Microspectroscopieetimagerievibrationnelles:desoutilsphotoniquesd’intérêt

enbiologieetdansledomainebiomédical

ValérieUntereiner

CHU DE REIMS

BioSpecT(BioSpectroscopieTranslationnelle),EA7506Plate-formed’ImagerieCellulaireetTissulaire(PICT)

UniversitédeReimsChampagne-Ardenne

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Wavenumber(∝Frequency∝Energy)(cm-1)

Absorbance(u

.a.)

Interestofvibrationalbiospectroscopy

2

CH2:asymetricstretching

CH2:symetricstretching

CH3:umbrellabend

CH2:scissorbend

CH2:rocking

Nondestructivelight/matterinteraction

Label-free

Micrometerresolution

Vibrationalmicro-spectroscopies

Ramanscattering

Sourcelaser

Infraredabsorption

SourceIR

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Insituanalysisofthechemicalbonds,withoutalteringtheintegrityofthesample

Highmolecularandstructuralspecificity

IRspectrumofabiologicalsample

1656cm-1 1669cm-1 1665cm-1

Secondarystructureofproteins

Interestofvibrationalbiospectroscopy

Ourcoreexpertise:vibrationalspectroscopyforbiologicalapplicationsandbiomedicaltranslation

Vibrationalspectrum=Molecularsignatureofthesampleinaspecificphysiologicalorpathophysiologicalstate

Exvivoandinvivosamples

Cell,tissue,biofluid

Live,frozenorfixed

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Acquisition Dataprocessing

Detectionandcharacterizationofphysiologicalor

pathologicalconditions

Identificationofspectroscopicbiomarkers

Discriminantvibrationsindependentofthebiologicalvariabilityusingmultivariatestatisticaldataprocessing

Ramanspectralanalysisonlivercells

Infraredspectralanalysisoncolontissue

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Vibrationalspectroscopyforbiologicalapplicationsandbiomedicaltranslation

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Ramanimagingofasingleliversinusoidalendothelialcell(LSEC)

SzafraniecEetal.,2019

Ø  Colon-lastpartofthedigestivesystem

Ø  Cecum,colon,rectumandanalcanal-largeintestine

ColonanatomyandhistologyHEstainedsections(non-tumoral)

(a)Transverse(b)Longitudinal

Source:BlueHistology,Univ.ofWesternAustralia

(a)

(b)

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(Source:cancerinformaiton.org.uk)

HEstained

ColorectalcancerØ  Lossoffunctionalcontrolinthenormalcellsliningthecolonorrectum

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Candidatemethod:infraredspectralimaging

Ø  Screeningmethods:Fecaloccultbloodtest(FOBT),colonoscopyandsigmoidoscopy,etc

Currentscreeninganddiagnosticmethods

Presentneed:Newerclassificationmethodsprovidingbiomolecularinformationfromcellsandtissuescomplementarytohistopathology

Diagnosisconfirmation:HISTOPATHOLOGY

Automatedapproachesforrapiddiagnosis

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10

X

σ

Y

hyperspectralimageDatacube(X!Y!σ)!

Spectralimaging

Tensofthousandsofspectra/image

Pixelsize:1–10µm

Uncolouredtissuesection,

possiblyparaffin-embedded

Millimeter

Millim

eter

HPSstainedtissue(5Xmagnification)

K-meansclusteredIRimage

Non

-tum

oraltissue

Tu

moraltissue

K-meansclusteringresultsoftheIRspectralimagesusingrandompseudo-colors

Crypts(innerpart)Crypts(outerpart)LaminapropriaSubmucosa

TumorTumorassociatedstroma

Others-notattributedtoany

histologicalclass

Histologicalattribution

Annotationbyanexpertpathologist

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Libraryofspectralmarkers

Spectrafromdifferentcolonichistologicalclasses

1 Tumorepithelium

2 Crypt-outerpart

3 Crypt-innerpart

4 Muscularispropria

5 Tumorassociatedstroma

6 Laminapropria

7 Submucosa

8 Bloodvessel

Confusionmatrix

1080-1300cm-1

Constructionandapplicationofthepredictionmodelbasedonlineardiscriminantanalysis

Internalvalidation

(1/3rdspectra)

Trainingset(2/3rdspectra)

Predictionmodel

Testset(identificationofunknownsamples)

(N=71samples)

posteriorprobability0.5

(N=9samples)

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EXTERNALVALIDATIONIdentificationofunknowncolonictissuesbyspectralhistopathology

(No.ofspectratested:3620287)

HPSstainedcolontissue(5Xmagnification)

IR-LDApredictedimage

Nontumoral

Finaldiagnosis

Adenocarcinoma

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Individualsatriskofthedisease Diseaseatan

earlystage

Overtdisease

Reponsetotreatment

Personalizedtreatment

Recurrence

Spectroscopicbiomarkers Diagnosis

Prognosis

Predictionofthetherapeuticresponse

Monitoring

ScreeningScreening

Translationaldevelopmentofvibrationalspectroscopy

Monitoring

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EquipeBioSpecT:BioSpectroscopieTranslationnelle

Plate-formeIBiSAd’ImagerieCellulaireetTissulaire(PICT)