Toolbox for in vivo imaging of host–parasite interactions at multiple scales

Toolbox

for

_In

_Vivo

Imaging

of

_{Host–Parasite}

Interactions

at

Multiple

Scales

Mariana

De

Niz,

* Florentin

Spadin,

Matthias

Marti,

Jens

V.

Stein,

Martin

Frenz,

and

Friedrich

Frischknecht

Animalmodelshaveforlongbeenpivotalforparasitologyresearch.Overthe

last few years, techniques such as intravital, optoacoustic and magnetic

resonance imaging, optical projection tomography, and selective plane

illuminationmicroscopydevelopedpromisingpotentialforgaininginsights

intohost–pathogen interactionsbyallowingdifferentvisualization formsin

vivo and ex vivo. Advances including increased resolution, penetration

depth,andacquisition speed, togetherwithmore complex imageanalysis

methods, facilitate tackling biological problems previously impossible to

study and/orquantify. Here wediscussadvances andchallenges in thein

vivo imaging toolbox, which hold promising potential for the field of

parasitology.

ImagingToolbox inParasitology

Imagingtechniquesdevelopedforbiomedicalapplicationshave hadanimportantimpactin parasitologyresearch.Suchtechniquesincludeplatformsdevelopedtoimagehost–pathogen interactionsat variousscales, rangingfrom molecules to wholeorganisms (summarizedin

Table1).Thesetechniqueshavecomplementaryadvantageswithrespecttoeachother.This

reviewfocusesonthetechnologicaladvancesusedforvisualizationofhost–pathogen inter-actionseither invivo, orex vivo inwhole organismsinseveral imaging techniques. These techniques are based onnonionizing radiationsuch as intravital microscopy (IVM), optical projection tomography (OPT), bioluminescence imaging, optoacoustic imaging (OAI), and magneticresonanceimaging (MRI),oronionizingradiationsuchas X-raymicro-computed tomography (micro-CT), positron emission tomography (PET), or single-photon emission computedtomography(SPECT).

Advanced FluorescenceMethodsAppliedtoIVM

IVMisa powerfultechniquefor investigatingdynamiccellularprocessesandhost–parasite interactionswithinfunctioningorgans.OrgansstudiedbyIVMinthecontextofparasitology includethebrain[1–4],theskin[5–12],thelungs[13],theliver[14–18],thespleen[19,20],the intestines[21,22],thelymphnodes[23,24],thebonemarrow[25],theadiposetissue[20],and theplacenta[26,27],(summarizedinTable2).Importantadvancesinparasitologyhavebeen achievedusingwide-fieldepifluorescence,confocal,spinning-disc,ortwo-photonIVM.Recent developments,whichhaveexpandedtheapplicationsofIVMincludethegenerationoflonger wavelength excitation lasers; a wider range of fluorescent reporters for dyes; transgenic fluorescencereportermiceandparasites;fluorescencelifetimemeasurements(see Glos-sary);adaptiveoptics;imagingwindowsandmicroendoscopes,butalsosoftware advan-ces for high-throughput automated analyses and motion correction. Moreover, for fixed specimens,tissue-clearingtechniquesprovidingdeeperopticalpenetrationintoorganshave openednewavenuesofresearch[28].

Highlights

TechnologicaladvancesinIVMinclude

ultrafast tunable infrared lasers,

super-resolutionimaginginvivo,and

better access to all organs by

improvedopticsormotion-correction

mechanisms.

Opticalclearancemethodsforusein

OPT andSPIM allowimagingentire

organsandorganisms(suchasmice

orinsectvectors),whileachieving

cel-lularandsubcellularresolution.

OAIcombinesopticsandacousticsvia

thethermoelasticeffecttoinvestigate

molecularmechanisms.

KeyadvancesinMRIincludeimproved

contrast agents, multimodal

meso-scopic imaging, and functional MRI

tovisualizethemetabolicfunctionof

tissues.

Developments in bioluminescence

imaging include red-shifted probes

withhighsensitivity;alargerdiversity

ofprobeswithemissionpeaksat

var-iouswavelengths;ultrabrightprobes,

andtheuseofBRETinvivo.

X-ray-based and gamma-ray-based

imaging platforms are noninvasive

and enablethe studyofanatomical

structures, metabolism, biochemical

compositionoftissues,anddynamics.

1

InstituteofCellBiology–Heussler

Laboratory,UniversityofBern,Bern,

Switzerland

2

WellcomeCentreforMolecular

Parasitology,UniversityofGlasgow,

Glasgow,Scotland,UK

3

InstituteofAppliedPhysics,

UniversityofBern,Bern,Switzerland

4

TheodorKocherInstitute,University

http://doc.rero.ch

Published in "Trends in Parasitology 35(3): 193–212, 2019"

which should be cited to refer to this work.

LongerwavelengthexcitationlasersoffersubstantialadvantagesforIVM.Wavelengthsinthe 700–1000nmexcitation rangeareto some extentlimitedby penetrationdepthand back-groundemissionfromtissueendogenoussignals.Ultrafasttuneableinfraredlasersarenow availablewhichenableimagingat1300nm,overcomingbothproblems[29].Longer wave-lengthlightislessscatteredandabsorbedbytissues,aslongasthewavelengthisshorterthan thewaterabsorptionpeaks,resultingindeeperopticalpenetration.Thisnewgenerationof lasers, coupledwith novel far-redprobes, takeadvantageof wavelengthsthat elicit fewer autofluorescencesignals,thushelpingtoimprovethesignal-to-noiseratio.Moreover, photo-toxicitytotissuesisbelievedtobereducedatlongerwavelengths[30].Further,wavelength mixingofsynchronizedlasers,ormultiphotonexcitation–achievedeitherviadual-outputlaser sourcesorspecializedlaserssuchastherecentlyreportedfemtoseconddiamondRamanlaser

[31]–enablemulticolourimaging,expandingtherangeofstructuresthatcanbestudiedinvivo byIVM.Inparallel, awiderangeoffluorescentprobesintheform ofimmunofluorophores, genetic tags, quantum dots, and organic dyes have been developed to study dynamic processesinvivo,including protein–proteininteractions,andhost–pathogeninteractionsin healthyanddiseasedstates(reviewedin[32,33]).

Microscopytoolsthatare keytoinvestigatingcellorproteindynamics,andinteractionsin vitro,haveinrecentyearssuccessfullypermeatedinto IVM(reviewedin[34]).Examplesof thesemethodsincludeFörsterresonanceenergytransfer(FRET),fluorescencelossin photobleaching (FLIP), and fluorescence recovery after photobleaching (FRAP). FRETinmicehasbeenachievedbyeitheroftwomethods:generationoftransgenicanimals withchromophoresactingaspartnersforresonanceenergytransfer,ortransfectionofcells followedbytheirtransferinto livinganimals[35].FRETposes importantchallengesinIVM, which are usually not encountered in in vitro conditions. These include signal strength limitations,photobleachingandphotodamage.Initially,FRETwasmostlyusedasa ratio-metrictechniqueinIVM,allowingthemeasurementofrelativechangesofdifferent param-etersofinterestathighspeed,generallyusingthefluorescentproteinsceruleanandcitrineas donorandacceptorfluorophores,respectively.DespitethevalueofratiometricFRET,oneof itsmainlimitationsisitsdependenceonrelativequantificationsratherthanabsolute measure-ments.Sofar,FREThasnotbeenreportedinvivoinparasitology,yetcouldbeveryusefulto monitorthe activity of kinases,proteases,and GTPases [36];calcium changes[37]; lipid concentrations[35,38–40]duringinfection;andevenphysicalstressessuchastemperature, mechanical stressors, or electromagnetic fields [41], as well as molecular interactions between host and pathogens; host cell subsets, or parasite molecules. Likewise, there hasbeensignificantprogressinthedevelopmentofsoftwaretoallowseparatequantification ofsignalsfromsinglecellsincomplex3Denvironments,suchasthosepresentinorgansof livinganimals[42,43].

AnothersetoffluorescencemethodsincreasinglyadaptedtoIVMareFRAPandFLIP.Both techniques are generally used to monitor the molecular movement within cells, and are amenabletorepeateduseinvivowithoutcausingtissuedamage.Theyareparticularlyuseful toassessmacromolecularfluxacrosscellsandtissues.Bothmethodsyieldinformationsuch ashalf-timetorecovery,rateofmovementoffluorescentmoleculeswithinandbetweencellular compartments,andmoleculartransferbetweenregionsindependentofrateofmovement.Like FRET,FRAPandFLIPhavebeenlargelyexploredin2Denvironmentsusingcellcultures,but haveonlyrecentlybeenincorporatedintoinvivoresearch[34].Thishasbeenpossibledueto thegenerationoftransgenicmicewithfluorescentreportersexpressedinspecificcelltypes, cell–celljunctions,orotherstructures.Particularlyusefulforthestudyofparasitecellmigration, invasion, and development, wouldbe reporter micethat enable quantification of dynamic

ofBern,Bern,Switzerland

5

DepartmentofOncology,

MicrobiologyandImmunology,

UniversityofFribourg,Fribourg,

Switzerland

6

IntegrativeParasitology,Centrefor

InfectiousDiseases,Heidelberg

UniversityMedicalSchool,Heidelberg,

Germany

7

Currentaffiliation:Institutode

MedicinaMolecular–JoãoLobo

Antunes,FaculdadedeMedicina,

UniversidadedeLisboa,Lisbon,

Portugal

8

Theseauthorscontributedequally

*Correspondence:

[email protected]

(M.DeNiz).

Glossary

Adaptiveoptics:anopticalmethod

wherebydeformablemirrorscan

correct,inrealtime,opticaleffects

causedbyincomingwavefront

distortions.

Elastography:amedicalimaging

methodthatcreatesadistortionin

tissue,andthenmeasuresthetissue

response(e.g.,mechanicalwaves)to

theoriginaldeformation.

Fluorescencelifetime:thetime

thatafluorophorecanspendinan

excitedstatebeforeemittinga

photonandreturningtotheground

state.

Fluorescencelossin

photobleaching(FLIP):animaging

methodwherebyafluorescently

labelledregion(usuallyamembrane)

isbleachedmultipletimesusingthe

laserbeamofaconfocal

microscope,toensurethatall

fluorophoresarebleached.

Measurementoffluorophore

exchangebetweennonbleachedand

bleachedareaovertimeprovides

informationonmembranedynamics.

Fluorescencerecoveryafter

photobleaching(FRAP):an

imagingmethodthatconsistsof

targetingasampleareawithasingle

high-intensityillumination,resultingin

theelapseofthefluorophore’s

fluorescencelifetime.Overtime,

fluorescentprobeswilldiffuse

throughthesampletoreplacethose

innonfluorescentareas,thus

allowingquantificationoflateral

diffusion.

Försterresonanceenergy

transfer(FRET):amechanismof

energytransferbetweenadonorand

anacceptorchromophore.Efficiency

ofenergytransferishighlylinkedto

(i)thedistancebetweentwo

fluorophores,(ii)thespectraloverlap

ofdonorandacceptor,and(iii)the

relativeorientationofdonorand

acceptordipolemoments.

Gradedindex(GRIN)lenses:

lensesofdifferentmaterialsthat

reduceopticalaberrationsbyhaving

arefractiveindexgradient,andaflat

surface.

Imagingwindows/optical

windows:surgicallyimplanted

windowsthatallowvisualizationof

internalorgansinsidealivingmouse.

Windowscanbeacute/terminal,or

chronic,allowingvisualizationover

days–weeks.

regulationofcell–celljunctions(e.g.,E-cadherin–GFP)[44],orstudyingmicrovesselleakiness asaproxyofendothelialbarrierfunctionusingdyessuchasfluoresceinisothiocyanate-dextran oralbumin[45].

Super-resolutionmicroscopyhasgainedsignificantmomentumforuseininvitroimagingover thepastdecade,anditsapplicationinIVMcontextsbringsaboutequallyexcitingprospects. OnelimitationthatpreventeditsfasteradaptationtoIVMisthelightscatteringwhichresultsin depth-dependentdeteriorationofspatialresolution,duetosphericalaberrationstothepoint spreadfunctionincomplextissue[46].Althoughnotyetusedinthefieldofparasitology,other fieldshavedevelopedmethodstoachievesuper-resolutionmicroscopywhileimagingliving animals.Examplesincludearecentlydevelopedsetupwhichincorporatesmultibeamstriped illumination,spatialmodulationofexcitation,andlaserscanningmicroscopy[47],ortheuseof stimulatedemissiondepletion(STED)toachievesuper-resolutiontoobserve morphologi-calchangesinactinwithinthebrain[48].

InadditiontomicroscopymethodsapplicabletoIVM,theuseofimplantedopticalwindowsto replacetheskull,andmicroendoscopes,hasgreatlybenefitedvariousresearchfields,including parasitology,toenablelong-termimagingoforgansthroughoutmultipledays(reviewedin[49]). While most optical windows are limited to the surface of the surgically exposed organs, gradient index (GRIN) lenses and microendoscopes enable access to deeper regions

[50], otherwise inaccessible, including deeper structuresin the brain, the spinal cord, the bonemarrow,thelungs,andtheheart[50–53].Bothmicroendoscopesandtemporaryoptical windowshave benefitedtheimaging ofparasites,giving insightsintophenomenaincluding parasitecrossingofendothelialbarriersenablingtheirdisseminationacrosstissues,dynamics relevanttoparasitesurvival,andmechanismsoftransmissiontoandfromtheinsectvectors. Furtherprogressinpenetrationdepthandresolutioninlivingsampleshasbeenachievedby adaptiveoptics,whichiswidelyappliedinastronomy.Biologicalspecimenshave refractive-indexinhomogeneitieswhichaffectresolution.Adaptiveopticsallowsreversing signal/resolu-tion degradation by predistorting the wavefront ofthe incoming lightto cancel distortions occurringinthelightpath[54].

Finally,asignificantchallengeforIVMismotioninducedbybreathing,peristalsis,and circula-tion,whichmostlyaffects organsinthechestandabdomen.A keyachievement forimage analysis in IVM is automation for motion artefactremoval [55,56]. Thisincludes triggering imagingatspecifictimeperiodstocoincidewiththeheartbeatorrespiration,ortriggeringheart contractionstocoincidewithimageacquisition.Conversely,variouspiecesofsoftwarehave beengeneratedforimageanalysis,whichallowscorrectionofsamplemotionfollowingimage acquisition[57].Thesemethodsareapplicabletodifferentlaser-scanningmodalities,including confocalandmultiphotonmicroscopy.

OpticalProjection TomographyandLightSheetFluorescence Microscopy Photonscatteringintissueshindersimagingatdepthsexceedingafewhundredmicrometres invivo,makingimaging ofwholeorgansimpossible.Hence, imagingatdepthrequires the physical sectioningof tissues due to photon scattering. Theimaging limitof conventional microscopyintermsofpenetrationdepthissetbyaphysicalparameterofphotonsknownas themeanfreepath(MFP)(reviewedin[58]).TheMFPtranslatesasthenumberofscatteringor collisioneventsthataphotonundergoes,each ofwhichmodifies thephoton’sdirectionof travel,ultimatelyresultinginimageblur.Forwidefieldepifluorescencemicroscopyatraditional thicknessfortissuesectionsis10–50mm,whichensuresahigh-qualityimageand diffraction-limitedresolution.Confocalandmultiphotonmicroscopyallowgreaterpenetrationdepthsofup

to 0.5–1mm.Nevertheless, to acquirea 3Dimage of an entiresample usingconfocal or multiphoton microscopy, automated methods for the digital reconstruction of thin serial sectionsrequiretheacquisitionofhundredsofindividualsectionsand/orlongimagingperiods underhigh-energyillumination.Thisultimatelyrendersconfocalandmultiphoton techniques impracticalforimagingintact,largespecimens.

Mesoscopicimaging techniques,suchas light-sheetfluorescence microscopy[LSFM;also knownasselectiveplaneilluminationmicroscopy(SPIM)]andopticalprojectiontomography (OPT),enablevisualizationoflargerfieldsofviewacrossentireorgans.Theprerequisiteisthat theseorgansmustbetransparentoropticallycleared.Tissueclearancehasgainedsignificant momentum inrecent years,asshown bya risingnumberofclearing methodswhichoffer differentadvantages,despitetheirusebeinglimitedtofixedtissues[59–61].Altogether, tissue-clearancemethodsmustsatisfythreebasiccriteria:(i)alltissuesmustbeefficientlycleared,(ii) cellularandsubcellularstructuresmustbeadequatelypreserved,and(iii)tissueclearancemust becompatible withfluorescence detection. Current techniques includethe useof organic solvents[60,62–70],water[59,71–76],andelectrophoresis-basedprotocols[77–79].A sum-maryofrelevantmethodsfortissueclearanceisshowninTable3.

Oncethesampleistransparent,OPTimagingisachievedviatissuetransilluminationand epi-illuminationovermultipleprojections[80].WiththeOPTsetup,thespecimenisrotatedthrough 360inangularstepsaroundasingleaxiswhilebeingheldinpositionforimaging(Figure1A). Virtual sections are independently reconstructed from the acquired images using a back-projectionalgorithm[81].Thismethodallowshigh-resolutionimagingatpenetrationdepthsof upto15mm[80].Asaresult,high-resolutiondigitalsections,and3Dimagereconstructionsof thesampledspecimen’svolume,canbeobtained.

LSFM,bycontrast,usesathinplaneoflight(orlightsheet),thatisshapedbyacylindricallens oralaserscannertoexclusivelyilluminatethefocalplaneofthesampleatanyonetimeand has been used extensively to image living,if small,organisms (Figure 1B) [82]. While, in conventionalmicroscopes,illuminationanddetectionfollows thesamepath,thedetection pathwayinLSFMisrotatedby90.Altogether,thesecharacteristicsmakeitapowerfultool for live-cellimagingoflarge samplesduetoitshighimagingspeed,reducedtoxicity, and photobleaching (reviewed in [83]). 3D image formation is based on raw images being assembled after translation or rotation of the entire sample. Multiple LSFM setups have beendeveloped,includingilluminationfrommultipledirections,enablingdoublingthe pene-trationdepth[84],orcombiningdifferentmesoscopictechniques,includingOPTiSPIM[85],a hybridsetupwhichbenefitsfromthe highresolutionof SPIMwiththe possibilitytoimage fluorescentandnonfluorescentcontrastsofOPT.Thefirstcommercialdevicesweremade available, and an open-source, custom-built version named OpenSPIM was produced

[86,87].OPTand/orLSFMhavebeenusedtoimagemulticellular culturemodels[88–91],

zebrafish[92],sparsecellpopulations[93],thedevelopmentofplants[94],livingembryos, andgenemapping[80,95],aswellasmultiplerodentorgansinhealthanddiseaseconditions

[63,66,96,97].OPTandLSFMwerebothsuccessfullyusedtoobtaindetailedinsightofthe

anatomyoftheflightmusculatureofaDrosophilafly,itsnervousanddigestivesystems,and b-galactosideactivityatwhole-bodylevel[98,99].Whileinitsorigins,theapplicationoftissue clearing andimagingwaslimitedtorodentorgans orsmallwhole bodies(such asthat of Drosophila flies), a build-up on previous hydrogel embedding, tissue-clearing techniques [passive clarity technique (PACT)], imaging reagents [refractive index matching solution (RIMS)],anddelivery routes[perfusion-assistedagentrelease(PARS)]made it possibleto imagewholeopticallyclearedmice[100].

Lateralresolution:theminimum

distancethatcanbedistinguished

betweentwocontiguousreflectors,

perpendiculartotheultrasound

beam.

Mesoscopicimagingmethods:

branchofimagingreferringto

measurementsofsamplesof

intermediatesizes(intherangeof

tensofmicrometrestomillimetres).

Microbubbles:intravenously

injectedmaterialsthatserveas

contrastagentsinultrasound.They

consistofalow-solubilitycomplex

gassurroundedbyaphospholipid

shell.Theyresonateinanultrasound

field,andgenerate‘signature’

harmonicsignals.

Opticalresolution:theshortest

distancebetweentwopoints,within

aspecimen,thatcanbe

distinguishedasseparateentities.

Photobleaching:aprocess

wherebytherepeatedcyclingofa

fluorophorebetweengroundand

excitedstatesleadstomolecular

damageandgradualreductionin

fluorescence.

Stimulatedemissiondepletion

(STED):amethodin

super-resolutionimagingthatreliesonthe

selectivedeactivationoffluorophores

whileleavingonlyasmallfocalspot

active.

Ultrasoundtransducer:adevice

thatproducessoundwaveswhich

interactwithbodytissuesand

produceechoes.Thissignalis

receivedandusedtogeneratea

sonogram.

Table1.ImagingScalesandCurrent/PotentialApplicationsinParasitology

Imagingmethod Resolution Penetration

depth(max)

Advantages Potentailparasitologyapplicationsinin

vivoimaging

Electronmicroscopy

(andcombinationswith

fluorescence microscopy)

2–5nm 1mm(TEM) Visualizesnotjustthefluorophore.Can

nowbecombinedwithfluorescence

microscopy(correlativelightandelectron

microscopy:CLEM)andintravital

imaging.

Observationofultrastructuraldetailsof

host–pathogeninteractions,cells,and

infection-relatedchanges.

STED 20nm(lateral) 100–200mm Enablesfluorescentimagingata

nano-scaleresolution.

Observationofhighlyresolvedstructures

andhost–parasiteinteractionsat

subcellularresolution.

IVM:Advanced

fluorescencemicroscopy

–FRET

2–10nm 100–200mm Enablesquantificationofprotein–protein

interactionsinvivo.

Monitorenzymeactivities,ionchanges,

host–pathogeninteractions,lipid

changes,mechanicalstress,and

moleculequantifications.

IVM:Advanced

fluorescencemicroscopy

–FRAP

2–10nm 100–200mm Enablesquantificationoflateraldiffusion

dynamics.

Monitorhost–pathogeninteractionsatthe

subcellularlevelwithincellsharbouring

intracellularparasites,forexample,

organellehijacking,nutrientacquisition.

IVM:Advanced

fluorescencemicroscopy

–FLIP

200nm 100–200mm Enablesquantificationofmembrane

dynamics.

Monitorhost–pathogeninteractionsatthe

subcellularlevel,forexample,intracellular

parasitemembranefusionandorganelle

segregationduringreplication.

IVM:Confocalimaging 200nm 100–200mm Compromisebetweenhighresolution

andfastacquisitionratesinvivo.

Monitorhost–pathogeninteractionsat

subcellularandcellularlevels.

IVM:Multiphoton

microscopy

400nm 500–800mm Thehighestadvantageoverother

methodsishighpenetrationdepth.Low

backgroundautofluorescence.

Monitoringparasitedynamicsindeep

organstructures.

IVM:Spinning-disc

microscopy

200nm 100–200mm Fastimageacquisitionspeedinvivo. Monitoringdynamicsofhost–pathogen

interactions,membranedynamics,tissue

changes,vasculatureatnearrealtime.

OPT 20mm cmincleared

samples

3Dreconstructionoflarge,optically

clearedsamples.

Visualizingparasiteandtissuedistribution,

vascularchanges,andsystemicchanges

atwhole-tissueandevenwhole-animal

level.

SPIM 200–500nm cmincleared

samples

3Dreconstructionoflarge,optically

clearedsamples.Littlephototoxicityinlive

imaging.

Visualizingparasiteandtissuedistribution,

vascularchanges,andsystemicchanges

atwhole-tissueandevenwhole-animal

level.

OAI Sub-100mmin

deeptissues

cm Imagesnoninvasivelyopticalabsorption

contrastoverawiderangeofspatial

scalesathighspeed.Abletoimage

structuresandfunction.

Noninvasiveimagingofeffectsofparasite

presenceintissues.Visualizationof

vascularandmetabolicchangesin

variousorganslongitudinally.

MRI 100–500mm Fullbody Noninvasiveimaging,applicableto

clinics.Providesdetailedinformationon

tissuearchitecture,morphology,

pathology.

Canbeappliedtoorgansrangingfrom

deeporganstoskin,tostudyparasite

effectsattissuelevel.FunctionalMRIcan

beusedtostudyfunctionintissues

affectedbyparasitepresence.

Radiography cm Fullbody Noninvasiveimaging.Providesdetailed

informationontissuearchitecture.

Applicabletostudytissuediffraction

propertiesuponinfectionwithdifferent

parasites.

Ultrasound Sub-100mmin

deeptissues

Fullbody Noninvasiveimaging,applicableto

clinics.Providesdetailedinformationon

tissuearchitecture,morphology,

pathology.

Canbeappliedtoorganstostudyparasite

effectsattissuelevel,includingmetabolic

changes.

Inparasitology,uses haveincludedthe generationofopticallyclearedgutfrom tsetseflies (vectorsofTrypanosomabrucei)[101](Figure1C)andopticallytransparentAnopheles mos-quitoes(vectorsofPlasmodium)(Figure1D,E).Importantly,theuseofmesoscopicmethods inparasitologyhasremainedscarce,despitetheirrelativelyeasyimplementationanduse.The potentialofthesetechniquestostudyinteractionsathighresolution,andhighthroughput,for thephenotypiccharacterization ofparasite-inducedchanges invectors,rodents,andother modelorganismsishigh,andworthexploringandimplementinginvariousareasofparasitology inyearstocome.

BioluminescenceImaging

Photonproductionisachievedprimarilythroughluminescenceandfluorescence.In lumines-cence,thephotonistheproductofexothermicchemicalreactions,whereanenzyme(e.g., luciferase) oxidizes a substrate (e.g., luciferin),leading to photon emission. Conversely,in fluorescencetheexcitedstates arecreatedbyabsorption andemissionoflight. Themain advantageofluminescenceassaysisaccuratequantificationwithhighsensitivity,whichcanbe appliedinhigh-throughputstudies,bothinvivoandinvitro.

Bioluminescenceimagingreliesonprocessesoccurringinnatureviathegenerationoflightby lower organisms, including beetles, bacteria, molluscs, algae, crustaceans, annelids, and coelenterates[102].Variousbioluminescentsubstrateshavebeenisolatedfromthese organ-isms, and their biochemical properties have been defined (Figure 2). Across parasitology research, the main bioluminescent probe used in vivo is firefly luciferase (reviewed in

[103,104]),withGaussia,NanoLucandRenillaluciferasebeinglesscommonlyused.

Trans-genicPlasmodiumspp.,Toxoplasmaspp.,Leishmaniaspp.,Trypanosomabrucei,and Try-panosomacruziexpressingbioluminescentprobeshavebeenusedforinvestigatingparasite growth and dissemination, virulence,coinfections, parasite stage-specific promoters,drug screening,andmonitoringhost–pathogeninteractions(reviewedin[103,104]).

Inrecentyears,animportantaimhasbeentoachieveultrabrightluminescencetodetectsignals withhigh sensitivityindeeptissues.Thishasbeeninitiallyachievedbygeneratingparasites expressingcodon-optimizedred-shiftedfireflyluciferases[7,105]andultrabrightluminescent probessuchasNanoLuc[106,107].AlthoughNanoLucluciferasehasbeenextremelyusefulin vitroduetoitsbrightsignal[108],itsperformanceinvivoisverypoorduetoitsshortemission

Table1.(continued)

Imagingmethod Resolution Penetration

depth(max)

Advantages Potentailparasitologyapplicationsinin

vivoimaging

CT mm Fullbody Noninvasive3Dimaging,applicableto

clinics.

Canbeappliedtoorganstostudyparasite

effectsattissuelevel.

Bioluminescence imaging

cm Severalcm Enableswhole-bodyimagingina

high-throughputmanner.Consistentwith3Rs

onanimalusage.

Visualizingluminescentparasite

distribution.PotentialtoperformBRETin

vivohasnotbeenexplored.

PET mm Fullbody Allowsobservationofmetabolic

processeswithinthebody,aswellas

parameterssuchasbloodflow,

metabolism,andneurotransmitters.

Observationoftheeffectsofparasitic

infectionsonhostmetabolism,bloodflow

perturbations,orneurotransmissionata

whole-bodylevel.

SPECT Severalmm Fullbody Allowsobservationofmetabolic

processeswithinthebody,aswellas

parameterssuchasbloodflow,

metabolism,andneurotransmitters.

Observationoftheeffectsofparasitic

infectionsonhostmetabolism,bloodflow

perturbations,orneurotransmissionata

whole-bodylevel.

Table 2. Key Examples of Organs and Parasites Imaged by IVM.

Organs/ pathogen

Central/peripheral nervous system

Skin Lungs Heart Stomach/

pancreas

Liver Spleen Intestines Kidneys/

bladder Lymph nodes Bone marrow Adipose tissue Placenta Vector-borne Plasmodium spp. [1] [5,6] [13] – – [14,15] [19,20] – – [23] [25] [20] [26,27] Trypanosoma brucei [2] [7,8] – – – – – – – – – – – Trypanosoma cruzi – [10,11] – – – – – – – – – – – Onchocerca – – – – – – – – – – – – – Leishmania – [9] – – – [16] – – – [24] – – – Wuchereria/Brugia – – – – – – – – – – – – – Schistosoma – [12] – – – [17] – – – – – – – Theileria – – – – – – – – – – – – – Babesia – – – – – – – – – – – – – Food/water/soil-borne Toxoplasma [3,4] – – – – – – [21,22] – – – – – Entamoeba – – – – – [18] – – – – – – – Echinococcus – – – – – – – – – – – – – Ascaris – – – – – – – – – – – – – Ancylostoma – – – – – – – – – – – – – Necator – – – – – – – – – – – – – Strongyloides – – – – – – – – – – – – – Paragonimus – – – – – – – – – – – – – Giardia – – – – – – – – – – – – – Cryptosporidium – – – – – – – – – – – – –

Table3.TissueClearanceMethodsforSPIMandOPT

Clearingmethod Rationale Advantages Limitations Refs

BABB(Murray’sclear;Benzyl

alcohol:benzylbenzoate)

Benzylalcoholandbenzyl-benzoate. Quicktissueclearance.Successful

forclearingconnectivetissues.

Strongquenchingof

fluorescence.Solutionsusedare

toxic.

[62]

3DISCO(3Dimagingof

solvent-clearedorgans)

Tetrahydrofluorane,

dichloromethane,anddibenzylether.

Successfullypreservesfluorescence

intissues(brain)whileenabling

adequatetissueclearance.

Fluorescencequenchinginsome

tissues.Clearancenotveryfast.

[63]

iDISCO

(immunolabelling-enabled3Dimagingof

solvent-clearedorgans)

Usesdibenzylether. Enablesdeepantibodydiffusionin

largeorgans.Compatiblewithuseof

AlexaFluordyes:doesnotrequire

immediateimagingasthereisno

diffusionovertime.

Minor. [64]

PACT(passiveclaritytechnique),

PARSandRIMS

Passivetissueclearingand

immunostainingofintactorgans.

Usesacrylamide,bis-acrylamide,and

SDS(sodiumdodecylsulfate).

Preservesfluorescencesuccessfully.

Lowmaintenanceprocedure.

Longincubationtimerequired.

Cancauseaberrationsintissue

morphology.

[60]

CUBIC(clearunobstructed

brain-imagingcocktailsand

computationalanalysis)

Urea,N,N,N0,N0-tetrakis

(2-hydroxypropyl)ethylenediamine,

TritonX-100,sucrose,and2,20,200

-nitrilotriethanol.

Relativelyfastprocedure,preserves

tissuemorphologyandfluorescence

intensity.Whole-bodyclearanceis

possibleviatrans-cardialperfusion

withreagents.

Minor. [65,66]

RTF Rapidclearingmethodbasedon

triethanolamineandformamide.

Canrendertissuestransparentwithin

hours.Isuserfriendly.Preserves

fluorescencesignal(includingGFP)

andtissuestructure.

Minor.Notyetvalidatedin

multipleorgans.

[67]

TDE(tetrachlorodiphenylethane) Basedon2,20-thiodiethanol,aglycol

derivativeoftenusedasmounting

medium.

Reporteduseinbrainwithsuccessful

clearanceandfluorescence

preservation.

Inhighconcentrations,TDE

quenchesfluorescence,and

affectscellshape.

[68,69]

Spalteholz Benzylalcohol,benzylbenzoateand

methylsalicylateisosafrole.

Quicktissueclearance.Successful

forclearingconnectivetissues.

Strongquenchingof

fluorescence.Solutionsusedare

toxic.

[70]

SWITCH(system-widecontrolof

interactiontimeandkineticof

chemicals)

SDSandcustomrefractive

index-matchingsolution.Requires

glutaraldehydeandthermal

delipidation,followedbyrefractive

index-matching.Increaseshybrid

porosity,facilitatingdiffusionfor

labelling.

Verystrongandfastclearing.Allows

clearingwhilepreserving

fluorescenceandantigenicityacross

thetissue.Bufferallowschemical

reactionsbetweenexogenousand

endogenousmolecules.Suitablefor

multiplexedproteomicimaging.

Canintroducecolouration

artefacts.

[71]

ScaleA2/U2 Urea,glycerol,andTritonX-100 Glycerolcounter-balances

urea-inducedtissueexpansion.Allows

targetinglipophilictissueregions.

Slowclearancerates.

Aberrationscanbeintroduced

duetoreagentsused,including

tissuefragilityandvolume

enlargementofspecimens.Not

goodinheavilymyelinated

tissues.

[72]

ScaleS OptimizedScaleprotocol.

Sorbitol-based.

Preservestissuemorphologyand

fluorescence.Sorbitoliseffectiveat

clearingtissuesotherwisedifficultto

clear.Allowsimagingathigh

resolutionandsignificantdepth.

Notalltissuesaresuccessfully

clearedusingthismethod.As

ScaleA2/U2,notsosuccessfulin

heavilymyelinatedtissues.

[59]

FRUIT SeeDB-derivedmethodthatusesD

(–)-fructoseandurea.

Water-solublemethodhasadditive

clearanceeffects.Allowsarterial

perfusionforenhancedclearance.

Compatiblewithyellowfluorescence

Minor.Usefulnessnotyet

reportedinmultipletissuetypes.

[73]

wavelength.Recently,anovelred-shiftedluciferase–luciferinpairbasedonthecoelenterazine analogue diphenylterazine (DTZ) and a NanoLuc mutant fused to a fluorescent reporter (resultinginanultrabrightprobe knownas Antares2),provedto behighlysuccessfulforin vivo imaging [109,110]. Similarly, another highly successful combination has been that of AkaLuminehydrochloride(AkaLumine-HCl –asyntheticD-luciferinanalogue),which,when catalysedbyfireflyluciferase,generatesnear-infraredemission,andhasfavourabledistribution in deep organs [111]. Randommutagenesis of firefly luciferase-based libraries led to the generationofAkaluc,whichdisplayedhighthermostabilityandthebrightestemissionwhen combined with AkaLumine [112]. The Akaluc/AkaLumine-HCl combination has allowed a revolutionaryadvance,namelyvisualizationofsinglebioluminescentcellsindeeptissuesof freelymovinganimals[112].

Furthertechnologicaladvancesinthefieldofbioluminescenceincludedual-probereporters whichallowthedetectionoftwosignalssimultaneously,atdifferentwavelengths.Thesehave beenuseful inthecontext ofcharacterization ofdifferentpromotersexpressed atdifferent parasitelifestages[113,114].Equally,bioluminescencecanbeusedinthecontextof biolumi-nescenceenergytransfer(BRET),orhybridbiosensors(BRET–FRET,orhyBRET)for investi-gationofprotein–proteininteractions,analysisofxenografts,andoptogenetics,invivo[115]. OptoacousticandUltrasoundImaging

Allopticalimagingtechniquesmentionedabovehavebeenshowntoimagebiological struc-tureswithpotentialsubcellularresolution(intherangeofthewavelengthused)andremarkable

Table3.(continued)

Clearingmethod Rationale Advantages Limitations Refs

proteins.Compatiblewithuseof

lipophilictracers.

ClearT Basedonformamide. Sinceitdoesnotusedetergentsor

solvents,allowstheuseoflipophilic

dyesandfluorescenttracers.

Clearingspeedofsometissues.

Themaincomponentistoxic.

Insufficienttissuetransparency

forfullvolumeimaging.

Quenchesfluorescence,

especiallyGFP.

[74]

ClearT2 Basedonformamideand

polyethyleneglycol(PEG).

Allowstheuseoflipophilicdyesand

fluorescenttracers.Clearingspeedof

sometissues.PEGstabilizesprotein

conformation,thusinhibitsGFP

quenching.

Insufficienttissuetransparency

forfull-volumeimaging.

[75]

SeeDB(SeeDeepBrain) Combinationoffructoseand

a-thioglycerol.

Fastclearingwithoptimal

preservationoftissueintegrityand

fluorescencesignal.Allows

large-scaleimaging.

Viscosityofsaturatedfructoseis

veryhigh.Difficulttopermeate

organs.Arterialperfusionnot

possible.

[76]

CLARITY(clearlipid-exchanged

acrylamidehybridizedrigid

imagingcompatibletissue

hydrogel)

Bioelectrochemicalclearing

technique.Buildsan

acrylamide-basedhydrogelhybridfromintact

tissues.Useselectrophoresis,

acrylamide,bis-acrylamide,andSDS.

Preservesfluorescencesuccessfully. Electrophoresisbasecanbe

challengingtoimplement.Can

damagetissuesorintroduce

artefacts.

[77,78]

MAP(magnifiedanalysisofthe

proteome)

BasedonCLARITYmethod,uses

densergeltoprovidethetissuewith

morestability.

Allowspreservationofthetissue

integritywhileallowingsuccessful

tissueclearance.Allowslineartissue

expansion,andthereforehighly

resolvedimaging.Allowsmultiple

roundsofimmune-labelling.

Minor. [79]

P Tr E OPT LSFM DetecƟon IlluminaƟon IlluminaƟon RotaƟng sample (A) (B) Camera Transmission Objec Ɵve lens Excita Ɵon Ex.Įlter Dichroic Emission Įlter Tube lens (C) (D) (E)

OpƟcally transparent Anopheles mosquito 3D rendered Anopheles mosquito

Figure 1.

(Figurelegendcontinuedonthebottomofthenextpage.)

Optical Projection Tomography (OPT) and Light Sheet Fluorescence Microscopy (LSFM).

(A)PrincipleofOPT.Theopticallyclearedspecimenisembeddedinagarose,attachedtoametalliccylinderwithina

rotatingstage,andsuspendedinanindex-matchingliquidtoreducescatteringandheterogeneitiesofrefractiveindex

throughoutthespecimen.Whenthespecimenisrotatedtoaseriesofangularpositions,imagesarecapturedateach

orientation.Thesetupisalignedtoensurethattheaxisofrotationisperpendiculartotheopticalaxis,sothatstraight-line

projectionsgoingthroughthesamplecanbegenerated,andcollectedbypixelsonthecharge-coupleddeviceofthe

camera.(B)Principleoflight-sheetfluorescencemicroscopy(LSFM).Theopticallyclearedsampleisembeddedinagarose

andsuspendedwithinasampleholderinsideanindex-matchingliquid.Athin(nm–mm)sliceofthesampleisilluminated

perpendicularlytothedirectionofobservation.Scanningisperformedusingaplaneoflight,whichallowsveryfastimage

acquisition.(C)Surfacerenderingmodelofisolatedinfectedflygut.Theintestinaltissueisvisualizedbyautofluorescence

(grey).ThePMisstainedwithrhodamine-labelledWGA(cyan)andthetrypanosomenucleuswithaGFP-reporter(yellow).

contrastbutarelimitedbystronglightscatteringintissue.Optoacoustic(OA)imaging,also referredtoasphotoacousticimaging,offerstheabilitytonotonlyovercomethislimitationbutto providestructural,functional,metabolic,andevenmolecularinformation[116,117].OAisa hybridimagingmodality,combiningopticsandacousticsviathethermoelasticeffect[118,119]. Thethermoelasticeffectconvertsopticallyabsorbedenergyintoacousticwaves,whichare recordedatthetissuesurfacebymeansofultrasounddetection.Opticalabsorptionprovides theimagingcontrastforOA,whichmeansthatanychromophoreormoleculethatselectively absorbstheilluminationwavelengthcanbeimaged[120].Chromophorescanbeendogenous orexogenous.Viatheirspectralfingerprint,endogenouschromophores–suchashaemoglobin –providequantitativeandphysiologicalinformationofthevasculature.Exogenous chromo-phores–suchasantibodiesfunctionalizedbyabsorbingmetallicnanoparticlesordyes–are suitable for the assessment of tumours and other pathologies. These features make OA imaginganinterestingtechniqueforstudyinghost–parasiteinteractions.

Imagingcanbeperformedbothinatomographicmacroscopicsetup,visualizingthewhole animal,orinamicroscopicsetup. Tomographyusesan arrayofultrasound sensors,often partiallyorcompletelysurroundingthetarget,toachieveaxialimageresolutionoftypicallyafew hundred micrometres, determined by the bandwidth of the ultrasoundtransducer, and imagingdepthsofuptofewcentimetres[121].Bycontrast,microscopysetupsuseasingle mechanicallyscannedfocused transducer(Figure3A)oratightlyfocuseddiffraction-limited laserbeamtogenerateanimage.Intheformercase,termedacousticresolutionmicroscopy, the lateral resolution is given by the properties of the acousticlens and the ultrasound transducer(40mmat50MHztransducerfrequency).Inthisconfiguration,OAmicroscopy hasbeenshowntoresolvefinestructuresinthedermisandassessdermalpapillae[122].Ithas

Scalebar:50mm.Figurereproduced,withpermission,from[101].(D)3Dreconstructionandrenderingofafemale

Anopheles mosquito clearlyshowing abdominalsegments,thorax, and head features.Scale bar:70mm.(E) 3D

reconstructionandclearviewofallbodycavitiesofanopticallyclearedAnophelesmosquito.Scalebar:100mm.

Abbreviations:PM,peritrophicmatrix;WGA,wheatgermagglutinin.

Deep-Sea shrimp (NanoLuc) 460 nm Gaussia (GLuc) 470 nm Renilla (RLuc) 480 nm Click beetle >600 nm 560 nm Railroad worm 630 nm Antares FireŇy (FLuc) >600 nm 613 nmRFLuc AkaBLI 675 nm Red-shiŌed Renilla (RLuc8) 535 nm 400 450 500 550 600 650 700 750 800 nm Bioluminescence (emission, nm)

Figure2.BioluminescenceImagingProbes.Bioluminescenceprobescommonlyusedininvivoimaginginclude

Gaussialuciferase,NanoLuc,Renillaluciferase,clickbeetleluciferase(anditsred-andgreen-shiftedforms),red-shifted

fireflyluciferases,railroadwormluciferases,andnovelprobeswithbrightluminescencetoimagedeepintotissuessuchas

AkaBLIandAntares/Antares2.Isolatedfromdifferentorganisms,theseprobeshaveavariedrangeofmolecularweights

andmaximumpeakofemissionwavelengthsasschematicallyillustratedhere,allowingfordualluminescencesystemsto

beused.

alsobeenusedtovisualizethecerebralvascularanatomyinmiceuptoadepthof3.7mmat sub-millimetre resolution (sub-100mm at 1mm depth), greatly surpassing what can be achievedwithpurelyopticaltechniques[123].In thelattercase,calledopticalresolution microscopy,thelateralresolutionisdeterminedbythefocalspotofthelaser,andimaging depthislimitedbyopticalscatteringtoamaximumof1mm,asitisinthecaseofpureoptical microscopy[121,124].Inlow-scatteringtissue,suchasthatfoundinmouseears,theresolution canbepushedto2.5mmatadepthof150mm[125].

Both contrast andpenetration depth are affectedby the choice of excitation wavelength. Shorterwavelengths(typicallynoshorterthan532nm)arestronglyabsorbedbyhaemoglobin andthushighercontrastcanbeachieved,attheexpenseofpenetrationdepth.Forlonger wavelengths (typically up to 1300nm), penetration depth is increased at the expense of contrast.Sincethepropagationspeedofultrasoundwavesinbiologicaltissueiscomparatively low,depthinformationisobtainedbyrecordingthepressurewaveinatime-resolvedmanner. Thus, all OA imaging techniques have the potential to record three-dimensional images. Furthermore,differencesinthewavelength-dependentabsorptioncoefficientsofoxygenated anddeoxygenatedhaemoglobincanbeutilizedforoxygenationmeasurementsinboth tomo-graphic[126]andmicroscopic[123,125]setups.

Currently, only a few commerciallyavailable invivo whole-animal OA imaging devices are capableofcapturingsinglecross-sectionaltomographicimagesoflivingsmallanimalswith estimatedin-planeresolutionsof150mm[127–129].OAimaginghasalsobeencombined withecho-ultrasoundimaginginawhole-bodylivemousetomographysystem,withreported in-planespatialresolutionsof150mm(OA)and350mm(ultrasound)[130].

UsingacousticresolutionOAmicroscopy(Figure3A),wehaveimagedthebrainofmiceaged 4–6weeksinfectedwithPlasmodium.Themicehadtheirscalpremoved(theskull wasleft intact)priortoimagingandwerekeptunderanaesthesiaduringimaging.Wehavefoundthat imagesofhealthymice(Figure3B)showclearlydiscerniblehemispheresandcerebellum,while

AcousƟc wave Water

(A) Photo-detector CL ns laser (532 nm) US transducer FL (B) (C) Rhomboid prism AcousƟc lens 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 8 7 6 5 4 3 2 1 0 0 0 1 2 3 4 5 6 7 8 x (mm) x (mm) y (mm) y (mm)

Figure3.OptoacousticMicroscopySetupandImagingoftheBrain.(A)Optoacoustic(OA)microscopysetup(acousticalresolution).Thelaserillumination

inducesthermoelasticpressurewavesinthesampleandtriggerssignalacquisition(photodetector).Thegeneratedpressurewave(red)iscollectedbytheacousticlens

anddirectedtotheultrasoundtransducer.(B)OAmicroscopicimageofthebrainofahealthymouseshowsclearlydistinguishablehemispheres.(C)Thesameimageof

amouseinfectedwithPlasmodium(5daysafterinfection)showsaberrantstructureofthevasculature.

ininfectedmice(5daysafterinfection)themajorvascularlandmarkscouldhardlybeidentified. Thevasculatureshowedalargenumberofaberrantlydistributedscrew-likebloodvessels,and thetwohemispherescouldnolongerbedistinguished(Figure3C).Inlaterstagesofthedisease (7+daysafterinfection),asignificantdecreaseinoptoacousticsignalamplitudewasdetected, possiblyduetoprogressiveanaemia.

Altogether,OAimaging,whichcombineshighcontrastandspectralsensitivitywiththehigh spatialresolutionobtainedinultrasoundimaging,providesexcellentvisualizationof micro-vasculature, making it a powerful tool for basic parasitological research and preclinical applications.Combinedwithexogenousopticallyabsorbingagentsorgeneticallyexpressed reporters it has been proven to provide molecular information with high sensitivity and specificity.

Ultrasoundimagingitselfhasalsobeensuccessfullyusedinthefieldofparasitologyandin medicalimaging.Ultrasounduseshigh-frequencysoundwaves,whicharereflectedoffbody structures,renderingan image.Key advancesinultrasoundover thepast decade include elastography [131],ultrasound contrast agentimaging usingmicrobubbles (reviewedin

[132]),super-resolutionimaging,2Darraytransducer,andultrafastultrasoundimaging[133].

Whiletheseadvanceshavebeenwidelyexploredinotherfieldsofresearch,theyremaintobe usedininvivoparasitology,andhaveenormouspotential.Elastographyisatechniquereferred to as ‘palpatingbyimaging',enablingmeasurement andvisualizationoftissuemechanical propertiesinhealthanddisease.Intermsofcontrastagents,microbubblesprovide consider-ableadvantagestoultrasoundimaging,includingthepossibilityofstudyingbloodrheologyand tissueperfusion,andofenhancingvascularpermeability, drugandgenedelivery,andlocal heating (reviewed in[134]). Finally, ultrafastand super-resolution ultrasound imaging were achievedusingintravenousinjectionofmicrobubbles,the propertiesofwhichallowed hae-modynamicquantificationandimagingstructuresat10mmunderthecranium.Singleechoes fromindividualmicrobubblescouldbedetected,allowingaccuratequantificationofbloodflow speedsatanimagingrateof1000framespersecond[133].Altogether,technological improve-ments in ultrasound speed and resolution, as well as echosonography applications, hold enormouspotentialforparasitologyintermsofstudyingchangesinrheology,vascular per-meability,tissueperfusion,andchangesinthephysiologyofimportantstructuressuchasthe brain–bloodbarrier.

MagneticResonanceImaging

Magneticresonanceimaging(MRI)isanoninvasiveimagingtechniqueuniquelysuitedtoimage deepinsidetissuewithhighspatialresolutionandwithoutionizingradiation.MRIisbasedon themagneticpropertiesofprotonsandneutronswithinatomicnucleiofthebody,particularlyof hydrogen,aswaterisamajorcomponentofthehumanbody.Thespinofhydrogennucleican bemeasuredastheyshowanunevennumberofprotons.Therefore,afterplacingthebody inside the magnetic field of the MRI scanner, the spins start to process with the Lamour frequencyandalignwiththemagneticfield(inbothdirections,howeverslightlymoreinthefield direction).Upon applicationof a radiofrequency (RF)pulse withexactly the same Lamour frequency,thenucleicanabsorbenergyandtransitionfromlowertohigherenergystates(so calledspinexcitation).AftertheRFpulseisswitchedoff,thenucleireturntotheirequilibrium state. Thisprocess requires timeand depends onthe surrounding tissue(also knownas relaxationtime).Duringtherelaxation process,asignalcanbedetected byRFcoilsinthe scanner,whichcanbereconstructed byadvancedimagingtechniques toanimage ofthe humanbody(reviewedin[135]).MRIoffersextraordinaryadvantages,includinghighspatial resolution, and the opportunity of simultaneously extracting physiological, molecular, and

anatomicalinformationfromthebody.Conversely,itslimitationsincludelowsensitivity,long scanningtime,andprobequantityrequiredforimaging.

MRIhasimproved ourunderstandingofdiseasesinparasitologybydetectingpathological alterations,forexample,inthebrain,causedbyToxoplasma[136](Figure4A),Trypanosoma brucei[137](Figure4B),andPlasmodium[138](Figure4C,courtesyofAngelikaHoffmann).In thissectionwediscusstheadvancesinthetechniquerelevanttoparasitology,inthreespecific aspects: MRI probes, multimodal mesoscopic imaging, andthe uses offunctional MRIto addresstranslationalquestions.

Contrastagentsareexogenouscompoundsadministeredtoimprovetheimagecontrastand detectpathologicalalterationsmoreaccurately.Significanteffortshavebeenmadetoimprove MRIbydevelopingcontrastagentsforuseasprobesandsensors.Theseincludegadolinium agents (Gd3+) and derivatives including gadolinium hexanedione [139], gadofluorine and liposomal-basedgadoliniumnanoparticles[140].Thesecontrastagentsareused,forexample, inthestudyofbrainnervebarrierpermeability[141],blood-vesselremodelling[142],orcellular uptake.Otheragentsincludemanganese-basedagentssuchasmanganesechloride(MnCl2)

[143],manganese oxide(MnO) [144],and silica-coatedparticles, whichallow detection of

(B)

T. brucei

(C1) (C2)

Control

P. berghei ANKA

(A1) (A2)

T.

gondii

Co

nt

ro

l

hrFM

hrFM

Figure4.MagneticResonanceImaging(MRI)oftheBrain.(A)T2*-weightedimagesandhigh-resolutionfibremapsof(A1)uninfectedand(A2)Toxoplasma

gondii-infectedmousebrains.Arrowsininfectedbrainsshowinjuriesinthesomatosensorycortex(T2*-weightedimages),andaffectedfibredensityandcortical

connectivitypattern(hrFMimages).Figurereproduced,withpermission,from[136].(B)MRIscanofamouseat28dayspostinfectionwithTrypanosomabrucei,

followingadministrationofcontrast.Arrowheadshowsmeningealenhancementofthehind-mostsectionofthebrain.Figurereproduced,withpermission,from[137].

(C)T1subtractionmapstoillustrateblood–brainbarrier(BBB)disruptionwithrostralpredominanceinaPlasmodium-infectedmouse(C1)comparedtoamousenot

displayingBBBdisruption(C2)(courtesyofAngelikaHoffmann).

specificcellpopulationsinvivoeitherontheirown,orbycombiningwithothercontrastagents. Equally,paramagneticironoxide(SPIO)agents,perfluorocarbons(PFCs),andhighly-shifted protonMRI[145–147],havebeenparticularlyusefulforsensitivelytrackingandvisualizingcell homing in vivo[148–150]. Altogether, advances in MRI contrast agents and probes have allowedthe monitoringofcellfunctions, includingenzymatic activity,geneexpression,and death,aswellassensitivetrackingofcellpopulations(reviewedin[151]).

WhiletheoutputofMRIisimagingofanatomicalchanges,functionalMRI(fMRI)isbasedon allowingvisualizationofmetabolicfunctionbytissues.fMRIallowsmeasurementofbloodflow

kBq/cc

0 800

0 700

X-ray source

Animal/specimen RotaƟon and acquisiƟon 360°

ScinƟllaƟon screen ObjecƟve lens CCD MulƟple projecƟons 3D reconstrucƟon (A) (B)

Foetal brain vasculature

18F-Ňuorodeoxyglucose uptake

HounsĮeld units

Detector ring

Collimator ScinƟllaƟon site for y rays EmiƩed y rays AbsorpƟon in collimator Positron-emiƫng radionuclide e+ e-AnnihilaƟon (C) _(D) y ray y ray y y

Figure5.X-Ray-BasedandPositronEmissionTomography(PET)-BasedImaging.(A)Principalcomponentsofamicrocomputertomographyscannerinclude

anX-raysourcethatproducesX-rayswhichtravelthroughthesampleandarecollectedontheothersidebyanX-raydetector(scintillationscreen,lens,andcharge-coupled

devicecamera).RotationoftheX-raysourceanddetectorallowsacquisitionover360,allowingthegenerationofaseriesofprojectionimages.Theseimagescanbe

processedto producea3Dimageofthe specimen.(B) Maximum-intensity projectionfromanX-raymicro-computedtomography(micro-CT) study ofthe brain vasculatureof

foetusesfrommalaria-infectedrodentmothers.Thebrainvasculatureshownisthatofafoetusfromanuninfectedmouse.Figureadaptedfrom[154].(C)Radioactivetracers

areinjectedintothemouseandwillemitpositronsbyradioactivedecay.Upondecay,positronsarereleasedfromthenucleusoftheradionuclideandannihilatewithelectrons

inthetissue,releasinggamma-photonsthatcanbedetectedwithinacoincidencedetectorring(composedofacollimatorandscintillationcrystals).Thisallowsmappingthe

anatomicallocalizationofthetracer.(D)3DreconstructionofamouseimagedbyPET-CTinastudyinvestigatingglucoseuptakeduringthedarkphaseofa24hlight–dark

cycle.(18)F-fluorodeoxyglucose(FDG)wasusedasatracer,andshowshighestaccumulationinthebrain,heart,andbladder,followedbythekidneysandbrownadipose

tissue.ScalebarsrepresentCTintensity(greyscale),andFDGuptake(colourscale,radioactivitypertissuevolume).Figureadaptedfrom[155].

changes,orbloodoxygenlevelfluctuations,providingapictureofoxygenconsumption.Itis basedontheuseofblood-oxygen-level-dependent(BOLD)contrast,basedonincreasedor decreased concentrationsofparamagnetic deoxyhaemoglobinassociatedwithchanges in neuronalactivity(reviewedin[152]).Theuseofthistechniquehasbeenrelativelyscarcein parasitology,yetithaspotentialforawidenumberofparasites,whichinducemetabolicand/or functionalchanges intissues. Similarly,the generationof MRIscannerssuch as the9.4T equipment[153]offersimprovedimagingresolution.

X-Ray-BasedandGamma-Ray-BasedNoninvasiveImagingMethods

Otherimagingmethodswhichhaverelevantclinicaltranslationpotentialincluderadiography, computedtomography,X-raymicro-CT(Figure5A,B)[154],andradioisotope-basedimaging suchasSPECTandPET(Figure5C,D)[155].

TwocategoriesofX-ray-basedimagingarestructuralandfunctionalimaging,enablingstudies ofanatomicalstructuresandofchangesinbiologicalfunctionssuchasmetabolism,bloodflow, andbiochemicalcompositionoftissues,respectively.Functionalanalysisoftissuesandtheir compositionusingX-rayshasseenprogresswiththeuseofexogenouscontrastagents,andis basedonthepossibleinteractionsbetweenX-raysandmatter.TheseincludeX-ray attenua-tion,X-rayfluorescence,andX-ray-excitedopticalluminescence(reviewedin[156,157]). Equally noninvasive is imaging based on gamma-ray emission from radioisotope-labelled biomolecules within samples upon radioactive decay, namely PET and SPECT. In both methods, radioactive biomolecules, synthesized from radio-nucleotides, are administered, and,followingbiologicalinteractionsinspecifictissues,thereisdecay,resultingintheemission ofgammaphotons.ThesephotonscanbedetectedbyPETandSPECTscannersandcanbe tomographically reconstructed to generate 3D images of functional tissue activity [158]

(Figure5C,D).Theuseofradiography-basedandnuclear-imaging-basedmethodshasbeen

relativelylimitedinparasitology,yethaspotentialforthestudyofhost–pathogeninteraction effectsonorganfunctions,tissuecomposition,andmetabolicchangesuponinfection. ConcludingRemarks

Altogether, multiple methods forin vivo imaging as stand-alone techniques have allowed improved resolution(e.g.,advancedmicroscopymethodssuchassuper-resolutionwithIVM),decreased invasiveness(e.g.,bytheuseofOAI),increasedpenetrationdepthallowingforimagingoforgan regionspreviouslyimpossibletoobserveinvivo(e.g.,throughtheuseofGRINlensesforIVM),and functionalimagingwithhighresolution(e.g.,fMRI,MRI,PET,SPECT).Manyofthesetechniques havesuccessfullyreachedtheparasitologyfield,particularlyIVM.However,theadvancesinIVM methodsandothertechniquesaltogether,stillholdenormouspotentialandapplicability,which needtobeexplored(seeOutstandingQuestions).Forinstance,theseadvancesopenupthe possibilityto imagetheheart upon infection with T. cruzi,the digestive organsupon infection with T. gondii,anddeep-brainstructuresuponinfectionwithT.brucei.FunctionalMRIandadvancesin X-ray-basedandgamma-ray-basedmethodscouldenablethestudyoffunctionalandanatomical changesintissuesandorgansuponinfection,aswellasfluiddynamicssuchasbloodflow.Aside fromtheseadvancesinstand-aloneplatforms,equallyrelevantwillbetheuseofhybrid, multi-modalitysystemswhichcouldprovideinterestingopportunitiestostudyhost–pathogen inter-actionsinvivoatvariouslevelssimultaneously.

Acknowledgments

WearegratefultoVolkerHeussler(IZB,UniversityofBern)forhelpfuladviceandintellectualinputonthetechniques

relevanttoOPT,LSFM,andOAI,andtraininganddiscussionskeytothesemethods,andIVM.WethankalsotheMICat

OutstandingQuestions

Cantheparasitologyfieldtake

advan-tage of motion-correction

mecha-nismstostudyparasitesintheheart

orgutbyIVM?

IVM, OPT, SPIM, and MRI allow

accesstothegastrointestinalsystem.

Whatcanthesetechniquesteachusin

thecontextoffood-borneand

water-borneparasites?

CannewimprovementsinIVM

appli-cations, allowing visualization of

deeperorgan structures,enablethe

imagingofrareinfectioneventssuch

asT.bruceiandT.gondiicrossingthe

blood–brainbarrier?

HowcanOPTandSPIMbemodified

to studythe impactofparasiteson

vector biology, and host–pathogen

interactionswithinvectorhosts?

Can OPT, SPIM,andother relevant

hybrid platforms, help to address

questionsrelevanttoparasite

dynam-icsandspread?

Will MRIandOAI,as wellashybrid

imaging platforms, enable us to

address questionsrelevant to

meta-bolic dysregulation by parasitic

infections?

How can technological advances in

imaging methods,applied toanimal

models,bebettertranslatedtohuman

medicine in the context of

parasitology?

Can experiments be repeated

fre-quently enough to generate robust

statistics?

theUniversityofBern,forprovidingaccessandtraininginallrelevantimagingmethodsherebydiscussed.Wethank

FedericaMoalli,RenzoDanuser(TKI,Bern);EmmanuelG.Reynaud(UniversityCollege,Dublin)andJessicaKehrer(Centre

forInfectiousDiseases,HeidelbergUniversityHospital)forhelpfulinputforOPTandLSFMtechniquesanddiscussions.We

thankRobertNuster(IAP,Bern)forexperimentalhelpduringtheestablishmentofOAIforexperimentalcerebralmalaria

imaging.WearegratefultoAngelikaHoffmann(DepartmentofNeuroradiology,HeidelbergUniversityHospital)forher

helpfulinputonMRItechniques,forkindlyprovidingFigure4C(MRIofaPlasmodium-infectedbrain),andforcarefully

proofreadingthismanuscript’ssectionsrelevanttoMRI.MDNisfundedbyEMBOfellowshipALTF1048-2016,andby

EVIMalaR(242095FP7)andRSHTM(000529)fellowshipswithVolkerHeussler(IZB,Bern)relatedtoOPTandOAIstudies,

aswellasSwissNationalScienceFoundationfellowships310030_159519and316030_145013fundingworkcarriedout

inhislaboratory. FSand MF acknowledgethe financial supportofthe Swiss NationalScienceFoundation(No:

205320_179038/1).WorkinthelaboratoryofFFisfundedbyaHumanFrontierScienceProgramgrant(RGY0066)

andtheGermanScienceFoundation(SFB1129).Weapologizetothemanycolleaguesfornotcitingtheirworkdueto

restrictionsbythejournalandthebroadscopeofthereview.

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