cellvizio lab

Hedi GHARBI, MSc.Product Manager

IN VIVO MOLECULAR IMAGING AT CELLULAR RESOLUTION

Prepared by H Gharbi | © Mauna Kea Technologies 2013

Agenda

2

INTRODUCTION SYSTEM DESCRIPTION APPLICATIONS

CANCER RESEARCH NEUROSCIENCE JOIN US

MKEA_CORP v0.9 © 2011 Mauna Kea Technologies

Our Strength, Our Mission

Mauna Kea Technologies is a global medical device company commercializing tools to advance the field of OPTICAL BIOPSY

Optical biopsies enable physicians to visualize tissue in real time at the microscopic level at the point of care so they can make more informed diagnoses and decisions at the patients’ bedside.

A fast growing Medical Device company

Leader in the endomicroscopy field

Cellvizio: a unique probe-based confocal laser endomicroscopy solution

Based in Paris and Atlanta, presence in Europe and Asia.

Listed on NYSE-Euronext, compartment B under the ticker MKEA.

4

Mauna Kea Technologies

french!


• Biliary structures• Colon, IBD• Lung• Barrett’s Esophagus• Urinary tract• FDA, CE, CPT Codes

Research• Functional Imaging• Colocalization• Structure-Function• Neuroscience• Cancer research• ...

Clinical

5

What is Cellvizio ?

• World’s smallest Flexible Microscope

• Designed for in vivo imaging

• For Researchers & Clinicians• Point Of Care Imaging Station

What is ? Image behavior in REALTIMEEmpower your experiments withCellvizio® is the smallest video-microscope.

It o!ers High-resolution, confocal imaging and provides in vivo & in situ imaging.

Neuron activity as it happens in vivo !Deep network activity monitoring.Real-time imaging of neurons in animals achieving basic behavioral tasks.

Deep brain imaging in freely-moving mice !

LOW INVASIVENESS

TECHNOLOGICAL BREAKTHROUGH

EASY-TO-USE, TURN-KEY SOLUTION

Laser Scanning UnitConfocal Microscope488 or 660 nm excitation beamSingle-photon detection (APD)Handy, turn-key, easy-to use

ImageCell™ SoftwareReal-time image processingQuantification featuresFramerate up to 200 fpsLSU control

ProFlex™ MiCROprobesDesigned for di!erent applicationsHigh Resolution: up to 1.4 ȝmThin diameter: down to 300 ȝm

CONSCIOUS, FREELY-MOVING MICE Functional Fluorescence imaging

LONGITUDINAL STUDIESMake your results more relevantImage more with fewer animals

www.cellviziolab.com

LIGHTEST IMPLANTS Only 0,3g

LOW INVASIVE MICROPROBE Only 350ȝm in diameter*

*Available in 350 ȝm or 470 ȝm (hardened tip)

A FULL SOLUTIONDedicated set of toolsFits the workflowWorks with anystereotaxic set-up

1

1

3

3

2

2

From bench to bedside


Carrefour Pathologie 2010 v3

The Histopathology laboratory today Challenges

Many Steps in Pathology Laboratories

Increasing needs:

Safety, Workflow, Quality Management

Many Opportunities of error

The Histopathology Lab today...

6

Challenges

Source: Roche

Subject Subject


• Real-Time answers• High-resolution • in vivo & in situ• Point Of Care station• Easy-to-use• Turnkey solution





LOW INVASIVENESS













1

1

3

3

2

2

7

Carrefour Pathologie 2010 v3

Many Steps in Pathology Laboratories

Increasing needs:

Safety, Workflow, Quality Management

Many Opportunities of error

6

How about Real-time information?


TRANSVERSAL VIEWEx-vivo

MicroscopicInvasive

Late imaging

FRONT VIEWIn-vivoMicroscopicMinimally invasiveReal-time imaging

Optical Biopsy Tissue Biopsy

Turn to Optical Biopsy

8


Turn to Optical Biopsy

9

Transverse section of a villus, from the human intestine. X 350.

a. Basement membrane.b. Lacteal.c. Columnar epithelium.d. Its striated border.e. Goblet cells.f. Leucocytes in epithelium.f’. Leucocytes below epithelium.g. Bloodvessels.h. Muscle cells cut across.

http://en.wikipedia.org/wiki/Intestine

http://en.wikipedia.org/wiki/Intestine

http://en.wikipedia.org/wiki/Basement_membrane

http://en.wikipedia.org/wiki/Basement_membrane

http://en.wikipedia.org/wiki/Lacteal

http://en.wikipedia.org/wiki/Lacteal

http://en.wikipedia.org/wiki/Columnar_epithelium

http://en.wikipedia.org/wiki/Columnar_epithelium

http://en.wikipedia.org/wiki/Goblet_cells

http://en.wikipedia.org/wiki/Goblet_cells

http://en.wikipedia.org/wiki/Leucocytes

http://en.wikipedia.org/wiki/Leucocytes

http://en.wikipedia.org/wiki/Bloodvessels

http://en.wikipedia.org/wiki/Bloodvessels

http://en.wikipedia.org/wiki/Muscle_cells

http://en.wikipedia.org/wiki/Muscle_cells


Kératinocyte

TRIPLE MARQUAGE

DAPI CHROMOSOMES BODIPY FL TUBULINE TEXAS RED ACTINE

Microscopie à fluorescence

22

1 2

• High-speed Live Cell Imaging a

• The Fastest True Confocal Sys

• Leading in Multispectral Imagi

• Intelligent and Intuitive Interfa

2

INVASIVE NON INVASIVEMINIMALLY INVASIVE

1 µm

10µm

100 µm

1 mm

0,5 µm

Confocal2-photonSTEDCARS

10

Cellvizio bridges an Imaging GAP

UltrasoundMRIPET/CTSPECTFluo/VisibleNear IRFMT

in vivoin vivo

in vivo

in vitroin vivo

in vitro

in vitro

in vivo

in vivo

in vitroin vitro

in vitro

in vitro

in vivo in vivoin vivo

in vitro in vivoin vivo

in vivo in vivo

in vivoin vivoin vivo

in vivo

in vivoin vivo

Resolution


Perfect Imaging Modality doesn’t exist

11

Depth of Penetration

Real Time

Field of view

Sensitivity

Ease of Use

Resolution Ideal ImagingModality

0

5


Cellvizio imaging

12

Real Time

Field of view

Sensitivity

Ease of Use

Resolution

Cellvizio



PET-CT/SPECT

13

Real Time

Field of view

Sensitivity

Ease of Use

Resolution


PET-CTSPECT


Whole body optical imager

14

Real Time

Field of view

Sensitivity

Ease of Use

Resolution


Opticalfull body


Whole body optical imager

15

Real Time

Field of view

Sensitivity

Ease of Use

Resolution


MRI


Multimodality gives the perfect combination

16

Real Time

Field of view

Sensitivity

Ease of Use

Resolution



SYSTEM DESCRIPTIONLaser Scanning Unit | Confocal Microprobes | Quantification features


Confocal Laser Endomicroscopy

19

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

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 di!erences 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 di!erent 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

“AgenceNaônalepourlaRecherche”[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 limitaôns of such imaging is that 1‐

samplesneedtobeplacedconvenientlyonaconvenônal

microscope stage which require extensive surgical

preparaôn, and 2‐ rapid image collecôn 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.Eachfiberisusedforexcitaôn

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 collecôn

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 biodistribuôn 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

recommendaôns of the European Community (86/609/CEE) and

theFrenchNaônalCommioee(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‐injecônusingaprototypeop^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 excitaôn 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,anda1.4µmlateralresoluôn.

Results

MacroscopicimagingofAngiostamp®usingfDOT/CT

The biodistribuôn 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

preferenâlly invades the^ssuebelow.The tracer seems

tohaveahigheruptakeinthatzonethatshouldberichin

newbloodvessels.

However,althoughfDOTcannowdetectfluorescence in

thenanomolarrange,ithassEllalow(afewmm)spaEal

resoluEonthatcannotpermit tohaveaprecise ideaof

thebiodistribuEonoftheprobeatthecellularscale.

Conclusions Usingtheendoscopicsystem,wedemonstratedthatwecansimultaneouslyobservethebiodistribuônofAngiostamp®with

bloodvessels.WeobservedahighaccumulaônofAngiostamp®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]

[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


Angiostamp®isnotsurroundingthebloodvesselsofmuscle



Angiostamp®isnotaccumulatedinliver

Angiostamp®isnotaccumulatedinspleen


Angiostamp®iseliminatedbyglomerulusofkidney



Angiostamp®isnotsurroundingthebloodvesselsofmuscle



Angiostamp®isslightlyaccumulatedinliver

Angiostamp®isslightlyaccumulatedinspleen


Angiostamp®iseliminatedbyglomerulusofkidney


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.






INTRODUCTION



























12Hz frequency,




time point.































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 !

[email protected]

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 di!erent signals using a new fibered confocal microscopy system, WMIC 2011

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.

Each fiber acts as a point source and a point detector or pinhole« »


Confocal Laser endo-microscope

20





LOW INVASIVENESS













1

1

3

3

2

2

CONFOCAL MICROPROBES

• Designed for di!erent applications• High Resolution: up to 1.4 µm• Diameter down to 300 µm• Flexible

ACQUISITION SOFTWARE

• Real-Time video recording• Quantification features• Frame rate 12-200 fps• LSU control• Various exports

LASER SCANNING UNIT

• Confocal Microscope• 488 and 660 nm excitation beam• Dual Band excitation/Detection• Highly sensitive (APD)• Handy, turn-key, easy-to-use


LINE UP

21

Single Band488 nm

Single Band660 nm

Dual Band488 + 660 nm

Range505 - 700 nm

Range680 - 900 nm

First Line502 - 633 nm

Second Line673 - 800 nm

Real-time recordingFrame rate:

8 - 12 fpsHyperScan®:

Up to 200 fps


8 - 12 fpsHyperScan®:

Up to 200 fps


9 fpsAdvanced:

Up to 50 fps

Image: png, bmp, jpeg, ti!, mhd

Video: avi, mpeg, mp4, swf





488 660

EXCITATION

DETECTION

FRAME RATE

EXPORT





LOW INVASIVENESS













1

1

3

3

2

2

MKEA_CORP v0.9 © 2011 Mauna Kea Technologies22

CONFOCAL MICROPROBES


Optical Sectioning(CONFOCAL THICKNESS)

Working DistanceWD

WD+ WD++

Tailored to YOUR application needs

HighResolution

1,4 µm

ThinDiameter300 µm

DeepWorking Distance

Series

MSeries

ZSeries

S

10µm

Working Distance100µm

15µm

70µm

55µm

65µm 65µm

135µm

Working Distance

60µm

Working Distance

0µm

Series

MSeries

ZSeries

S

ex S1500 ex UltraM ex Z1800

Optical sectioning

Working Distance (Focal point)

Incident Laser light (488 nm)

Legend

Tissue

Resolution 1,4 µm Resolution 3,5 µmResolution 3,3 µm


Model ApplicationsTip

Diameter (mm)

Lateral Resolution

(µm)

Optical Sectioning

(µm)

Working Distance

(µm)

Max Field Of view

(µm)

Deep brain imaging, designed for permanent implantation on freely moving mice

Part of the NeuroPak™ solution

0,350,47 3,3 15 0 325

Brain, deep brain in mice, other organs at depth if low invasiveness is mandatory 0,3 3,3 15 0 300

Surface Imaging

Brain, deep brain in rats, other organs at depth if low invasiveness is mandatory 0,65 3,3 15 0 600

General applicability, can be used to check fluorescence sensitivity in most targets 1,5 3,3 15 0 600

Vessels, angiogenesis, cell fate, cell morphology, utility depends on cell layer thickness and invasiveness

2,6 1,4 10 60 240

Hi-ResImaging


4,2 1,4 10 30 240

ImagingVessels, angiogenesis, cell fate, cell morphology, utility depends on cell layer thickness and invasiveness

4,2 1,4 10 100 240

Blood flow through the vessel (without penetration) image deeper cell layers of tumor, organ or tissue

1,8 3,5 70100/170

at488/660

600

Depth Imaging

Cavities, eye 0,94 3,5 30

50/70at

488/660 325

SSeries

MSeries

S-300*

S-0650*

S-1500

UltraMiniO

MiniO/30

MiniO/100

Z-1800

Mini-Z

ZSeries

CerboFlex™CerboFlex™ J

25

Confocal Microprobes

> Made of thousands fiber-optics

Series

Series

Series

S

M

Z

Model ApplicationsTip

Diameter (mm)

Lateral Resolution

(µm)

Optical Sectioning

(µm)

Working Distance

(µm)

Max Field Of view

(µm)

Deep brain imaging, designed for permanent implantation on freely moving mice

Part of the NeuroPak™ solution

0,350,47 3,3 15 0 325

Brain, deep brain in mice, other organs at depth if low invasiveness is mandatory 0,3 3,3 15 0 300

Surface Imaging

Brain, deep brain in rats, other organs at depth if low invasiveness is mandatory 0,65 3,3 15 0 600

General applicability, can be used to check fluorescence sensitivity in most targets 1,5 3,3 15 0 600


2,6 1,4 10 60 240

Hi-ResImaging


4,2 1,4 10 30 240

ImagingVessels, angiogenesis, cell fate, cell morphology, utility depends on cell layer thickness and invasiveness

4,2 1,4 10 100 240

Blood flow through the vessel (without penetration) image deeper cell layers of tumor, organ or tissue

1,8 3,5 70100/170

at488/660

600

Depth Imaging

Cavities, eye 0,94 3,5 30

50/70at

488/660 325

SSeries

MSeries

S-300*

S-0650*

S-1500

UltraMiniO

MiniO/30

MiniO/100

Z-1800

Mini-Z

ZSeries

CerboFlex™CerboFlex™ J

Series

Series

Series

S

M

Z


Quantification TOOLBOX

27

Improved SNR and sampling

Get bigger resolution

Wider field of view

Follow the probe’s track

Advanced Mosaïcing™

00:00:04.50

50 µm

Vessel Detection®

Automatic vessel segmentation

Measure neoangiogenesis

Evaluate anti-angiogenic drugs

Quantify fluorescence over time

Manage multiple ROIs

Vector format graph

Export in csv, txt, png and pdf

Kinetic Analysis

IC Viewer


GFP mouse brain

Neuron bodies

Vessel branches


Features

• Automatic Segmentation• Compatible with Mosaicing• Functional Capillary Density• Total Area measurements• Total Length measurements• Export

30

Vessel Detection™ Module

Mouse Kidney Vasculature

Mouse MesentericVasculature Mosaic


Total Vessel Length: 3552 µmTotal Vessel Area: 47716 µm!Total Area: 131476 µm!Mean Vessel Diameter: 13.4 µmDiameter Standard Deviation: 2.7Functional Capillary Density - Length: 0.027 µm·"Functional Capillary Density - Area: 0.36

Vessel Detection™ Module

Features

• Automatic Segmentation• Batch processing• Compatible with Mosaicing• Functional Capillary Density• Total Area measurements• Total Length measurements• Export


A Wealth of Applications


Infectious Diseases: Parasitology

35

Plasmodium falciparum in the mouse liver • WT infected mouse• GFP PLasmodium• Alexa 647- BSA

• Blood vessels• Liver structure

Courtesy of Rogerio Amino, Institut Pasteur, Paris

Series

S

Release of Merozoïtes

A Wealth of Applications

Periph Nervous System

In Research : !


Drug Biodistribution - Pharmacokinetics

11

In vivo monitoring of a drug candidate uptake in the intestine

• Wistar rat• FITC Dextran 2000 kDa

• Intestine vasculature

• VivoTag 680 conjugated pharma compound• Biodistribution• Uptake and colocalization• Absorption, distribution

and metabolism (ADME) Series

SCellvizio Dual Band, S1500 microprobe


Vascular and Perfusion Studies

• WT Hamster

• Evans Blue (Red): 250µl• Vasculature• Blood flow• Velocity

• Acriflavine topical (Green)• Cell labelling

• Pericytes

• Endothelium

• Sarcocytes (muscle)

• Cellvizio Dual Band• S1500 microprobe

• UltraM microprobe

• Abdomen• Kidney capsule/glomeruli

S

M


Vascular and Perfusion Studies

S

M

• WT Hamster

• Evans Blue (Red): 250µl• Vasculature• Blood flow• Velocity

• Acriflavine topical• Cell labelling

• Pericytes

• Endothelium

• Sarcocytes (muscle)

• Cellvizio Dual Band• S1500 microprobe

• UltraM microprobe

• Abdomen• Kidney capsule/glomeruli20 µm


Cancer Research and Angiogenesis CEA (F. Duconge)

Multimodal biomarker distribution evaluation

• RAFT-RGD nIR marker: AngioStamp®

• αvβ3 integrin molecular marker

• FITC-Dextran 500 kDa

• Endothelial pattern over-expressed in tumor neovessels

• Angiogenesis indicator

• Uptaken by Tumor Cells and Associated Macrophages

• Endothelial wall & cells

Molecular imaging

TUMORKIDNEY CLEARANCE

CONTROLHINDLIMB


Pancreatic Cancer Eser et al, PNAS 2011

In vivo diagnosis of murine pancreatic intraepithelialneoplasia and early-stage pancreatic cancer bymolecular imagingStefan Esera,1, Marlena Messera,1, Philipp Eserb, Alexander von Werdera, Barbara Seidlera, Monther Bajbouja,Roger Vogelmanna, Alexander Meininga, Johannes von Burstina, Hana Algüla, Philipp Pagelb, Angelika E. Schniekec,Irene Espositod, Roland M. Schmida, Günter Schneidera, and Dieter Saura,2

aII. Medizinische Klinik, Technische Universität München, 81675 Munich, Germany; bLehrstuhl für Genomorientierte Bioinformatik and cLivestockBiotechnology, Technische Universität München, Wissenschaftszentrum Weihenstephan, 85354 Freising, Germany; and dInstitute of Pathology, TechnischeUniversität München, 81675 Munich, Germany

Edited by David A. Cheresh, University of California at San Diego, La Jolla, CA, and accepted by the Editorial Board May 6, 2011 (received for review January18, 2011)

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease withpoor patient outcome often resulting from late diagnosis in ad-vanced stages. To date methods to diagnose early-stage PDACare limited and in vivo detection of pancreatic intraepithelial neo-plasia (PanIN), a preinvasive precursor of PDAC, is impossible.Using a cathepsin-activatable near-infrared probe in combinationwithflexible confocalfluorescence lasermicroscopy (CFL) in agenet-ically defined mouse model of PDAC we were able to detect andgrade murine PanIN lesions in real time in vivo. Our diagnosticapproach is highly sensitive and specific and proved superior toclinically established fluorescein-enhanced imaging. Translation ofthis endoscopic technique into the clinic should tremendously im-prove detection of pancreatic neoplasia, thus reforming manage-ment of patients at risk for PDAC.

molecular in vivo imaging | early detection | carcinoma in situ | geneticallyengineered mouse model | cathepsin

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC)is one of the deadliest human malignancies, with an extremely

poor 5-y survival rate below 5% (1). Because patients generallypresent with symptoms relating to late stages of the disease, di-agnosis of resectable PDAC is achieved in less than 15% of cases(1). However, investigators have reported that diagnosis and re-section of early-stage PDAC (<2 cm in size) can result in a 4-ysurvival rate of up to 78% (2–5). These data suggest that earlydetection of PDAC can improve patient outcome. In addition, ithas been shown that resection of PDAC combined with adjuvantchemotherapy can improve 5-y survival rates to 25%. Thus, in-creasing the chance of resection will lead to improved survival.Of the more than 33,000 new cases of PDAC diagnosed in the

United States every year, !10% occur in families with a highprevalence of PDAC and are thus referred to as familial (6, 7). Ina cohort study of twins, it was proposed that inherited factorsmay be responsible for up to 36% of pancreatic cancers, sug-gesting that the incidence of familial pancreatic cancer may beeven higher than presently assumed (8). To date, several con-ditions associated with familial PDAC have been described andgroups of individuals at low, moderate, or high risk for developingthe disease have been defined (9, 10). Although clinical trials arecurrently testing screening protocols for the high-risk group,aimed at detecting curable precursors of PDAC such as intra-ductal papillary mucinous neoplasia, pancreatic intraepithelialneoplasia (PanIN), and early-stage PDAC (10–12), the results ofthese trials show that early lesions are often missed. Furthermore,false positive findings can lead to overtreatment of a significantfraction of the screened population (12). These findings show thatbetter diagnostic tools for the detection of preneoplastic lesionsand early-stage PDAC are urgently needed. Recent data dem-onstrate that preinvasive precursors progress slowly over many

years to decades to invasive pancreatic cancer (13). The parentalpancreatic cancer founder clone then requires more than 5 y toacquire the capacity to metastasize (13). Thus, there is a timeframe of several years for the diagnosis of curable disease, im-plicating the need for sensitive diagnostics.In the current study, we investigated a relevant genetically

engineered KrasG12D-dependent endogenous mouse model ofmurine PanIN (mPanIN) development and progression to PDAC,which accurately recapitulates the human disease (14, 15). By usinga cathepsin-activatable near-infrared (NIRF) probe in combina-tion with confocal fluorescence lasermicroscopy (CFL) we wereable to detect early-stage PDAC as well as mPanIN lesions in vivoon the cellular level. Furthermore, it was possible to differentiatebetween normal pancreatic tissue and low-grade mPanINs on theone hand and high grade mPanINs and early-stage PDAC on theother, illustrating the great potential of this technique in the di-agnosis of curable precursors and early-stage PDAC.

ResultsIdentification of Cathepsins as Targets for Molecular Imaging ofPanIN Lesions and Early-Stage PDAC. To identify targets for mo-lecular imaging of PanIN lesions and early-stage PDAC we usedgenomewide pancreatic gene expression analyses of Ptf1aCre/+;LSL-KrasG12D/+ mice, littermate controls, as well as mice withacute caerulein-induced pancreatitis. Expression profiles of over30 individual mice revealed high levels of cathepsins B, H, L, andS in all mice with mPanIN lesions or early-stage PDAC (Fig. 1A).In contrast, expression of these cathepsins was low in normal andinflamed pancreatic tissue (Fig. 1A). To validate expression ofcathepsins and to examine the localization of the correspondingproteins, we performed immunohistochemistry. Strong stainingfor cathepsins B, H, L, and S was detected in preinvasive mPanINlesions and PDAC (Fig. 1B). In line with the microarray mRNAexpression data, minimal cathepsin B, H, L, and S staining wasobserved in normal and inflamed pancreatic tissue (Fig. 1B).Furthermore, immunohistochemical staining of human pancre-atic tissue sections also revealed high expression of cathepsins B,H, L, and S in PanIN lesions and PDAC (Fig. S1 and Table S1).

Author contributions: D.S. designed research; S.E., M.M., A.v.W., B.S., M.B., and D.S. per-formed research; P.E., H.A., P.P., I.E., and R.M.S. contributed new reagents/analytic tools;S.E., M.M., P.E., A.v.W., B.S., M.B., R.V., A.M., J.v.B., P.P., A.E.S., I.E., G.S., and D.S. analyzeddata; and S.E., G.S., and D.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. D.A.C. is a guest editor invited by the EditorialBoard.1S.E. and M.M. contributed equally to this work.2To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1100890108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100890108 PNAS Early Edition | 1 of 6

MED

ICALSC

IENCE

S

In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging

Longitudinal, real-time in vivo imaging

Fluorescein:

Morphology and extent of fibrosis

ProSense 680:

Cathepsin activityPtf1aCre/+; LSL-KrasG12D/+ mice

Molecular imaging

EUS FNA

19G

S650

normal pancreast0

low grade PanINs3 months

high grade PanINs6 months

PDAC9 months


Cardiovascular Imaging

41Courtesy of Pasdois et al, LYRIC, Bordeaux

S

• Real Time Imaging of the beating perfused heart

• S300/B microprobe (300 µm)• In situ minimally invasive access• Monitoring of ventricular arrhythmia in

the beating heart• Use of a voltage sensitive fluorescent

dye (Safranin O) accumulating into mitochondria.



42 Courtesy of Pasdois et al, LYRIC, Bordeaux

M


• MiniO/30 microprobe: high res/WD• In situ NON-INVASIVE access • Monitoring of ventricular arrhythmia in

the beating heart• Use of a voltage sensitive fluorescent

dye (Safranin O) accumulating into mitochondria.



43 Courtesy of Dr Hitchcock et al, Unive of UTAH, USA


• UltraMiniO microprobe: high res/WD• Microdosing with an innovative dye

carrier• Imaging of atrial sub-epicardial

myocardium >10 mins with su!cient image quality to identify cellular structures.

ImagingMicroprobe

Magnetic Holder

3D Manual Micromanipulator

Fiber-opticalBundle

Perfusion Pump

Real-time Visualization

Leica FCM 1000

Imaging Setup

ImagingMicroprobe

Endo/epicardium

Myocardium

Extracellular Dye

* Imaged Region

Pipet

DyeDelivery

LASHER et al.: TOWARDS MODELING OF CARDIAC MICRO-STRUCTURE WITH CATHETER-BASED CONFOCAL MICROSCOPY 1159

Fig. 3. Imaging with catheter-based confocal microscopy system (Leica FCM1000). (a) M/30 confocal microprobe with hydrogel carrier loaded with dye. (b)Image of atrial tissue acquired with catheter-based confocal microscopy systemand the modified microprobe. Scale: 5 mm in (a) and 50 m in (b).

Fig. 4. Exemplary raw XY images from a three-dimensional stack of atrialtissue. The images are from the (a) epicardial surface and a depth of (b) 10 m,(c) 20 m, and (d) 30 m into the myocardium. Scale: 50 m in (a) applies to(a)–(d).

rapidly through the endo- or epicardial layers and into the my-ocardium. The dye was immediately available in sufficient con-centration for confocal imaging of the cardiac microstructure.

Exemplary two-dimensional images of atrial and ventriculartissue sections acquired with the BioRad confocal microscopeare shown in Figs. 4 and 5, respectively. These images orig-inate from three-dimensional stacks covering approximately1 m outside of the tissue surface and up to 80 m into themyocardium. Fluorescence appeared to be associated withclefts between cells (interstitial space), collagen fibers, trans-verse tubules and capillary vessels; whereas darker regionsappeared to be associated with cells. Image slices through theepicardial and endocardial network of thin collagen fibers inatrial and ventricular tissue are shown in Fig. 4(a) and Fig. 5(a),respectively. The fibers are brighter than their surroundingsand appear to be, to some degree, orientated parallel to themyocytes. The image through the ventricular endocardium[Fig. 5(a)] includes endothelial cells. Image slices into atrialand ventricular myocardium are presented in Fig. 4(b)–(d) andFig. 5(b)–(d), respectively. These image slices are from depthsof 10, 20, and 30 m into the myocardium with respect to theepicardial or endocardial surface layer [Fig. 4(a) and Fig. 5(a)].The density of the network of collagen fibers appeared to belarger in the endo- and epicardium than within the myocardium.Furthermore, images extending further into the myocardiumexhibited less overall fluorescence.

Optical properties of the BioRad confocal microscopy systemwere characterized by measurement of PSFs as described above.The PSF exhibited full widths at half maximum of 0.30 m in

the XY plane (transverse to the laser-beam) and 1.85 m in thedirection (parallel to the laser beam).We also acquired two-dimensional images with a catheter-

based confocal microscope (FCM1000, Leica Microsystems,Wetzlar, Germany). The dye carrier was attached to the cathetertip and gently pressed on the epicardial surface of the atria andventricles of a Langendorff-perfused heart. An exemplary two-dimensional image of atrial tissue is shown in Fig. 3(b). As inour studies of tissue sections with the BioRad confocal micro-scope, the dye was immediately available for imaging. High andlow fluorescence intensities were associated with the extra- andintracellular spaces, respectively.

B. Image Quantification and Modeling of Tissue

We applied methods of digital image processing and analysisto quantitatively describe and model cardiac tissue microstruc-ture from three-dimensional image data. For this purpose, weacquired 19 image stacks from a total of nine rabbits for sub-sequent analysis. Fourteen of these stacks were rejected fromanalysis due to low signal-to-noise ratios, discontinuities withinthe image stack by motion and/or poor tissue quality. Signal-to-noise ratios below 3 were considered low. We removed back-ground signals, corrected for depth-dependent attenuation, anddeconvolved the image stacks as described. Fig. 5(e) and (f) il-lustrates the effect of this processing on the image stacks. Pro-cessed image stacks exhibit fine details of myocytes such asthe transverse tubular system [Fig. 5(f)], which were difficultto identify in the unprocessed image data [Fig. 5(e)].

Individual myocytes were segmented from three-dimensionalimage stacks (Fig. 6), which allowed for subsequent spatialmodeling (Figs. 7 and 8) and quantitative analysis of myocytes(Tables I and II). Segmentation was performed on 50 atrialmyocytes and 36 ventricular myocytes. Quantitative analysiswas only performed on whole myocytes, which included 28atrial myocytes and 20 ventricular myocytes.

An exemplary segmentation of a single myocyte from athree-dimensional stack of atrial tissue is shown in Fig. 6. Themanually deformed surface mesh is illustrated in three orthog-onal planes in Fig. 6(a)–(c). Threshold values to distinguishbetween intramyocyte and extracellular space were chosen tobe the mode plus two standard deviations of signal intensityfor each segmented myocyte. Fig. 6(d) shows the segmentedmyocyte after thresholding and in a bounding box aligned to theprincipal axes of the myocyte. The dimensions of the boundingbox determined the length, width and height of the myocyte.Three-dimensional spatial models of segmented myocytesfrom three-dimensional stacks of atrial and ventricular tissueare shown in Figs. 7 and 8, respectively. Fig. 7(d) shows athree-dimensional visualization of the atrial model overlaidwith orthogonal confocal images.

Quantitative analysis revealed mean and standard deviationof lengths, widths and heights of atrial myocytes

to be , and m, respec-tively, and ventricular myocytes to be

, and m, respectively (Table I). Average vol-umes of atrial and ventricular myocytes were and

m , respectively. Furthermore, the myocytevolume fractions for atrial and ventricular tissue were

10 mmLens

Fiber-optical Bundle

Imaging Microprobe

Dye Carrier

Dye CarrierDesign

M

Cellvizio488


Multimodal Drug Delivery

Derieppe et al, 2012Courtesy of Dr Chrit Moonen, UMC Utrecht

Real-Time Assessment of Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Transport across the plasma membrane is a critical step of drug delivery for weakly permeable compounds with intracellular mode of action.

Local Drug Delivery

The FCFM device consists of a laser-based optoelectronics unit,a Z microprobe (Z1800, Mauna Kea Technologies, Paris, France)composed of a bundle of 12,000 optical fibers that is the linkbetween the laser scanning unit and a distal micro-objective. Thetwo-dimensional bundle is scanned by the laser with a 4-kHzoscillating mirror and a galvanometric mirror. Each fiber has anillumination–emission cycle, and the same path is followed forexcitation light and fluorescence emission; the latter is diverted by adichroic filter to a photodetector. The Z probe used has a 100-μmworking distance, which allows placing the optical section beyondthe 75-μm-thick culture chamber membrane, at the cell monolayer.The Z probe provides 70 μm axial resolution, 3.9 μm lateralresolution, and 600×500 μm field of view, allowing observation ofa large number of cells, and, thus, the computation of statistics overa cell population.

FCFM System Linearity

Signal linearity of the FCFM system was studied in a dilution seriesof SYTOX Green in salmon sperm DNA solution (dilution 1 mg/ml, Invitrogen Life Technologies, Saint-Aubin, France): 11Eppendorf tubes containing SYTOX Green concentrations of 0 to

70 μM were obtained by serial dilution. Then, for each concentra-tion, the tip of the FCFM microprobe was immersed for 5 s tomeasure fluorescence intensity. The system response was consid-ered linear when Pearson’s correlation coefficient r² was greaterthan 0.99.

Live Cell Microscopic Imaging

To compare nuclei detection with FCFM to epifluorescencemicroscopy, live cell imaging was performed using an epifluor-escence microscope (Leica DMR, Leica Microsystems, Wetzlar,Germany) equipped with a×10 objective (HC PL Fluotar 10X dry0.3 NA) and a set of filters appropriate for SYTOX Green. Imageswere acquired with a CoolSnapHQ camera (Roper Scientific, Evry,France), using a 5-ms exposure time.

Experimental Protocol

The regions with US-exposed cells were identified by drawingcircles on the membrane of the culture chamber [22]. The tip of theFCFM microprobe was placed in the center in line with the

Fig. 1. a In vitro setup for ultrasound (US)-mediated cell uptake monitoring by fibered confocal fluorescence microscopy(FCFM). The cell monolayer was positioned horizontally so that the adherent cell monolayer was attached to the uppermembrane, which was in direct contact with the FCFM Z microprobe. For all studies, the mono-element US transducer wasplaced below the culture chamber at a distance from the cell monolayer equal to the maximal measured acoustical pressure (8mm) of the transducer. b Time line of the experiments. SYTOX Green and microbubbles were injected immediately before theimaging. The first video was acquired continuously during 9 min and started 10 s before US exposure. US exposure lasted 30 s.After the first 9-min video, 5-s videos were acquired every minute to limit photobleaching effect.

M. Derieppe, et al.: Real-Time Fluorescence Imaging of US-Mediated Drug Delivery

The FCFM device consists of a laser-based optoelectronics unit,a Z microprobe (Z1800, Mauna Kea Technologies, Paris, France)composed of a bundle of 12,000 optical fibers that is the linkbetween the laser scanning unit and a distal micro-objective. Thetwo-dimensional bundle is scanned by the laser with a 4-kHzoscillating mirror and a galvanometric mirror. Each fiber has anillumination–emission cycle, and the same path is followed forexcitation light and fluorescence emission; the latter is diverted by adichroic filter to a photodetector. The Z probe used has a 100-μmworking distance, which allows placing the optical section beyondthe 75-μm-thick culture chamber membrane, at the cell monolayer.The Z probe provides 70 μm axial resolution, 3.9 μm lateralresolution, and 600×500 μm field of view, allowing observation ofa large number of cells, and, thus, the computation of statistics overa cell population.

FCFM System Linearity

Signal linearity of the FCFM system was studied in a dilution seriesof SYTOX Green in salmon sperm DNA solution (dilution 1 mg/ml, Invitrogen Life Technologies, Saint-Aubin, France): 11Eppendorf tubes containing SYTOX Green concentrations of 0 to

70 μM were obtained by serial dilution. Then, for each concentra-tion, the tip of the FCFM microprobe was immersed for 5 s tomeasure fluorescence intensity. The system response was consid-ered linear when Pearson’s correlation coefficient r² was greaterthan 0.99.

Live Cell Microscopic Imaging

To compare nuclei detection with FCFM to epifluorescencemicroscopy, live cell imaging was performed using an epifluor-escence microscope (Leica DMR, Leica Microsystems, Wetzlar,Germany) equipped with a×10 objective (HC PL Fluotar 10X dry0.3 NA) and a set of filters appropriate for SYTOX Green. Imageswere acquired with a CoolSnapHQ camera (Roper Scientific, Evry,France), using a 5-ms exposure time.


The regions with US-exposed cells were identified by drawingcircles on the membrane of the culture chamber [22]. The tip of theFCFM microprobe was placed in the center in line with the

Fig. 1. a In vitro setup for ultrasound (US)-mediated cell uptake monitoring by fibered confocal fluorescence microscopy(FCFM). The cell monolayer was positioned horizontally so that the adherent cell monolayer was attached to the uppermembrane, which was in direct contact with the FCFM Z microprobe. For all studies, the mono-element US transducer wasplaced below the culture chamber at a distance from the cell monolayer equal to the maximal measured acoustical pressure (8mm) of the transducer. b Time line of the experiments. SYTOX Green and microbubbles were injected immediately before theimaging. The first video was acquired continuously during 9 min and started 10 s before US exposure. US exposure lasted 30 s.After the first 9-min video, 5-s videos were acquired every minute to limit photobleaching effect.

M. Derieppe, et al.: Real-Time Fluorescence Imaging of US-Mediated Drug Delivery

B World Molecular Imaging Society, 2012DOI: 10.1007/s11307-012-0568-9

Mol Imaging Biol (2012)

RESEARCH ARTICLE

Real-Time Assessment of Ultrasound-MediatedDrug Delivery Using Fibered ConfocalFluorescence MicroscopyMarc Derieppe,1,2 Anna Yudina,1,2 Matthieu Lepetit-Coiffé,1

Baudouin Denis de Senneville,1,3 Clemens Bos,2 Chrit Moonen1,2

1Laboratory for Molecular and Functional Imaging: From Physiology to Therapy, FRE 3313–CNRS and University Bordeaux Segalen, 146,rue Léo Saignat, Case 117, 33076, Bordeaux cedex, France2Imaging Division, University Medical Center Utrecht, Heidelberglaan 100P.O. Box 85500,3508 GA, Utrecht, Netherlands3Institut de Mathématiques de Bordeaux, UMR 5251–CNRS–Université Bordeaux 1–INRIA, Bordeaux, France

AbstractPurpose: Transport across the plasma membrane is a critical step of drug delivery for weaklypermeable compounds with intracellular mode of action. The purpose of this study is todemonstrate real-time monitoring of ultrasound (US)-mediated cell-impermeable model druguptake with fibered confocal fluorescence microscopy (FCFM).Procedures: An in vitro setup was designed to combine a mono-element US transducer, a cellchamberwith amonolayer of tumor cells together with SonoVuemicrobubbles, and a FCFMsystem.The cell-impermeable intercalating dye, SYTOX Green, was used to monitor US-mediated uptake.Results: Themajority of the cell population showed fluorescence signal enhancement 10 s after USonset. The mean rate constant k of signal enhancement was calculated to be 0.23±0.04 min!1.Conclusions: Feasibility of real-time monitoring of US-mediated intracellular delivery by FCFMhas been demonstrated. The method allowed quantitative assessment of model drug uptake,holding great promise for further local drug delivery studies.

Key Words: Drug delivery, Biological barrier, Plasma membrane permeabilization, Ultrasoundbioeffects, Fibered confocal fluorescence microscopy, Pharmacokinetic parameters, SYTOXGreen, US-mediated drug delivery

Introduction

Delivery to the target site at high concentration, while

minimizing accumulation in healthy organs, is a crucialfactor in determining the efficacy of a drug [1]. It is of aparticular interest when treatment involves compounds with

toxic side effects, for example chemotherapy agents [2].Efficient delivery requires the therapeutic agent to crossbiological barriers such as the endothelium for intravenouslyinjected molecules, gastrointestinal barriers for orally ad-ministered drugs, or the plasma membrane for drugs thatneed to reach the intracellular compartment in order to exerttheir action. The permeability of biological barriers to a drugis influenced by the physico–chemical properties of the drugsuch as molecular weight, solubility, lipophilicity, andionization [3].

It has been demonstrated in vitro [4-6] and in vivo [4, 6]that ultrasound (US) can enhance both vascular and plasmamembrane permeability, thus locally facilitating compounddelivery. However, the recent literature indicates that the

Electronic supplementary material The online version of this article(doi:10.1007/s11307-012-0568-9) contains supplementary material, whichis available to authorized users.

Correspondence to: Chrit Moonen; e-mail: [email protected]

Quantitative assessment of model drug uptake, holding great promise for further local drug delivery studies.

Sytox Green 1 µM

Time(minutes)


Mucosal microcirculation in sepsis

45

the mean vessel diameter was reduced significantly in septicpatients (13.4 vs 14.0 mm; P,0.01) and FCD was reduced ac-cordingly (0.27 vs 0.30; P,0.05).

DiscussionIn severe sepsis, major alterations in microvascular perfusionhave been described in several clinical studies in children and

adults19 20 and are of pathophysiological and clinical rele-vance.10 Disturbed microcirculation of the GI tract is of majorimportance because it not only represents a complication ofsepsis but also facilitates infection and inflammation by pro-moting translocation of bacteria from the gut into the blood-stream.21 – 24 We showed a significant decrease in the FCDand mean vessel diameter throughout duodenal, ileal,gastric, and rectal mucosal beds in a porcine model of septic

00:00:00.25

00:00:07.07

00:00:04.50

00:00:00.25

00:00:07.07

00:00:04.50

50 µm 50 µm

50 µm 50 µm

50 µm 50 µm

A

B

C

Fig 2 Representative images of the mucosa of the gastric antrum (A), duodenum (B), and rectum (C) obtained by cLE after administration ofFITC-labelled dextran before (left panel) and after (right panel) detection of mucosal vessels by the image detection software for quantitativeanalysis.

cLE detection of mucosal microcirculation in sepsis BJA

Page 5 of 8

at TULB

Jena on June 25, 2013http://bja.oxfordjournals.org/

Dow

nloaded from

Confocal laser endomicroscopy reliably detects sepsis-relatedand treatment-associated changes in intestinal mucosalmicrocirculationC. Schmidt1,3†, C. Lautenschlager1†, B. Petzold2, Y. Sakr2, G. Marx4‡ and A. Stallmach1,3‡*

1 Clinic for Internal Medicine IV and 2 Department of Anesthesiology and Intensive Therapy, Jena University Hospital, Germany3 Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), Jena, Germany4 Department of Intensive Care, RWTH University Hospital, Aachen, Germany

* Corresponding author: Clinic for Internal Medicine IV, Gastroenterology, Hepatology and Infectious Diseases and Integrated Researchand Treatment Center, Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, 07740 Jena, Germany.E-mail: [email protected]

Editor’s key points

† Confocal laser endoscopywas used both in patientswith sepsis and a pigmodel.

† Microcirculation indifferent areas of the gutmucosal bed was imaged.

† Functional capillarydensity (FCD) in theduodenum was decreasedin patients.

† In pigs, FCD decreasedafter sepsis and improvedwith volume therapy.

† Confocal laser endoscopymay be useful clinically.

Background. Microcirculatory alterations play a central role in the pathophysiology of sepsis.We investigated probe-based confocal laser endomicroscopy (pCLE) to assess alterations inmucosal microcirculatory perfusion in vivo in a porcine model of septic shock and in patientsfulfilling consensus criteria for severe sepsis.

Methods. Septic shock was induced using a faecal peritonitis model in anaesthetized,mechanically ventilated pigs. Mucosal microcirculation was assessed using pCLE in thestomach, duodenum, terminal ileum, and rectum. Duodenal microcirculation was furtherevaluated in 10 patients with severe sepsis and in 8 healthy controls to quantify capillarydiameter, capillary length, and functional capillary density (FCD).

Results. In the animal model, FCD was markedly decreased in duodenal (P,0.001), ileal(P,0.001), gastric (P,0.001), and rectal mucosa (P,0.005) 4 h after induction of sepsis.After volume therapy, FCD partially recovered to 90.0% (duodenum), 94.4% (ileum), 95.4%(gastric), and 97% (rectum) of baseline values, indicating decoupling of microvascular andmacrovascular flow. In septic patients, the mean capillary diameter (P,0.01) and FCD(P,0.05) in duodenal mucosa were decreased compared with healthy controls.

Conclusions. pCLE reliably detected and quantified microcirculatory alterations in thegastrointestinal mucosa in a porcine model of sepsis and in patients with severe sepsis. Ourdata suggest that pCLE is a promising tool to assess the efficacy of therapeuticinterventions on mucosal microcirculation in real-time, even in the clinical context.

Keywords: drug therapy; microscopy, confocal; perfusion; plasma substitutes; shock

Accepted for publication: 17 April 2013

Microcirculatory alterations are common in sepsis1 2 and playan important role in the pathophysiology of sepsis-associatedorgan dysfunction. The mechanisms of sepsis-related micro-vascular haemodynamics are complex and involve localchanges in blood flow and alterations of microvascular regula-tory mechanisms because of the effects of cytokines on endo-thelium and vascular smooth muscle cells.3 4 The splanchnicbed and the gut mucosa, in particular, are prone to earlyinjury in the course of septic shock.5 Several experimentalstudies have confirmed the early occurrence of splanchnichypoperfusion in the course of endotoxaemia, before the de-velopment of hypotension.6 7

Improvement of the microcirculation is an emerging noveltherapeutic target in sepsis treatment, requiring bedsideevaluation of microcirculation. Orthogonal polarization spec-troscopy (OPS) and sidestream dark field (SDF) imaging techni-ques have been used to directly see the microcirculation inaccessible mucosal surfaces in humans.8 – 10 However, themucosal beds accessible for imaging using these techniquesremain limited. Therefore, studies using OPS or SDF in clinicalsettings have largely focused on the sublingual region.Although these studies have resulted in important observa-tions on the prognostic implications of microcirculatory altera-tions in critically ill patients,11 it remains unknown whether

† Both authors contributed equally.‡ Both senior authors contributed equally.

British Journal of Anaesthesia Page 1 of 8doi:10.1093/bja/aet219

& The Author [2013]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved.For Permissions, please email: [email protected]

BJA Advance Access published June 25, 2013

at TULB


Dow

nloaded from

microcirculatory events observed in the sublingual region ad-equately reflect microcirculatory changes in other vascularbeds. Data on human intestinal microcirculatory alterationsin sepsis are scarce and mainly derived from measurementsin ileal stomas of patients with abdominal sepsis.12

Confocal laser endoscopy (cLE) is a newly introduced endo-scopic tool that allows the observation of not only living tissuebut also the vascular networks in the bronchial and gastro-intestinal (GI) tract during endoscopy.13 – 16 This technique deli-vers high-quality images that are magnified up to 1000-fold,enabling the visualization of the capillary architecture in themucosal layer after injection of a contrast agent (fluorescein).

The aim of the current pilot study was to evaluate the feasi-bility of probe-based confocal laser endomicroscopy (pCLE) forin vivo imaging of microcirculatory alterations in various GImucosal beds and to quantify the influence of volumetherapy in the early phase of shock in a porcine model ofseptic shock. Moreover, we investigated microvascular archi-tecture in the early phase of severe sepsis in patients toassess the feasibility of studying microcirculatory failure inhumans.

MethodsAnimalsSeven female, white cross pigs (source: GbR Fraatz, Polzig,Germany) with a mean [standard deviation (SD)] weight of29.8 (3.5) kg were investigated. Animals were adapted toclimate- and light-cycle-controlled environment for at least7 days before experiments. All animals were allowed standardlaboratory food and water ad libitum.

Care and handling of the animals were in accordance withNational Institutes of Health Guidelines, and the principles oflaboratory animal care were followed. A veterinarian con-firmed a good clinical condition of all animals, and the studywas performed under a protocol approved by the local commit-tee of animal use and care.

Anaesthesia and monitoringAnimals received i.m. premedication with 5 mg kg21 ketamine(Ratiopharm, Ulm, Germany). After placement of a peripheral

venous cannula into an ear, vein anaesthesia was induced byi.v. propofol (AstraZeneca, Wedel, Germany). Pigs were thenorally intubated and placed in the supine position. Anaesthesiaand analgesia were maintained with an infusion of 10 mg kg21

h21 propofol and 0.02 mg kg21 h21 fentanyl (Actavis Deutsch-land, Langenfeld, Germany). Animals were mechanically venti-lated with an inspiratory oxygen fraction (FIO2

) of 0.3, positiveend-expiratory pressure of 5 cm H2O, and a tidal volume of 8ml kg21. The respiratory rate was adjusted between 12 and15 min21 to maintain arterial CO2 partial pressure between4.6 and 5.9 kPa. Body core temperature was kept at .378C byusing an infrared lamp and warmed solutions.

Surgical procedure and sepsis inductionThe right jugular vein and the right femoral artery were surgi-cally exposed. For drug and fluid administration, a centralvenous catheter was inserted into the superior vena cava anda balloon-tipped thermodilution pulmonary artery catheter(139HF75, 7.5-Fr, Edwards Lifesciences, Irvine, CA, USA) wasinserted via the right jugular vein. A 4-Fr catheter with an inte-grated thermistor and fibreoptic module (Pulsiocath 4F PV2024L, Pulsion Medical Systems, Munich, Germany) wasinserted into right femoral artery. Catheters and infusionsystems were not heparinized. The abdominal cavity wasopened via median laparotomy. Ileostomy was constructedusing an opened loop of the ileum. A caecal incision of 2 cmwasmade and faecal material was aspirated. Then, the caecot-omy was closed and a catheter was positioned intra-abdominally before the abdomen was closed with a suture.After the surgical preparation, towels were placed to avoidheat loss and animals were allowed to recover for 120 minbefore baseline measurements were performed. Continuousinfusion of Ringer’s solution (10 ml kg21 h21 intravenously)was given during surgery and the post-surgical period untilthe induction of sepsis. To induce sepsis, autologous faeces(0.75 g kg21) were suspended in 150 ml of isotonic NaCl(378C) and injected through the abdominal catheter. After in-duction of peritonitis, the catheter was used to drain ascites.The time course of the experiment is shown in Figure 1. I.V.volume therapy was performed by infusion of 30 ml kg21 gel-atine (MW 30 kDa; Serumwerk Bernburg, Germany) within 90

Intubation,preparationof ileostomy,

arterialand

venouscannulation

Baselineendoscopy

andpCLE

imaging

Sepsis–2 0 0.5 4.5 5 7

Therapy

Induction ofseptic shock

by faecalperitonitis

(0.75 g kg–1

bodyweightautologousfaeces)

Endoscopyand

pCLEimagingduringsepsis

Volumetherapy

(gelatine)

Endoscopyand

pCLEimaging after

therapy

Fig 1 Time course of the animal model of septic shock.

BJA Schmidt et al.

Page 2 of 8

at TULB


Dow

nloaded from

the first application of the substance even during the secondand third examination. Because of the limitations and tech-nical difficulties of this technique, the use of pCLE may not besuitable for routine application in clinical settings in thecurrent form. The time-consuming analysis should be replaced

by fast, automated analysis for pCLE to become a suitableapproach in daily clinical practice.

In conclusion, pCLE is a promising tool to evaluate GI micro-circulation and to assess influences of therapeutic approachesin sepsis treatment, such as volume therapyor vasopressors. Inaddition to furtherevaluation of this promising methodology inanimal studies, subsequent larger studies in humans are ne-cessary to define the role of pCLE to assess microcirculationin septic patients.

Supplementary materialSupplementary material is available at British Journal ofAnaesthesia online.

Declaration of interestNone declared.

FundingThis work was supported by grants from the Deutsche For-schungsgemeinschaft (DFG Sta 295/9-1) and the Federal

00.25 0.26 0.27 0.28 0.29

FCD

0.30 0.31 0.32 0.33

20

40

60M

AP

80

100

120

300.24 0.26 0.28 0.30 0.32

FCD

0.34 0.36 0.38 0.40

40

50

60

70

MA

P

80

100

90

110

300.30 0.31 0.32 0.33 0.34

FCD

0.35 0.36 0.37

40

50

60

70

MA

P

80

100

90

110

300.26 0.28 0.30 0.32

FCD

0.34 0.38 0.38

40

50

60

70

MA

P

80

100

90

110

A B

D(C)

Fig 4 Uncoupling of MAP and FCD in different parts of the GI tract (A, stomach; B, duodenum; C, ileum; D, rectum) at baseline (triangle), duringseptic shock (circle), and after volume therapy (square). Arrows indicate the sequence of measurements.

Table 2 Haemodynamic parameters of septic patients at the timeof microvascular imaging. Data are presented as mean values (1 SD).

Mean SD Normal range

Temperature (8C) 37.0 1.1

MAP (mm Hg) 70 9

Heart rate (bpm) 99 17

Central venous pressure(mm Hg)

11 6

pH 7.38 0.08 7.32–7.43

PaCO2 (kPa) 5.5 0.7 4.8–5.9

PaO2 (kPa) 9.8 2.8 9–14

Haemoglobin concentration(mol litre21)

6.0 1.2 Males: 8.7–10.9Females: 8.6–12

Arterial lactate (mmol litre21) 2.0 1.3 0.5–2.2


Page 7 of 8

at TULB Jena on June 25, 2013http://bja.oxfordjournals.org/

Downloaded from

pCLE is a promising tool to

evaluate GI micro-

circulation and to assess

influences of therapeutic

approaches in sepsis

treatment, such as volume

therapy or vasopressors.

pCLE reliably detects sepsis-related and treatment-associated changes in intestinal mucosal microcirculation

PIGSFITC-Dextran 70 kDa, 5 ml Injection IV into septic animal

Vess

el D

etec

tion®




00:00:00.25

00:00:07.07

00:00:04.50

00:00:00.25

00:00:07.07

00:00:04.50

50 µm 50 µm

50 µm 50 µm

50 µm 50 µm

A

B

C



Page 5 of 8

at TULB


Dow

nloaded from




00:00:00.25

00:00:07.07

00:00:04.50

00:00:00.25

00:00:07.07

00:00:04.50

50 µm 50 µm

50 µm 50 µm

50 µm 50 µm

A

B

C



Page 5 of 8

at TULB


Dow

nloaded from

Schmidt et al., 2013

© Mauna Kea Technologies 2014

46

Dynamic in vivo histology

Mouse colonAcriflavine staining

Protocol• Colon washing (PBS)• Syringe + catheter

insertion• Topical dye application

Normal mucosaRound CryptsRegular size.

Tubulous adenomaLarger CryptsIrregular size.

} }

Endoscope view

• MiniZ or S-1500 insertion• Endoscope combination

1

2





LOW INVASIVENESS













1

1

3

3

2

2

7

© Mauna Kea Technologies 2014

47

Inflammatory state: Cellvizio Dual Band

Acriflavine:Cellular architecture

AngioSPARK: Macrophages

S1500

Mouse Colon

13


Colon Cancer dynamic monitoring

48

Colitis | Colon Cancer | IBD

4

Waldner et al. Nature Protocols, 2011

©20

11 N

atur

e A

mer

ica,

Inc.

All

righ

ts r

eser

ved.

PROTOCOL

NATURE PROTOCOLS | VOL.6 NO.9 | 2011 | 1473

CLE was used for the specific analysis of the microvascular archi-tecture following the administration of a plasma marker in order to evaluate successful antiangiogenic therapy. CLE is a ground-breaking tool for the more detailed in vivo characterization of various aspects of intestinal pathophysiology, as it can be used with a large range of available fluorescent dyes, genetically engi-neered mice expressing a fluorophore and fluorescence-labeled antibodies (Table 2 describes frequently used fluorescent dyes). CLE can be used to analyze subcellular or even molecular changes in the intestine, as recently shown for the expression of epider-mal growth factor receptor, vascular endothelial growth factor (VEGF) and CD44v6 in colorectal cancer19–21 Once it is trans-ferred to patients, molecular CLE could improve initial diagnosis of disease and predict or monitor therapeutic success in various clinical settings.

This manuscript provides a detailed protocol for performing NBI endoscopy and CLE in living mice on the basis of the methods used in our recent publication17.

Comparison with other endoscopic methods in miceConventional endoscopy and chromoendoscopy in mice were introduced by Becker et al.4,5 for the analysis of colitis and colonic tumor development. Similarly to NBI endoscopy, these methods allow the evaluation of the same animal at repeated time points, provide a good diagnostic accuracy and are increasingly being used. In contrast to NBI endoscopy, the detection of small dysplastic lesions requires the administration of a dye (e.g., methylene blue) for chromoendoscopy.

CLE has been previously used for the study of intestinal pathol-ogy in mice. However, the large diameter (>5 mm) of the con-focal laser probes required laparotomy and thus gut surgery19. Laparotomy with subsequent incision of the mouse colon for inser-tion of the laser probe, however, may affect the results obtained by CLE, as this surgical procedure may alter tissue perfusion and cell survival. Kim et al.22 presented the first study to address these problems through the use of a rotational side-view CLE device that allowed endoscopic imaging of the mouse intestine at repeated time points. However, the described device was too big to be used in combination with conventional or virtual chromoendoscopy during ongoing endoscopy in mice.

In addition to CLE, images of various tissues, including the intestine, of mice have been acquired using fluorescence-based intravital microscopy23–25. However, these devices, similar to those for autofluorescence endoscopy and endocytoscopy, are too large

(>5 mm) for use in mice without laparotomy and gut surgery, and thus cannot be used for repeated analysis of the same animal at different time points.

Advantages of NBI and CLEIn comparison with the technique described by Kim and co-workers22, the present protocol on the combined use of NBI and CLE provides faster direct access to pathological in vivo histology of the mucosa, as it combines an endoscopic red-flag technique (NBI) with an approach for CLE. Moreover, this pro-tocol can be repeated several times in the same animals during experimental protocols for chronic colitis or experimental colon cancer. This is an important advantage of the NBI-CLE pro-tocol, as it permits long-term monitoring of mice (e.g., over a period of 80 d in the AOM-DSS model of colitis-associated colon cancer) and may reduce the number of mice required for experimental studies17.

Limitations of NBI and CLEAs the endoscope cannot be inserted into the esophagus, the presented method is restricted to analysis of diseases of the colon. Following the insufflation of air into the colon, approxi-mately 3–4 cm of the distal colon can be investigated with the rigid endoscope.

During NBI endoscopy, the white-light source is reduced by a filter system to two specific spectral bands that are preferably absorbed by hemoglobin. Therefore, images obtained from NBI endoscopy can be darker than images obtained from conventional endoscopy. However, both modes can be interchanged on the device instantly during the investigation.

CLE is limited by the availability of suitable fluorescent dyes for a certain task. In particular, when using fluorescent antibodies, the ability of an antibody to bind to its target in vivo might limit its overall usability in the absence of cell permeabilization (for instance, antibodies for intracellular targets).

Concerning the CLE device, images can be captured at a rate of 12 frames per second. This frame rate might be too low for certain tasks, such as monitoring cell-cell interactions in the vasculature and so on. However, a Hyperscan module with frequencies up to 200 Hz is available.

As the image quality of the CLE device is dependent on the probe, the properties of the acquired images can vary. As the probe used in this protocol has a fixed image plane depth of 50 ± 15 m, only this plane of the mucosa can be analyzed.

aSuperficial capillaries

Submucosalveins

Digital recorder

Air pump

Camera

Light source CLE-device

bFigure 1 | Narrow-band imaging (NBI) endoscopy and system setup. (a) Principle of NBI endoscopy. During NBI endoscopy, a filter restricts the spectrum of the white-light source to two narrow bands of different wavelengths (blue at 415 nm and green at 540 nm), which are more readily absorbed by hemoglobin. The 415 nm channel is absorbed by vessels of the superficial capillary network, whereas vessels in the depth of the mucosa absorb the 540-nm channel. Image adapted from Olympus. (b) NBI endoscopy and CLE are used in combination for the analysis of intestinal pathologies in mice. The CLE device is combined with the NBI endoscope (schematic drawing of the setup).

©20

11 N

atur

e A

mer

ica,

Inc.

All

righ

ts r

eser

ved.

PROTOCOL

1478 | VOL.6 NO.9 | 2011 | NATURE PROTOCOLS

11| CLE should be performed by two investigators. Guide the tip of the laser probe with the endoscope to the designated area of the mucosa (investigator 1), while carefully pushing the laser probe forward until it touches the mucosa (investigator 2).

CRITICAL STEP During this step, both investigators have to cooperate very closely. Usually, the investigator controlling the endoscope (investigator 1) provides instructions to investigator 2.

12 Turn on the laser power on the CLE device and adjust the image settings for optimal quality (investigator 2). CRITICAL STEP The obtained image should have enough signal strength to allow a clear discrimination of the intended

structures without overexposure in order to prevent loss of details (Fig. 3).? TROUBLESHOOTING

13 If desired, start recording video images on the CLE device while directing the confocal probe with the endoscope along the designated area of the mucosa (Fig. 3).! CAUTION Avoid keeping the confocal laser probe at one position for more than 15 s and always turn off the laser power when it is not in use to prevent phototoxic damage to the mucosa.

14| Repeat Steps 11–13 to analyze all intended regions with CLE.

15| When the examination is completed, carefully pull the confocal probe out of the accessory channel of the endoscope in order to prevent damage to the probe and then withdraw the endoscope from the intestine of the mouse.

16| Place mice back into the housing cages, observe them until they recover from anesthesia and protect them from reduced body temperature with a heating light. As soon as the mice are awake again, they can be housed as they were before endoscopy.

Off-line analysis of the fractal dimension of vessels with ImageJ TIMING 5 min17| For off-line analysis of obtained images, choose a representative image of the designated area and open the image in ImageJ.

18| Delete any areas in the image that could lead to misinterpretation of the result (e.g., scale bars or legends).

19| Convert the image to an RGB stack (Image Type RGB stack). For further processing, you can choose any channel, as the information in each channel should be identical because of the black-and-white image source.

20| Open the threshold tool (Image Adjust Threshold…) and adjust the threshold to a value that shows only vessels in red. Click ‘Apply’.

CRITICAL STEP This step is important for comparability. When comparing various images, the threshold should be set to the same value.

Acriflavine GFP expressed in IECs Excessive FITC-dextran CLE probe not placed properly

NBI and CLE combined

Mucosa

Laser probe

SYTOX green FITC-dextran Insufficient FITC-dextranFigure 3 | Confocal endomicroscopy in living mice. The laser probe is fed through the accessory channel of the NBI endoscope and can be seen within the lumen of the colon. Depending on the fluorescent dye used, different aspects of the mucosa can be analyzed at a cellular or even subcellular level. These include the crypt structure (acriflavine: 100 l of a 0.05% (wt/vol) solution applied topically), non-living cells (SYTOX green: 100 l of a 1 mM solution applied topically) and the vessel architecture (FITC-dextran: 100 l of a 5% (wt/vol) solution applied i.v.). In addition, the expression of fluorophores can be detected as seen in the image of a mouse expressing GFP under the Villin promoter in intestinal epithelial cells. Possible sources of errors include incorrect concentrations of the fluorescent dye, which result in overexposed images or loss of detail, and misplacing of the confocal probe. IECs, intestinal epithelial cells. Scale bars, 50 m.

Karl Storz endoscope with Cellvizio 488nm

Virtual Chromoendoscopy • NBI• FICE• i-Scan

Dye-based ChromoendoscopyMethylene Blue

Classical endoscopy(White Light)

Cellvizio (pCLE) • Fluorescein (Non specific, structural marker)• FITC-Dextran* (Non specific, structural marker)• SYTOX Green* (Apoptosis)• Acryflavine** (Non specific, RNA labelling)

Molecular Imaging• VEGF Antibodies*• EGFR Antibodies*

*Not FDA approved**Approval may depends on countries

©20

11 N

atur

e A

mer

ica,

Inc.

All

righ

ts r

eser

ved.

PROTOCOL

1472 | VOL.6 NO.9 | 2011 | NATURE PROTOCOLS

characterized more precisely with CLE. To this end, following the topical or systemic administration of a fluorescence-based dye (fluorescein, acriflavine hydrochloride and cresyl violet), the CLE probe, which is either integrated into the tip of the endoscope or fed through the accessory channel of the endoscope, is placed on the area of interest. The CLE system then illuminates the fluorescence-stained tissue with a low-power laser and the excitation of the dye is detected at the appropriate wavelength. As CLE is based on confocal microscopy, this technique provides high-resolution images with a lateral resolution of up to 0.7 m and an axial resolution of up to 7 m; this enables even the subcellular detection of morphologi-cal alterations of the gastrointestinal mucosa13. Whereas currently approved fluorescent dyes provide detailed images of the mucosal architecture14–16, nearly any fluorescence-based dye could theoreti-cally be used in the future, and, therefore, the possible applications for CLE seem unlimited.

Of note, the combination of red-flag techniques of endoscopy with CLE has further improved the diagnostic accuracy of colorectal lesions in clinical studies14. In addition to these techniques,

several additional methods including autofluorescence endoscopy, endocytoscopy, spectroscopy and others are currently being investigated for possible usefulness in endoscopy of the human gastrointestinal tract.

Application of NBI and CLE to miceIn a recent study, our group has combined NBI endoscopy with CLE for the evaluation of mucosal inflammation and tumor development in a mouse model of colitis-associated cancer in vivo17. Generally, NBI endoscopy can be used as a ‘red-flag’ tech-nology with any mouse model that leads to colonic diseases such as inflammation (e.g., chemical-induced models of colitis, such as dextran sulfate sodium (DSS)-induced or 2,4,6-trinitroben-zenesulfonic acid (TNBS)-induced colitis) or tumor development (e.g., azoxymethane (AOM) + DSS colitis, APCmin mice). In our study, NBI endoscopy was especially helpful for the detection of small dysplastic lesions that might have been missed by conven-tional endoscopy, as NBI endoscopy improves the detection of aberrant vessel formations (Fig. 1a)17,18. After NBI endoscopy,

TABLE 1 | Endoscopic techniques used in the colon of humans and mice.

Endoscopic technique Example Mechanism Advantages Limitations Published applications

Standard endoscopy (colonoscopy)

Colon cancer screening

White-light endoscopy

Detection of polypoid lesions; widely used in humans and increasingly used in mice

Less image information and inferior vessel detection than in newer methods; difficulties in the detection of flat lesions

Human: standard method in gastrointestinal diagnostics for several decades Mouse: colitis and colorectal cancer4

Dye-based chromoendoscopy

Methylene blue staining

Local application of dye

Improved detection of dysplasia

Administration of dye during endoscopy; time consuming

Human: colorectal cancer screening38 Mouse: tumor development4

Virtual chromoendoscopy

NBI White light filtered; narrowing of spectra

Improved contrast of vasculature

Images eventually darker

Human: colitis and cancer development39–41 Mouse: colitis and tumor development17

FICE Image postprocessing

Improved contrast of vasculature and neoplastic tissue

Only limited data available so far

Human: colorectal cancer screening42 Mouse: none

i-SCAN Image postprocessing

Different modes for surface, vessel and pattern architecture

Only limited data available so far

Human: colorectal cancer screening43 Mouse: none

Confocal laser endomicroscopy

Fluorescein, acriflavine

Confocal laser scanning microscopy

In vivo acquisition of microscopic images, possibility of molecular imaging

Restricted to the use of one fluorescent dye at a time; limited field of view

Human: colorectal cancer screening12

FITC-dextran Mouse: analysis of angiogenesis17,22

Molecular imaging of VEGF and EGFR expression

Mouse: analysis of tumor tissue19,20

Crypts

Non living cells

Microvillosity

Vasculature Vasculature

Leakage


PERIPHERAL NERVESMotor endplates


Ex vivo Per iodic control epifluorescence microscopy can only be performed on fixed tissue and with multiple animals.

In vivo & in situ :

Post-crush outgrowth measurement, 4 days after injury

Peripheral Nervous System

Adapted from Vincent et al., EMBO 2006

Thy1.2-YFP16 mouse

Con

trol

C

rush

ed N

erve

Nerve regeneration after crush injury

4 days

Longitudinal monitoring

51

DEEP BRAIN IMAGINGCALCIUM WAVES

Lab


Current techniques can’t reach deep brain structures 2-photon Microscopy Limitation

Addiction

Adult neurogenesis

Alzheimer

ParkinsonHuntington

Light up Deep Brain Neurons

500 µm


Current techniques can’t reach deep brain structures 2-photon Microscopy Limitation

Addiction

Adult neurogenesis

Alzheimer

ParkinsonHuntington

Light up Deep Brain Neurons

500 µm





LOW INVASIVENESS













1

1

3

3

2

2

accesses deep neurons in situ...in a minimally invasive manner


A B

C

D

F G

E

Fig 2 | Fibred fluorescence microscopy allows non-invasive access to the olfactory epithelium and a minimally invasive approach to deep-brain nuclei.

(A) Stereotactic approach for the introduction of the fibre-optic probe (blue) into the brain and for its non-invasive introduction through a nostril

(green arrows). The red-coloured area on the surface of the brain (red arrows) shows the penetration depth achievable with a two-photon microscope,

which also cannot non-invasively access the olfactory epithelium. (B,C) Images from supplementary Movie 2 online showing individual olfactory

neurons (arrows in B) and bundles of the olfactory nerve (arrows in C) of CaMK–eGFP transgenic mice (CaMK, Ca2! /calmodulin-dependent kinase II;

eGFP, enhanced green fluorescent protein). Scale bars, 25 mm (B) and 30 mm (C). (D) In the striatum of CaMK–eGFP transgenic mice, the fibred

fluorescence microscopy probe shows the somata of medium-spiny neurons (arrow) and their surrounding neuropil. Penetration depth, 4mm.

Scale bar, 50 mm. (E) Lentivirus-transduced neurons in the ventral tegmental area (VTA) and individual dendrites (arrows). Penetration depth,

4.5mm. Scale bar, 25mm. (F) Single image from supplementary Movie 3 online showing dopaminergic neurons (arrows) in the VTA. Penetration

depth, 4.5mm. Scale bar, 25 mm. (G) Confocal image of a slice of VTA, sampled immediately after the imaging experiment, showing the trace

left by the 300mm probe used to image the TH–eGFP transgenic mouse used in (D). The arrow points to one GFP-labelled dopaminergic neuron.

Scale bar, 200mm.

Fibred fluorescence microscopy

P. Vincent et al

EMBO reports &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION

scientificreport

4

53 Vincent et al. EMBO, 2006

Response to bicuculline-induced epileptiform activity.

Corresponding EEG

Probe insertion into the Hippocampus

Calcium Imaging

DISCUSSIONWe have shown that FFM is a fibre-optic microscopy approachthat allows the in vivo imaging of fluorescently labelled neuralstructures in the anaesthetized rodent, including in regions that areinaccessible by other technologies. At present, FFM is the only

method for non-invasive access to the olfactory neuro-epitheliumand the olfactory nerve (Mehta et al, 2004); this relates specificallyto the characteristics of the flexible probes it uses. The probe com-prises many optical micro-fibres arranged in a bundle, and has alateral resolution well below 15mm (supplementary Figs 1,2 online).

2

43

65

1

1 2 3 4 5 6

3,000

2,000

1,000

0

1

0

0 50 100 150 200 250

!1

Time (s)

EEG

(mV)

Fluo

resc

ence

(AU

)

a b c

a

b

c

1 s

BicucullineA B

C D

Fig 3 | Fibred fluorescence microscopy monitors changes in intracellular calcium in the hippocampus in response to bicuculline-induced epileptiform

activity. (A) Time course of the fluorescence intensity of Oregon Green BAPTA-1 measured in the four regions indicated by the coloured outlines in

the inset. The radius of the inset image is 490mm. (B) Enlarged views of the recorded region outlined by the white angles in the inset of (A) and

corresponding to the time points indicated in (A). (C) An electroencephalogram (EEG) was recorded simultaneously and bicuculline was injected

intravenously at the time indicated by the arrow in (A). After bicuculline injection, epileptiform activity recorded on the EEG developed synchronously

with the increase in the fluorescence measured by fibred fluorescence microscopy at points 1–6 (A). (D) Magnified portions of the EEG as indicated

by the brackets in (C).


P. Vincent et al

&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports

scientificreport

5

DISCUSSIONWe have shown that FFM is a fibre-optic microscopy approachthat allows the in vivo imaging of fluorescently labelled neuralstructures in the anaesthetized rodent, including in regions that areinaccessible by other technologies. At present, FFM is the only

method for non-invasive access to the olfactory neuro-epitheliumand the olfactory nerve (Mehta et al, 2004); this relates specificallyto the characteristics of the flexible probes it uses. The probe com-prises many optical micro-fibres arranged in a bundle, and has alateral resolution well below 15mm (supplementary Figs 1,2 online).

2

43

65

1

1 2 3 4 5 6

3,000

2,000

1,000

0

1

0

0 50 100 150 200 250

!1

Time (s)

EEG

(mV)

Fluo

resc

ence

(AU

)

a b c

a

b

c

1 s

BicucullineA B

C D

Fig 3 | Fibred fluorescence microscopy monitors changes in intracellular calcium in the hippocampus in response to bicuculline-induced epileptiform

activity. (A) Time course of the fluorescence intensity of Oregon Green BAPTA-1 measured in the four regions indicated by the coloured outlines in

the inset. The radius of the inset image is 490mm. (B) Enlarged views of the recorded region outlined by the white angles in the inset of (A) and

corresponding to the time points indicated in (A). (C) An electroencephalogram (EEG) was recorded simultaneously and bicuculline was injected

intravenously at the time indicated by the arrow in (A). After bicuculline injection, epileptiform activity recorded on the EEG developed synchronously

with the increase in the fluorescence measured by fibred fluorescence microscopy at points 1–6 (A). (D) Magnified portions of the EEG as indicated

by the brackets in (C).


P. Vincent et al

&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports

scientificreport

5

Time course of the Oregon Green BAPTA-1 fluorescence intensity

After bicuculline injection, epileptiform activity recorded on the EEG developed synchronously with the increase in the fluorescence measured by fibred

fluorescence microscopy


Results presented at WMIC 2012 & 2013Dublin, Ireland and Savannah, GA

Calcium ImagingIm

ages

cou

rtes

y of

KU

Leu

ven,

Bel

giu

m

GCaMP3 loaded neural cellsin the olfactory bulb | AAV Vector

S300/B probeStereotaxic frame

Cellvizio 488 with HyperScanCalcium waves monitoring






INTRODUCTION



























12Hz frequency,




time point.































Stat

isti

cs V

alue

s

Time (s) 1206030

800

Kinetics Analysis

Imaging techniques, such as magnetic resonance imaging and positronemission tomography, have provided huge information about thestructure and function of the brain during the last years but the lowresolution and acquisition times limit the information that can beobtained with these techniques.Multiphoton microscopy allows fast and high resolution acquisition inlive rodents. However, it can only access the brain down to about 500µm. Thus, in vivo access has been limited to superficial layers of thecortex or olfactory bulb.A new technology developed by MaunaKea®, called confocal

CALCIUM IMAGING OF SEVERAL CELLS USING A CALCIUM DYE (OGB-1)





In vivo assessment of cell function with confocal endomicroscopyJesus Pascual-Brazo, Chris Van Den Haute, Phebe Van Wijk, Zeger Debyser, Veerle Baekelandt


INTRODUCTION IN VIVO IMAGING WITH CELLULAR RESOLUTION IN VIVO IMAGING OF CALCIUM LEVELS

Calcium imaging with OGB-1 AM. The dye was dissolved in calcium-freeHEPES buffer and injected in the olfactory bulb. One hour after injection,images with a frequency of 40 Hz were recorded.Calcium imaging with GCaMP3. Lentiviral vector expressing GCaMP3 wasstereotaxically injected in the hippocampus. Images were recorded at 40Hzfrecuency. Raw data of signal intensity was plotted for every time point.• The resolution is limited by fiber separation (3 µm)

• Deep brain images with cellular resolution in the alive animal • Confocal images: 15 µm optical sections

• Fast adquisition (up to 200 Hz)• Posibility of use in BEHAVING ANIMALS

A new technology developed by MaunaKea®, called confocalendomicroscopy, is trying to fill the gap between the existing brainimaging techniques. The Cellvizio microscope, based on a fiber opticprobe that transports the emission and fluorescent light to the scanningunit, is able to acquire confocal images with cellular resolution (3 µmaxial resolution) of deep brain regions in live animals. However,expression of reporter proteins with conventional viral vectorsgenerates background during the imaging process. Optimization ofviral vector technology accordingly with the characteristics of thetechnique can improve the quality of the images acquired with thismicroscope.

CALCIUM IMAGING OF SEVERAL CELLS USING GCaMP3 PROTEIN

CONFOCAL ENDOMICROSCOPY

STRIATUM HIGH TITERS STRIATUM LOW TITERS STRIATUM OPTIMIZED

OB HIGH TITERS OB OPTIMIZEDOB LOW TITERS

Hippocampus Olfactory Bulb Striatum

Conventional and optimized viral vectors were stereotactically injected withviral vectors engineered to express GFP in several brain regions, includingolfactory bulb, striatum and hippocampus. Comparison of the signal/noiseratio after conventional (high and low titers) and optimized viral vectorstransduction was carried out. Images were acquired at 12Hz frequency.

• Optimized viral vector technology increased the signal/noise ratio ofconfocal endomicroscopy images in several regions of mouse brain.

• GCaMP3 and OGB-1 allows to record calcium levels of several cellsin live mice.

• Mitochondrial redox state can be monitored in vivo using roGFP.

A plasmid for mito-roGFP was provided by S.J. Remington (University ofOregon,USA). GCaMP3 was obtained from Addgene (L. Looger). This workhas been supported by IWT-SBO/060838 Brainstim, SCIL programmefinancing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.

• Confocal images: 15 µm optical sections

Lentiviral vectors were originally designed for clinical gene therapy.Because their capacity to transfer genes efficiently and stably into mice,this technology has a value to express reporter proteins in the micebrain. Optimized viral vectors were designed and produced taking intoaccount the characteristics of this new microscopy technique.We carried out stereotacxic injections to transduce cells with greenfluorescent protein (GFP) in order to visualize neurons in deep brainregions of anesthetized mice. However, this strategy doesn't allow toobtain information about the cell function. Mutational modifications ofGFP have given rise to calcium sensitive fluorescent proteins asGCaMP3. or redox sensitive roGFP., which have the capacity toresponse with big changes in the emitted light to small variations in thelevels of calcium or the redox state.The combination of viral technology with confocal microscopy allows toobtain information in vivo that has been only reached in vitro.

Redox imaging. Lentiviral vector expressing mitochondria targeted roGFP wasinjected in the hippocampus. ImageCell® software was used to record imagesat a frequency of 12 Hz, for quantification and representation of the intensityof the fluorescent signal. Raw data of signal intensity was plotted for everytime point.

VIRAL VECTOR TECHNOLOGY IN VIVO IMAGING OF MITOCHONDRIAL REDOX STATE CONCLUSIONS

ACKNOWLEDGMENTS

Imaging techniques, such as magnetic resonance imaging and positronemission tomography, have provided huge information about thestructure and function of the brain during the last years but the lowresolution and acquisition times limit the information that can beobtained with these techniques.Multiphoton microscopy allows fast and high resolution acquisition inlive rodents. However, it can only access the brain down to about 500µm. Thus, in vivo access has been limited to superficial layers of thecortex or olfactory bulb.A new technology developed by MaunaKea®, called confocal

CALCIUM IMAGING OF SEVERAL CELLS USING A CALCIUM DYE (OGB-1)





In vivo assessment of cell function with confocal endomicroscopyJesus Pascual-Brazo, Chris Van Den Haute, Phebe Van Wijk, Zeger Debyser, Veerle Baekelandt


INTRODUCTION IN VIVO IMAGING WITH CELLULAR RESOLUTION IN VIVO IMAGING OF CALCIUM LEVELS

Calcium imaging with OGB-1 AM. The dye was dissolved in calcium-freeHEPES buffer and injected in the olfactory bulb. One hour after injection,images with a frequency of 40 Hz were recorded.Calcium imaging with GCaMP3. Lentiviral vector expressing GCaMP3 wasstereotaxically injected in the hippocampus. Images were recorded at 40Hzfrecuency. Raw data of signal intensity was plotted for every time point.• The resolution is limited by fiber separation (3 µm)

• Deep brain images with cellular resolution in the alive animal • Confocal images: 15 µm optical sections

• Fast adquisition (up to 200 Hz)• Posibility of use in BEHAVING ANIMALS

A new technology developed by MaunaKea®, called confocalendomicroscopy, is trying to fill the gap between the existing brainimaging techniques. The Cellvizio microscope, based on a fiber opticprobe that transports the emission and fluorescent light to the scanningunit, is able to acquire confocal images with cellular resolution (3 µmaxial resolution) of deep brain regions in live animals. However,expression of reporter proteins with conventional viral vectorsgenerates background during the imaging process. Optimization ofviral vector technology accordingly with the characteristics of thetechnique can improve the quality of the images acquired with thismicroscope.

CALCIUM IMAGING OF SEVERAL CELLS USING GCaMP3 PROTEIN

CONFOCAL ENDOMICROSCOPY

STRIATUM HIGH TITERS STRIATUM LOW TITERS STRIATUM OPTIMIZED

OB HIGH TITERS OB OPTIMIZEDOB LOW TITERS

Hippocampus Olfactory Bulb Striatum

Conventional and optimized viral vectors were stereotactically injected withviral vectors engineered to express GFP in several brain regions, includingolfactory bulb, striatum and hippocampus. Comparison of the signal/noiseratio after conventional (high and low titers) and optimized viral vectorstransduction was carried out. Images were acquired at 12Hz frequency.

• Optimized viral vector technology increased the signal/noise ratio ofconfocal endomicroscopy images in several regions of mouse brain.

• GCaMP3 and OGB-1 allows to record calcium levels of several cellsin live mice.

• Mitochondrial redox state can be monitored in vivo using roGFP.

A plasmid for mito-roGFP was provided by S.J. Remington (University ofOregon,USA). GCaMP3 was obtained from Addgene (L. Looger). This workhas been supported by IWT-SBO/060838 Brainstim, SCIL programmefinancing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.

• Confocal images: 15 µm optical sections

Lentiviral vectors were originally designed for clinical gene therapy.Because their capacity to transfer genes efficiently and stably into mice,this technology has a value to express reporter proteins in the micebrain. Optimized viral vectors were designed and produced taking intoaccount the characteristics of this new microscopy technique.We carried out stereotacxic injections to transduce cells with greenfluorescent protein (GFP) in order to visualize neurons in deep brainregions of anesthetized mice. However, this strategy doesn't allow toobtain information about the cell function. Mutational modifications ofGFP have given rise to calcium sensitive fluorescent proteins asGCaMP3. or redox sensitive roGFP., which have the capacity toresponse with big changes in the emitted light to small variations in thelevels of calcium or the redox state.The combination of viral technology with confocal microscopy allows toobtain information in vivo that has been only reached in vitro.

Redox imaging. Lentiviral vector expressing mitochondria targeted roGFP wasinjected in the hippocampus. ImageCell® software was used to record imagesat a frequency of 12 Hz, for quantification and representation of the intensityof the fluorescent signal. Raw data of signal intensity was plotted for everytime point.

VIRAL VECTOR TECHNOLOGY IN VIVO IMAGING OF MITOCHONDRIAL REDOX STATE CONCLUSIONS

ACKNOWLEDGMENTS




S300/BStereotaxic frame

HyperScan

S


Losartan prevents acquired epilepsy via TGF-β signaling suppression

55

Epilepsy - TGF-ß Modulation

Bar-Klein, Friedman et al, 2014

• WT Wistar• Evans Blue (Red)

• Vasculature• Alexa 488-Albumin

• Uptaken in Astrocytes

• Cellvizio Dual Band• Z1800 microprobe

• Epilepsy is frequently associated with dysfunction of the BBB

• Albumin-triggered TGF-ß signaling in astrocytes is epileptogenic

• Losartan is FDA-approved and acts as an anti-epileptogenic by blocking the TGF-ß pathway


Genetic Disruption of Dopamine Activity Patterns

56

8

Cellvizio demonstrates thathSK3D Increases Evoked

Calcium Signals and Dopamine ReleaseSoden et al. Neuron, 2013

• The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns.

• Expression of a human disease-related SK3 mutation (hSK3D) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity.

• hSK3D increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release.

• These results demonstrate the cell-autonomous e!ects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior.

this effect was reversed by acute treatment with the antipsy-chotic dopamine D2 receptor antagonist haloperidol (Figure 6F).No differences were detected in baseline startle responsesbetween groups (Figure S6).To establish whether disruption of sensory gating caused by

expression of hSK3D is related to changes in dopamine neuronactivity, we used a genetic mouse line in which activationof dopamine neurons can be induced rapidly and reversibly(Guler et al., 2012). In this model, TRPV1, a calcium-permeablecation channel, is expressed exclusively in dopamine neurons(TRPV1-DA mice; Figure 6G), and moderate doses of theTRPV1 agonist capsaicin, a component of chili peppers, in-crease dopamine burst firing (Guler et al., 2012). The effects ofcapsaicin are transient, lasting only 15–20 min (Guler et al.,2012); therefore, we assessed PPI in TRPV1-DA mice with anabbreviated protocol using only one prepulse volume (75 dB).Injection of capsaicin immediately prior to testing reduced PPIcompared to vehicle injections in the same animals (Figure 6H).As in hSK3DGFP-expressing mice, this reduction was blockedby haloperidol (Figure 6H).

Expression of hSK3D Potentiates PsychomotorActivationDisruption of sensory-motor gating in hSK3D-expressing mice isconsistent with an alteration in corticostriatal dopamine signaling(Swerdlow et al., 1994). In addition to impairing PPI, enhanceddopamine release can also potentiate behavioral responses topsychomimetic drugs, such as MK-801 (Gainetdinov et al.,2001), in part through a glutamate- and dopamine-dependentcorticomeso feedback loop (Moghaddam et al., 1997). To estab-lish whether altered dopamine activity and potentiated dopa-mine release associated with hSK3D expression alters sensitivityto a psychomimetic drug, we monitored locomotor responses inhSK3DGFP- or GFP-expressing mice before and after adminis-tration of low doses of MK-801. Systemic administration ofMK-801 significantly enhanced locomotion in hSK3DGFP micerelative to GFP controls. This effect was blocked by pretreatmentwith haloperidol (Figures 7A and 7B). Consistent with the dopa-mine-independent nature of high doses of MK-801 (Chartoffet al., 2005), we did not observe a significant effect of hSK3DGFPexpression on locomotion at 0.5 mg/kg MK-801 (Figure S7).

Figure 5. hSK3D Increases Evoked Calcium Signals and Dopamine Release(A) Pseudocolor image obtained with fiber-optic probe of VTA dopamine neurons expressing GCaMP3. Scale bar, 40 mm. Right (top to bottom): single neuron

before, during, and after PPTg stimulation. Scale bar, 10 mm.

(B) Average changes in GCaMP3 fluorescence intensity in the VTA after PPTg stimulation at indicated stimulus intensities (stimulus onset at time 0; control: n = 26

cells/3 mice; hSK3DHA: n = 14 cells/3 mice).

(C) Quantification of area under the curve of the fluorescence signal (two-way RM ANOVA, virus 3 time, F(3,114) = 9.74, p < 0.001; Bonferroni post hoc analysis:

****p < 0.0001).

(D) Example pseudocolor plot depicting changes in redox currents in the nucelus accumbens evoked by PPTg stimulation as a function of applied potential over

time. Right, example voltammograms of stimulated DA release in mice expressing GFP (black) or hSK3DGFP (red).

(E) Average DA oxidation currents after PPTg stimulation at indicated stimulus durations (400 mA stimulus intensity; stimulus onset at time 0; stimulus artifacts

removed for ease of viewing; n = 3 mice per group).

(F) Peak DA oxidation currents (two-way RM ANOVA, virus 3 duration, F(6,24) = 3.77, p < 0.01; Bonferroni post hoc analysis: ***p < 0.001, **p < 0.01, *p < 0.05).

Bars represent mean ± SEM. See also Figure S5.

Neuron

Genetic Disruption of Dopamine Activity Patterns

Neuron 80, 1–13, November 20, 2013 ª2013 Elsevier Inc. 7

Please cite this article in press as: Soden et al., Disruption of Dopamine Neuron Activity Pattern Regulation through Selective Expression of a HumanKCNN3 Mutation, Neuron (2013), http://dx.doi.org/10.1016/j.neuron.2013.07.044

PPTg stimulation and imaging of VTA dopamine neurons in an anesthetized mouse

VTA dopamine neurons expressing GCaMP3.

VTA

this

effe

ctwasreve

rsed

byacute

treatm

entwith

theantip

sy-chotic

dopamineD2receptorantagonist

haloperid

ol(F

igure

6F).

No

diffe

rences

were

detected

inbase

line

startle

resp

onse

sbetw

eengroups(Figure

S6).

Toesta

blish

whetherdisru

ptio

nofse

nso

rygatin

gcause

dby

exp

ressio

nofhSK3Dis

relatedto

changesin

dopamineneuron

activity,

we

use

da

genetic

mouse

line

inwhich

activa

tion

ofdopamine

neurons

can

be

induced

rapidly

and

reve

rsibly

(Guleretal.,

2012).In

this

model,TRPV1,acalcium-perm

eable

catio

nchannel,is

exp

resse

dexc

lusive

lyin

dopamineneurons

(TRPV1-D

Amice;Figure

6G),

and

moderate

dose

softhe

TRPV1

agonist

capsa

icin,a

componentofchili

peppers,

in-

crease

dopamineburst

firing(G

uleretal.,

2012).Theeffe

cts

of

capsa

icin

are

transie

nt,

lastin

gonly

15–2

0min

(Guleretal.,

2012);therefore,weasse

ssedPPIin

TRPV1-D

Amicewith

an

abbrevia

tedprotocolusin

gonly

oneprepulse

volume(75dB).

Injectio

nofcapsa

icin

immediately

prio

rto

testin

greducedPPI

comparedto

vehicle

injectio

nsin

thesa

meanim

als(Figure

6H).

Asin

hSK3DGFP-exp

ressin

gmice,this

reductio

nwasblocke

dbyhaloperid

ol(F

igure

6H).

Exp

ressio

nofhSK3D

Potentia

tesPsyc

homotor

Activa

tion

Disru

ptio

nofse

nso

ry-motorgatin

ginhSK3D-exp

ressin

gmiceis

consiste

ntw

ithanalte

ratio

nincortic

ostria

tald

opaminesig

naling

(Swerdlow

etal.,

1994).In

additio

nto

impairin

gPPI,enhanced

dopaminerelease

canalso

potentia

tebehavio

ralresp

onse

sto

psyc

homim

etic

drugs,

such

as

MK-801

(Gainetdinov

etal.,

2001),in

part

throughaglutamate-anddopamine-dependent

cortic

omeso

feedbackloop(M

oghaddam

eta

l.,1997).T

oesta

b-

lishwhetheralte

red

dopamineactivity

and

potentia

ted

dopa-

minerelease

asso

ciatedwith

hSK3Dexp

ressio

nalte

rsse

nsitivity

toapsyc

homim

etic

drug,w

emonito

redlocomotorresp

onse

sin

hSK3DGFP-orGFP-exp

ressin

gmicebefore

andafte

radminis-

tratio

noflow

dose

sofMK-801.Syste

mic

administra

tion

of

MK-801sig

nific

antly

enhancedlocomotio

nin

hSK3DGFPmice

relative

toGFPcontro

ls.Thiseffe

ctw

asblocke

dbypretre

atm

ent

with

haloperid

ol(F

igures7Aand7B).Consiste

ntwith

thedopa-

mine-in

dependentnature

ofhigh

dose

sofMK-801

(Charto

ffeta

l.,2005),w

edid

noto

bse

rveasig

nific

ante

ffecto

fhSK3DGFP

exp

ressio

non

locomotio

nat0.5

mg/kg

MK-801

(Figure

S7).

Figure

5.hSK3D

Increase

sEvo

kedCalcium

Signals

andDopamineRelease

(A)Pse

udocolorim

ageobtainedwith

fiber-o

ptic

probeofVTAdopamineneuronsexp

ressin

gGCaMP3.Scale

bar,40mm.Right(to

pto

botto

m):sin

gle

neuron

before,durin

g,andafte

rPPTgstim

ulatio

n.Scale

bar,10mm.

(B)A

veragechangesinGCaMP3flu

oresc

enceintensity

intheVTAafte

rPPTgstim

ulatio

natin

dicatedstim

ulusintensitie

s(stim

ulusonse

tattim

e0;c

ontro

l:n=26

cells/3

mice;hSK3DHA:n=14cells/3

mice).

(C)Q

uantific

atio

nofareaunderthecurve

oftheflu

oresc

encesig

nal(tw

o-w

ayRM

ANOVA,viru

s3

time,F(3,114) =

9.74,p<0.001;Bonferro

nip

ost

hocanalysis:

****p<0.0001).

(D)E

xamplepse

udocolorplotdepictin

gchangesinredoxcurre

nts

inthenucelusaccumbensevo

kedbyPPTgstim

ulatio

nasafunctio

nofa

ppliedpotentia

love

r

time.Right,exa

mple

volta

mmogramsofstim

ulatedDArelease

inmiceexp

ressin

gGFP(black)

orhSK3DGFP(re

d).

(E)Ave

rageDAoxid

atio

ncurre

nts

afte

rPPTgstim

ulatio

natindicatedstim

ulusduratio

ns(400mAstim

ulusintensity;

stimulusonse

tattim

e0;stim

ulusartifa

cts

remove

dforease

ofvie

wing;n=3micepergroup).

(F)P

eakDAoxid

atio

ncurre

nts

(two-w

ayRM

ANOVA,viru

s3

duratio

n,F(6,24) =

3.77,p<0.01;Bonferro

nip

ost

hocanalysis:

***p<0.001,**p

<0.01,*p

<0.05).

Bars

represe

ntmean±SEM.Seealso

Figure

S5.

Neuron

Genetic

Disru

ptio

nofDopamineActivity

Patte

rns

Neuron80,1–1

3,Nove

mber20,2013ª2013Else

vierInc.

7

Please

citethis

articlein

press

as:Soden

etal.,

Disrup

tionofDopam

ineNeuro

nActivity

Pattern

Reg

ulationthro

ughSelective

Exp

ressionofaHum

anKCNN3Mutatio

n,Neuro

n(2013),http

://dx.d

oi.o

rg/10.1016/j.n

euron.2

013.07.044

-85 350


Blood flow analysis in epilepsy

Leal-Campanario et al., Neurostereology: Unbiased Stereology of Neural Systems, 2013

Simultaneous hyperemic focus and hypoxia

ROIs

• Hippocampal vasospasms create a hyperemic influx, then discarded in hyperoxic draining veins

• Smaller diameter capillaries get blocked due to that increased flow and pericyte constriction

• Cells near ischemic capillaries become hypoxic and add to the cell death due to excitotoxicity-driven apoptosis

• In vivo observation of hippocampus capillaries vasospasms in awake mice.• Fluorescence changes measurement over time for each vessel.

• Kv1.1 KO mice: the only existing genetic model of human epilepsy(episodic ataxia type 1) (Zuberi et al., 1999)

• 9% vessels exhibit pericyte-driven vasospasms in KO (2% in WT)

• Vessels stained with fluorescein dextran

pericytes

BLOOD FLOW ANALYSIS IN EPILEPSY USING A NOVEL STEREOLOGICAL APPROACH 167

of vasospasms per vessel expressed as a percentage of the total recording time per vessel (t(1100) = 6.05; p < 1 × 10−8, labeled *8) and the average vasospasm magnitude (t(81) = 2.00; p = 0.049, labeled *). There was no difference between cohorts for the average vasospasm dura-tion (t(55) = 0.52; p = 0.61), or the average vasospasm onset (t(52) = 0.93; p = 0.36) and termina-tion speeds (t(36) = −0.084; p = 0.40). See Figure 12.5.

Kv1.1 KO Mice versus WT Stereology

We found AIF+ cells more numerous in KO mice than in WT mice (Figure 12.12a,b). AIF+ cells were, on average, 15.17% (+/−0.07% s.e.m.) nearer to blood vessels than AIF− cells (χ2(1,

Figure 12.10 Fiber-coupled laser-scanning confocal imaging of hippocampal capillaries. (a) Eight sequential images of a length of capillary, recorded at 87.5 Hz. The arrows indicate a pair of red blood cells (black) flowing as a cluster to the right at 219 μm/s. (b) An occluded capillary releases from vasospasm rapidly and then slowly reconstricts over a 10-second period. Recorded at 11.7 Hz. (c) A later vasospasm releases in the same vessel, now recorded at 26.8 Hz. (d) An occlusion of a capillary due to a pericyte contraction in a KO mouse. The arrows point to pericytes (1 and 2). Pericyte 2 at first causes the vasospasm, and then releases at ∼26 seconds. (e) High-speed (87.5 Hz) analysis of ictal vasospasms leading to red blood cell blockages in a capillary network. First frame (left) shows serum flow through the vessel network (black arrows). Eight seconds later, a vessel constriction appears (white arrow) due to an external (unstained) pericyte. Red blood cells clog the anastomosis (red arrows) over the fol-lowing 40 seconds. The vessel then reopens and flows freely for ∼5 minutes, followed by a second constriction event and a red blood cell blockage throughout the local network (yellow arrows). (f) Vasospasm of a WT mouse capillary (white arrow points to a pericyte shadow in the capillary), which begins the recording in vasospasm, releasing at time of 60 seconds, then vasospas-ming again at time of ∼86 seconds, to release again at time of ∼114 seconds. Scale = 20 μm (all panels).

a

d

e

f

b

c

An occluded capillary releases from vasospasm rapidly and then slowly reconstricts over a 10-second period. Recorded at 11.7Hz.


Ultrasound triggered BBB disruption

58

Application:Local Drug Delivery

BBB Disruption:MiniZ probeFITC-Dextran 30, 50, 70, 150 kDa rangeFocalized Ultrasound applicationSonoVue® Microbubbles injection


NZW Rabbit(WT)

End Sonication

Continuous Cellvizio recording

Time(min)


Ultrasound triggered BBB disruption

59

Application:Local Drug Delivery

Kinetic Analysis


NeuroPak™ : Freely-moving imaging!

60

• A full solution

• A set of tools, including a stereotaxic holder

• Compatible with any stereotaxic frame

• Ultra light implants: only 0,3 g

• Reach neuronal activity in situ in real time

• Chronical, logitudinal deep brain imaging

• Max depth: 10 mm

• Minimal invasiveness

• 4 m long flexible image guide


NeuroPak™Freely-moving imaging



SeriesS


( )

62


+ x

Hippocampal neuronsCa2+ imaging

Behavioral studiesex: open field

Rodent modelMouse and Rat

NeuroPak™Full solution

Mouse

Rat

=

Real time12-200 fps

Stereotaxic Frame

63

When access matters....

Druart at al, Reproduction Research, 2009

In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tractM


360+ Cellvizio systems worldwide

Regulatory approvals in 40+ countries

Proven E!ectiveness & Global Footprint

66

SCIENTIFIC & CLINICALPUBLICATIONS & STUDIES300 +


Take away messages

67

is the key modality that brings you the missing piece of information

• Cellular resolution brought to in vivo imaging • From surface to deep tissues• Minimally invasive, Maximally informative• Longitudinal studies• Easy-to-use, fastest learning curve• Cost e"ective

Deep Brain Angiogenesis GI disorders Stem Cells

Cellvizio Lab main areas of research

Functionalimaging Biodistribution

IMAGING SOLUTIONS OVERVIEW

VITROINVASIVETISSUE SAMPLE CELL CULTUREDIGITAL PATHOLOGY BIOPSY

NO IMMEDIATE ANSWER

PET/CT/SPECT

UltrasoundPhotoacousticsVEVO LAZR

FluoBeamnIR Camera

MRI/µMRI

WHOLE BODY OR ORGAN LEVEL

IVIS SPECTRUM

SURFACEDEEP ACCESS

2-PHOTON

OPTICAL BIOPSYAT CELLULAR RESOLUTION

pCLE

HIGH RESOLUTION


NON OR MINIMALLY INVASIVE

IN VIVOTRANSLATIONAL MEDICINE

DYNAMIC MOLECULAR IMAGING

LONGITUDINAL STUDIES

HIGH & SUPER RESOLUTION

CONFOCAL SLIDE SCANNER

FLUO&WHITE LIGHTMICROSCOPY

2 PHOTONMICROSCOPY

cellvizio lab

Documents