Invivogenedeliveryisgainingincreasingmomentumforgenetherapyasdeliverymethodsbecomemoreadvancedandsafe.Thisraisestheneedforprecise,sensitive,andeasyevaluationofgenetransfectionefficacy.Amethodthathasbecomeincreasinglypopulariswhole-bodyimagingoffluorescentproteins(1).Fluorescentproteinsprovideuniqueopportunitiesfornon-invasivelabelingandtrackingoftransfectedcellsinlivinganimalsinrealtime(2).Togetherwiththedevelopmentofnewsystemsforwhole-bodyimaging,advancementsinnewfluorescentproteinsofferthepossibilityfordirectvisualizationofgeneexpressioninvivo.
Fordeeptissueimaging,theopticalwindowforfavorablelightpenetrationisinthenear-infraredwavelengths(3),whichinvolvesproteinswithemissionspectrainthefar-redwavelengths.Mostphotonsinthevisualspectrumareabsorbedbymelaninandhemoglobininanimaltissue,whilephotonswithwavelengthslongerthan1,100nmareabsorbedbywater.Inaddition,considerablescatteringofthesignaloccursatlowerwavelengths(3).
Recently,anovelfar-redfluorescent,Katushka,wascharacterized(4).ThisproteinwasderivedfromtheseaanemoneEntacmaeaquadricolor,enhancedtoperformwithhigherbrightnessandexposedtosite-directedmutagenesistogenerateproteinswithemissionspectrainthefar-redregion.TheresultingKatushkawasreportedtobe7-to10-foldbrighterthanotherfar-redfluorescentproteins,e.g.,HcRedandmPlum(4),andischaracterizedbyhighpHstabilityandphotostability(4).FewstudieshavesofarusedKatushkaforinvivobio-imaging(3;5;6),thusinthisstudywewantedtocharacterizethepotentialofKatushkaasamarkerforgeneexpression,includinganevaluationofthekineticsandspatialdistributionoftheKatushkasignal.
Untilrecentyears,thepreferredmethodfordetectingfluorescencewasbyuseofacharge-coupleddevice(CCD)camera(1;7).Thisapproachlimitsthedegreeofdetailsonecanobtainasthecameraintegrateslightfromtheobject,resultinginaplanarpicture,whichisdominatedbythelightcontributionfromthefirst1–2mmoftissue.Usingnewtechnology,BIOLOGicalsamplescanbeexcitedwithsub-nanosecondlaser,whileemissionofindividualphotonsisdetectedusingarapidphotonmultiplier(PMT).Inthisway,thearrivaldistributionofphotonsasafunctionoftimeofexcitationcanbecollectedfromindividualpointsinthetissue;allowingforprecisespatialandtimedistributionoftheemittedlight.Oneadvantageofdeterminingthefluorescenceinrealtimeisaprecisedeterminationofthedecaytimeforthemolecularquantummechanicaltransition,whichisresponsIBLeforlightemission.ThislifetimeischaracteristicfordifferentFluorochromesandbyfilteringlightemissionbylifetime,onecanobtainaprecisemeasurement,wherebackgroundluminescenceiseliminated.Theprecisionalsoallowsforconstructionofa3Dvolumetricoptictomographicmapofthefluorescence.
OurchoiceofmethodforgenedeliveryisDNAelectrotransfer.Byexposinglivingcellstoshortandintenseelectricpulses,position-dependentchangesinthetransmembranepotentialareinduced,renderingcellsaccessibleforCDNAentry(8;9).TherearemanyreportsonsuccessfulinvivoDNAelectrotransfertomuscletissueasreviewedinMiretal.(9).Weperformedthetransferusingacombinationofonehighvoltage(HV)andonelowvoltage(LV)pulses.Apulsecombination,whichhasprovenhighlyefficientformuscletissue(10;11)andcausingminimaladverseeffectsonthemuscletissue(12;13).
Thus,usingtimedomainopticalimaging,wewantedtoinvestigatethenovelfar-redfluorescentKatushkaasamarkerfortransgenicexpressionwithrespecttointensity,lifetime,andspatialdistribution,andcompareittothewell-knownfluorescentmarkerGFP.
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