuEonoftheprobeatthecellularscale.
Conclusions Usingtheendoscopicsystem,wedemonstratedthatwecansimultaneouslyobservethebiodistribu^onofAngiostamp®with
bloodvessels.Weobservedahighaccumula^onofAngiostamp®suroundingbloodvesselsclose to tumor. Incontrast,no
Angiostamp®waslocalisedclosetobloodvesselsofhealthy^ssuesuchasmuscle,spleen, liverorkidney.Hence,thenew
Cellvizio®allowsustoconfirmthatthemacroscopicimageobtainbyfDOTcorrespondstotumorangiogenesisimagingand
maybe also to uptake by tumor associated macrophages expressing theαvβ3 Integrin. In conclusion, the simultaneous
monitoring of two fluorescent signals by endomicroscopy can be useful to validate fluorescent probes used for
macroscopicimaginganditopensanewavenuetomonitorinvivomoleculareventsatamicroscopicscale.
For further information Pleasecontact:[email protected]
Microscopic imaging of Angiostamp® using Cellvizio®
DualBand
FollowingfDOTimaging,themicewereinjectedwithFITC‐
Dextran before imaging with the fiber bundle‐based
fluorescence imaging prototype (Cellvizio® Dual Band).
Theinstrumentallowedtoacquiredinreal‐^meimageof
blood vessels labeled with FITC‐Dextran and the signal
from Angiostamp®. Thanks to the high flexibility of the
systemdifferentorganscaneasilybeenanalyzedaswellas
differentpartofthetumorxenogran(scheme2).
Fig.1:BiodistribuEonofAngiostamp®analyzedbyfDOT/CTimaging
Fluorescence signal reconstructed in 3D (colored) was fused to CT
imagingofthemouse(gray).
FITC-dextran AngioStamp ® Merge
Angiostamp®issurroundingthetumorbloodvessels
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotsurroundingthebloodvesselsofmuscle
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotaccumulatedinliver
Angiostamp®isnotaccumulatedinspleen
FITC-dextran AngioStamp ® Merge
Angiostamp®iseliminatedbyglomerulusofkidney
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotsurroundingthebloodvesselsofmuscle
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isslightlyaccumulatedinliver
Angiostamp®isslightlyaccumulatedinspleen
FITC-dextran AngioStamp ® Merge
Angiostamp®iseliminatedbyglomerulusofkidney
FITC-dextran AngioStamp ® Merge
3h post-injection 7h post-injection
Merge FITC-dextran
Angiostamp®issurroundingthetumorbloodvessels
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.
[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.
Scheme2:IllustraEonofdifferentpartofthetumorthatcanbe
imagedbytheCellvizio®DualBand
Scheme1:Cellvizio®DualBandsystem
Distal optics
Confocal microscope
Tissue
Fiber bundle
Real TimeImage Processing
488 nm 660 nm Merge
Colon cryptsAcryflavine
MacrophagesAminoSPARK 680
Macrophagesdistribution
Dynamic mouse colon imaging and macrophages targetting during inflammation
Animal model Balb/c mouse colon inflammation modelTopical spray of Acryflavine to reveal cypts structure0,5 mg AminoSPARK (Perkin Elmer) nIR fluorescent nanoparticle intravenous administration (tail vein)Vessels are visible by negative contrast
50 µm 50 µm 50 µm
In vivo quantification of Calcium spikes in olfactory bulb neurons using GCaMP3
Animal modelBalb/c mouse, GCaMP3 loaded AAV local transfection.A 300 µm bevelled probe is inserted into the olfactory bulb under a stereotaxic frame
Improvement of Fibered Fluorescence Microscopy images of
individual cells in the brain of live mice
MITOCHONDRIAL REDOX STATE IN LIVE MICE
Jesus Pascual-Brazo, Veerle Reumers, Sarah-Ann Aelvoet, Zeger Debyser, Veerle Baekelandt
Laboratory for Neurobiology and Gene Therapy. Department of Neurosciences. Faculty of Medicine, K.U. Leuven
INTRODUCTION
Imaging techniques, such as magnetic resonance imaging and
positron emission tomography, have provided huge information about
the structure and function of the brain during the last years but the low
resolution and acquisition times limits the information that can be
obtained with these techniques.
A new technology developed by MaunaKea®, called Fibered
Fluorescence Microscopy, is trying to fill the gap between the existing
brain imaging techniques. The Cellvizio microscope, based in a fiber
optic probe that transport the emission and fluorescent light to the
scanning unit, is able to acquire confocal images with cellular
resolution (3 μm axial resolution) of deep brain regions in live animals.
However, it is necessary to introduce a fluorescent dye or protein to
visualize the cells, which usually generates background during the
imaging process. Optimization of viral vector technology accordingly
with the characteristics of the technique can improve the quality of the
images acquired with this microscope.
FIBERED FLUORESCENCE MICROSCOPY
Microscope description. The system is composed of 2 main parts: laser
scanning unit and the fiber optic probe. The light from a photodiode
laser is injected in every microfiber optic of the probe, which transports
the light till the tissue. The emitted light is transported by the same
microfiber till the detector. The S-300 probe used for these experiments
contains 10.000 microfibers.
Procedure. Animals were anaesthetized by intra-peritoneal injection of
ketamine/medetomidine and placed in a stereotactic device. The probe
was slowly introduced in the target brain area and images acquired at
12Hz frequency,
Image processing. ImageCell® software was used to select regions of
interest, to quantify the intensity of the fluorescent signal and to
represent the data. Raw data of signal intensity was plotted for every
time point.
C O N C L U S I O N S
• Optimized viral vector technology increased the signal/noise ratio of Fibered Fluorescence Microscopy images in the hippocampus of live mice.
• GCaMP3 allows to record calcium levels of several cells in live mice using this new technique.
• Mitochondrial redox state can be monitored in vivo using roGFP in vivo with cellular resolution.
MOLECULAR VIROLOGY & GENE THERAPY
LEUVEN VIRAL VECTOR CORE - LVVC
NEUROBIOLOGY & GENE THERAPY
INCREASED SIGNAL/NOISE RATIO AFTER OPTIMIZATION
HIPPOCAMPUS HIGH TITERS HIPPOCAMPUS LOW TITERS HIPPOCAMPUS OPTIMIZED
Redox imaging. Lentiviral vector targeting redox sensitive protein (roGFP) to
the mitochondria was designed and produced. Image acquisition revealed
redox spikes of Individual cells in the hippocampus of live mice. ImageCell®
software was used to record images at a frequency of 12 Hz, for quantification
and representation of the intensity of the fluorescent signal. Raw data of
signal intensity was plotted for every time point.
CALCIUM IMAGING OF SEVERAL CELLS IN LIVE MICE
GCAMP3 IMAGING IN THE HIPPOCAMPUS
GCAMP3 IMAGING IN THE OLFACTORY BULB
Calcium Imaging of several cells in the hippocampus of live mice. Calcium
sensitive protein GCaMP3 was expressed employing AAV vectors. ImageCell®
software was used to record images at a frequency of 40 Hz,
Calcium Imaging of several cells in the olfactory bulb of live mice. Calcium
sensitive protein GCaMP3 was expressed employing AAV vectors. Sequential
activation of neighboring cells was plotted at frequency of 12Hz.
Conventional and optimized viral vectors were stereotactically injected with
viral vectors engineered to express GFP in the hippocampus. Comparison
of the signal/noise ratio after conventional (high and low titers) and
optimized viral vectors transduction was carried out.
ACKNOWLEDGEMENTS. A plasmid for mito-roGFP was provided by S.J. Remington (University of Oregon,USA). GCaMP3 was obtained from Addgene (L. Looger). This work has been supported by IWT-SBO/060838 Brainstim,
SCIL programme financing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.
Improvement of Fibered Fluorescence Microscopy images of
individual cells in the brain of live mice
MITOCHONDRIAL REDOX STATE IN LIVE MICE
Jesus Pascual-Brazo, Veerle Reumers, Sarah-Ann Aelvoet, Zeger Debyser, Veerle Baekelandt
Laboratory for Neurobiology and Gene Therapy. Department of Neurosciences. Faculty of Medicine, K.U. Leuven
INTRODUCTION
Imaging techniques, such as magnetic resonance imaging and
positron emission tomography, have provided huge information about
the structure and function of the brain during the last years but the low
resolution and acquisition times limits the information that can be
obtained with these techniques.
A new technology developed by MaunaKea®, called Fibered
Fluorescence Microscopy, is trying to fill the gap between the existing
brain imaging techniques. The Cellvizio microscope, based in a fiber
optic probe that transport the emission and fluorescent light to the
scanning unit, is able to acquire confocal images with cellular
resolution (3 μm axial resolution) of deep brain regions in live animals.
However, it is necessary to introduce a fluorescent dye or protein to
visualize the cells, which usually generates background during the
imaging process. Optimization of viral vector technology accordingly
with the characteristics of the technique can improve the quality of the
images acquired with this microscope.
FIBERED FLUORESCENCE MICROSCOPY
Microscope description. The system is composed of 2 main parts: laser
scanning unit and the fiber optic probe. The light from a photodiode
laser is injected in every microfiber optic of the probe, which transports
the light till the tissue. The emitted light is transported by the same
microfiber till the detector. The S-300 probe used for these experiments
contains 10.000 microfibers.
Procedure. Animals were anaesthetized by intra-peritoneal injection of
ketamine/medetomidine and placed in a stereotactic device. The probe
was slowly introduced in the target brain area and images acquired at
12Hz frequency,
Image processing. ImageCell® software was used to select regions of
interest, to quantify the intensity of the fluorescent signal and to
represent the data. Raw data of signal intensity was plotted for every
time point.
C O N C L U S I O N S
• Optimized viral vector technology increased the signal/noise ratio of Fibered Fluorescence Microscopy images in the hippocampus of live mice.
• GCaMP3 allows to record calcium levels of several cells in live mice using this new technique.
• Mitochondrial redox state can be monitored in vivo using roGFP in vivo with cellular resolution.
MOLECULAR VIROLOGY & GENE THERAPY
LEUVEN VIRAL VECTOR CORE - LVVC
NEUROBIOLOGY & GENE THERAPY
INCREASED SIGNAL/NOISE RATIO AFTER OPTIMIZATION
HIPPOCAMPUS HIGH TITERS HIPPOCAMPUS LOW TITERS HIPPOCAMPUS OPTIMIZED
Redox imaging. Lentiviral vector targeting redox sensitive protein (roGFP) to
the mitochondria was designed and produced. Image acquisition revealed
redox spikes of Individual cells in the hippocampus of live mice. ImageCell®
software was used to record images at a frequency of 12 Hz, for quantification
and representation of the intensity of the fluorescent signal. Raw data of
signal intensity was plotted for every time point.
CALCIUM IMAGING OF SEVERAL CELLS IN LIVE MICE
GCAMP3 IMAGING IN THE HIPPOCAMPUS
GCAMP3 IMAGING IN THE OLFACTORY BULB
Calcium Imaging of several cells in the hippocampus of live mice. Calcium
sensitive protein GCaMP3 was expressed employing AAV vectors. ImageCell®
software was used to record images at a frequency of 40 Hz,
Calcium Imaging of several cells in the olfactory bulb of live mice. Calcium
sensitive protein GCaMP3 was expressed employing AAV vectors. Sequential
activation of neighboring cells was plotted at frequency of 12Hz.
Conventional and optimized viral vectors were stereotactically injected with
viral vectors engineered to express GFP in the hippocampus. Comparison
of the signal/noise ratio after conventional (high and low titers) and
optimized viral vectors transduction was carried out.
ACKNOWLEDGEMENTS. A plasmid for mito-roGFP was provided by S.J. Remington (University of Oregon,USA). GCaMP3 was obtained from Addgene (L. Looger). This work has been supported by IWT-SBO/060838 Brainstim,
SCIL programme financing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.
In vivo neural activation in the olfactory bulb
Quantification of Calcium spikes into Regions of interest
In vivo biodistribution and kidney clearance of αvβ3 integrin molecular marker
Animal modelFemale nude mouse bearing MDA MB231 tumor xenograft underwent intravenous injection of 1 mg FITC-Dextran 500 kDa (Sigma-Aldrich) and 10 nmol Angiostamp® 700 (Raft RGD, fluOptics)
A, B | Optical biopsy of hindlimb vessels. Endothelial wall cells visible as well as blood flowC | Tumor vessels and tumor associated macrophages mixed with endothelial cells D | Optical slicing of the kidney, exhibiting AngioStamp® elimination beside vessels.
Kidney vasculatureFITC Dextran
AngioStamp® clearancein the glomerulus
Overlay of the two channels
488 nm 660 nm Merge
50 µm 50 µm 50 µm
D
A B C
50 µm 50 µm 50 µmHindlimb Hindlimb Tumor
Contact
us !
References1- Vercauteren et al., Multicolor pCLE, SPIE Bios 2013, 2- Brazo et al., Improvement of Fibered Fluorescence Microscopy images ofindividual cells in the brain of live mice, WMIC 20123- Ducongé et al, Simultaneous imaging of two different signals using a new fibered confocal microscopy system, WMIC 2011
H. Gharbi*, F. LacombeMauna Kea Technologies, Paris, France
Fig 1 Cellvizio bridges the gap between conventional miicroscopy and whole body imagers
Fig 2 Optical biopsy avoids tissue samples by providing dynamic in vivo microscopic images in a non or minimally invasive manner.Rea l t ime st ructure and function characterization and physiopathology diagnostics is therefore possible.