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1 Noninvsive multiphoton fluoresene mirosopy resolves retinol n retinl onenstion prouts in mouse eyes Grzyn Plzewsk 1, To Me 2,3, Yoshikzu Imnishi 3, Wenyu Sun 1, Yu Chen 3, Dvi R Willims 4, Dvi W Piston 5, Akiko Me 2,3 & Krzysztof Plzewski 1,3 21 Nture Ameri, In. All rights reserve. Multiphoton exittion fluoresene mirosopy (MPM) n imge ertin moleulr proesses in vivo. In the eye, fluoresent retinyl esters in suellulr strutures lle retinosomes meite regenertion of the visul hromophore, 11-is-retinl, y the visul yle. But hrmful fluoresent onenstion prouts of retinois lso our in the retin. We report tht in wil-type mie, exittion with wvelength of ~73 nm ientifie retinosomes in the retinl pigment epithelium, n exittion with wvelength of ~91 nm revele t lest one itionl retinl fluorophore. The ltter fluoresene ws sent in eyes of genetilly moifie mie lking funtionl visul yle, ut entute in eyes of oler wil-type mie n mie with efetive lerne of ll-trns-retinl, n intermeite in the visul yle. MPM, noninvsive imging molity tht filittes onurrent monitoring of retinosomes long with potentilly hrmful prouts in ging eyes, hs the potentil to etet erly moleulr hnges ue to ge-relte mulr egenertion n other efets in retinoi metolism. High-resolution noninvsive imging hs eome n essentil metho for unerstning omplex iologil systems in experimentl ell lines n is use with inresing frequeny in tissues n live nimls 1 5. These methos re lso eing slowly pte for ignosti testing n linil pplitions, where they show gret promise 2,6,7. Two-photon mirosopy (TPM) offers rel time, highresolution imges of enogenous fluoresent moleules in living tissues with little or no tissue mge Use of long-wvelength n nonliner exittion ypsses sorption y ultrviolet n visile light hromophores n llows imging t low lser power with suellulr resolution t tissue epths not ttinle with other noninvsive optil molities. Verterte eyes hve evolve to prevent short ultrviolet wvelength light from rehing the retin. Inste, infrre illumintion use in MPM is iel for visulizing fluoresent iomrkers tht exist enogenously in retinl pigment epithelil (RPE) ells of the eye. There re two types of fluoresent retinois initive of ertin funtionl n isese sttes of humn eyes. The intrinsi hromophore, ftty i esterifie retinol, hs wek fluoresene n hs propensity to luster with phospholipis n helper proteins into strutures lle retinosomes lote lose to the RPE plsm memrne Ftty i esterifie retinol is generte y sequentil retions of the visul yle (Supplementry Fig. 1). First, photoisomeriztion of 11-is-retinyliene, the light-sensitive hromophore of visul pigments in ros n ones, genertes ll-trns-retinyliene, whih is then hyrolyze n reue to retinol. Next, retinol is trnsporte from photoreeptors to the RPE n esterifie in leithin: retinol yl trnsferse (LRAT)-epenent retion; itionlly, these esters re forme from retinol importe from the irultion 15. As inite y noninvsive in vivo TPM imging through the sler, n y hemil nlyses of eyes of wil-type () n genetilly moifie mie, retinosomes umulte ftty i esterifie retinol essentil for 11-is-retinl proution. Thus, retinosomes serve s the reservoir of ll-trns-retinyl esters for ynmi trnsfer n onversion of retinois in the eye. Conenstion prouts of ll-trns-retinl re lso fluoresent, n their umultion within the RPE is initive of ging retin or retinl pthology. These stle onenstion prouts, inluing A2E (2 [2,6 imethyl 8 (2,6,6 trimethyl 1 ylohexen 1 yl) 1E, 3E,5E,7E ottetrenyl] 1 (2 hyroxyethyl) 4 [4 methyl 6 (2,6,6 trimethyl 1 ylohexen 1 yl) 1E,3E,5E hextrienyl] pyriinium), A2DHP-PE (phosphtiyl-ihyropyriine isretinoi) n ll-trns-retinl imers 16 tht result from inequte lerne n onversion of ll-trns-retinl k to 11-is-retinol through the visul yle 17,18, omprise the lipofusin of RPE. All-trns-retinl itself is retive ytotoxi lehye tht n mge the retin 17,18, ut the reltive toxiities of its onenstion prouts hve not een fully lrifie. Severl explntions for suh toxiity hve een propose, inluing omplement tivtion 19,2, estrution of memrne strutures 16 n photooxitive mge y ting s hromophore for lue light 21. Retinyl esters n ll-trns-retinl onenstion prouts must e spetrlly resolve to monitor their sttus inepenently. 1 Polgenix, Cleveln, Ohio, USA. 2 Deprtment of Ophthlmology n Visul Sienes, Cse Western Reserve University, Cleveln, Ohio, USA. 3 Deprtment of Phrmology, Cse Western Reserve University, Cleveln, Ohio, USA. 4 Center for Visul Siene, University of Rohester, Rohester, New York, USA. 5 Deprtment of Moleulr Physiology n Biophysis, Vnerilt University, Nshville, Tennessee, USA. Corresponene shoul e resse to K.P. (kxp65@se.eu) or G.P. (gplzewsk@polgenixin.om). Reeive 27 April; epte 3 August; pulishe online 14 Novemer 21; oi:1.138/nm VOLUME 16 NUMBER 12 DECEMBER 21 nture meiine

2 Figure 1 Multiphoton exittion of 6-month-ol mouse eye t 73 n 91 nm proue emission spetr initing more thn one fluorophore. () A series of TPM imges of n intt mouse eye were otine long the xis perpeniulr to the RPE lyer with n exittion wvelength of 73 nm. The min ox revels the en fe imge of RPE ells; the two ross-setion imges, one shown t the ottom n the other t the right ege, were ssemle from series of Z-slie imges. The yellow outline retngle represents the region from whih fluoresene ws ollete for spetrl nlysis with the exittion light fouse on the RPE. () Fluoresene emission spetr from the RPE of n intt mouse eye through the sler (ts) n from flt-mounte (fm) mouse RPE. The seon hrmoni signl (SH) shows shrp mximum t hlf of the 91-nm exittion wvelength. () A series of TPM imges of n intt mouse eye otine with n exittion wvelength of 91 nm. In the region of the lue outline retngle, strong seon hrmoni signl from the sler ws ominnt, s the urvture of the eye rought the sler more into fous. The yellow outline retngle represents the region from whih fluoresene ws ollete for spetrl nlysis shown in. () TPM imge of flt-mounte ex vivo RPE. Prt of the RPE is fole over, exposing sgittl view of retinosomes, inite with the yellow rrow. Sle r, 2 μm. 75 µm 75 µm nm, ts 73 nm, fm 91 nm, ts 91 nm, fm 91 nm, SH Nture Ameri, In. All rights reserve. Herein we esrie stuies tht exten the use of noninvsive multiphoton fluoresene mirosopy to the stuy of ntive tissues within the eye. Spetrl nlyses of fluorophores tivte t two ifferent wvelengths n hemil nlyses of the eyes in mie with ifferent geneti kgrouns revele istinguishing fetures etween retinosomes n onenstion prouts of ll-trnsretinl ssoite with retinl pthology. Imging methos esrie herein oul e highly useful to ssess the outome of phrmologil interventions. RESULTS Fluoresent imges of the RPE exite y n infrre lser Consistent with previously pulishe results 12 14, we ientifie retinosomes in the RPE of mie y using 73-nm femtoseon (fs) lser (Fig. 1). The emission spetrum nlyze from fluoresene otine from the re outline y the yellow retngle in Figure 1 showe two mxim t 48 nm n 521 nm (Fig. 1). This spetrum ws superimposle with tht of uthenti lltrns-retinol solution in ethnol otine uner ientil experimentl onitions (Supplementry Fig. 2). The two-photon spetr iffere from those erive from ultrviolet exittion nm 73 nm 76 nm 78 nm 78 nm 81 nm 85 nm 91 nm of retinol tht proue muh stronger emission t 48 nm, s ompre to the 521 nm pek, whih only ppere s shouler. This ifferene proly resulte from omplex eletron exittion-relxtion struture for this onjugte polyene, wherein the lower energy omponent is enhne y two-photon exittion. Retinol lso emits fluoresene t longer exittion wvelengths (Supplementry Fig. 2), so we nlyze the RPE with n exittion wvelength of 91 nm (Fig. 1). Although we oserve strong fluoresene, it iffere from tht of retinosomes y hving mximl emission t ~57 nm (Fig. 1), suggesting the presene of fluorophores other thn retinol within the RPE. We lso oserve very strong seon hrmoni signl erive from the sler (Fig. 1,) tht proly originte from ollgen-enrihe strutures 9. Fluoresene emission spetr were the sme whether imges were otine iretly from flt-mounte RPE or through the sler of the intt eye (Fig. 1,). Imges tken s funtion of exittion wvelengths with onstnt lser power n etetor setting revele tht the high-fluoresene emission oserve t 72 nm exittion erese until 85 nm n then inrese gin t 91 nm (Fig. 2). However, the well-efine fluoresent pttern of retinosomes otine with 73-nm exittion (Fig. 2) ws reple with more uniformly istriute ellulr strutures t the longer 91-nm exittion wvelength (Fig. 2). Moreover, the spetrum systemtilly shifte from shorter- to longer-wvelength emission s the exittion wvelength inrese from 72 nm to 91 nm (Supplementry Fig. 2). Figure 2 Multiphoton exittion of the RPE in n intt 6-month-ol mouse eye t ifferent wvelengths of exittion light. () Grph showing fluoresene s funtion of exittion light wvelength. Fluoresene intensities were otine s men pixel vlues from the re overe y t lest 2 RPE ells in fous n were normlize to the mximum vlue t 72 nm. Blue rrows (from left to right) inite fluoresene in response to 73-, 85- n 91-nm exittion. () Zoome-in two-photon imge of RPE ells otine with 73-nm exittion. Blue rrowhes enote retinosomes (white) lustering long plsm memrnes; the eges of one RPE ell re highlighte in yellow. Drk, mostly oule nulei re inite y pink rrowhes. Sle r, 25 µm. () Imges of RPE ells t the exittion wvelengths inite in eh imge. All imges were otine with 1 mw of lser power. Photomultiplier tue (PMT) gin n offset were kept onstnt for the imges in the top row. Then the gin ws rejuste n kept onstnt for the imges in the ottom row. Sle r, 38 μm. nture meiine VOLUME 16 NUMBER 12 DECEMBER

3 21 Nture Ameri, In. All rights reserve. Figure 3 Visuliztion of retinosomes y three-photon exittion spetrosopy in the intt 7-week-ol Rpe65 / mouse eye. () Grph showing fluoresene intensity s funtion of exittion light wvelength; fluoresene intensity ws lulte s men pixel vlue from the re overe y t lest 2 RPE ells in fous uring imging n normlize to the mximum vlue t 72 nm exittion. () Emission spetr from the RPE of n Rpe65 / mouse eyes exite with lser light t 73 nm n 91 nm. () Logrithmi plot of fluoresene intensity s funtion of exittion power shows eviene of three-photon exittion t n exittion wvelength of 91 nm. AU, ritrry units. () Logrithmi plot of fluoresene intensity s funtion of exittion power shows eviene of three-photon exittion t n exittion wvelength of 73 nm. In n, t points re shown s lk irles. Moeling of the pure seon-power epenene is shown y the lue otte line n moeling of the thir-power epenene is inite y the green she line. Insets epit res ontining the retinosomes from whih fluoresene ws quntifie. Sle rs, 2 μm. Three-photon exittion of retinosomes If the unientifie fluorophores emitting in response to 91 nm exittion were onenstion prouts of ll-trns-retinl proue from lehe visul pigments 16, they shoul e sent in retinl pigment epithelium speifi 65-kD protein (Rpe65)-knokout mie, whih hve efetive visul yle 22. Fluoresene s funtion of inresing exittion light wvelength in these mie showe monotoni ey with no inrese in signl oserve t 91 nm (Fig. 3, Supplementry Fig. 3). Exittion t either 73 nm or 91 nm eliite the sme emission spetrum (Fig. 3), suggesting tht retinyl esters re visile with oth 73 nm n 91 nm exittion. Moreover, the emission spetrum ws onsistent with those erive from mie with 73-nm exittion (Fig. 3). We further onfirme our ientifition of retinosomes in Rpe65 / mie y gvging these mie with rtifiil hromophore 23,24. We oserve expnsion of retinosomes n inrese retinyl ester ontent fter we trete these mie with 9-is-retinyl ette (Supplementry Fig. 4). All-trns-retinyl esters or retinol show mximl emission t ~48 nm, with shouler t longer wvelengths when exmine in mixture of orgni solvents (Supplementry Fig. 5) 25. The fluoresene of retinosomes in retins of Rpe65 / mie sujete to twophoton exittion with 91 nm shoul e negligile, given tht the optil ensity of retinol (or retinyl esters) t hlf this wvelength, tht nm 73 nm 78 nm 1.2.8, 73 nm.4, 73 nm, 91 nm, 91 nm Fluoresene (AU) Slope: 2.83 Seon power Thir power ROI Lser power (mw) is, 455 nm, is only.3% of tht t 365 nm (hlf of 73 nm) with singlephoton exittion in hexne (Supplementry Fig. 5). Moreover, the optil ensity t 33 nm for retinol shoul e 64% of its mximl sorption t 326 nm; thus, it is more likely tht the 91 nm inue signl is the result of three-photon exittion. Aoringly, we foun tht retinyl esters re inee exite y simultneous sorption of three 91-nm photons. The proility of exittion y three-photon exittion is proportionl to light intensity ue, wheres the proility y two-photon exittion is proportionl to the light intensity squre. At n exittion wvelength of 91 nm, fluoresene emission ws proportionl to the 2.83r power of the exittion intensity (Fig. 3), initing sustntil ontriution of the three-photon effet. The thir-power epenene, rther thn the seon-power epenene, showe goo greement with the t. At n exittion wvelength of 73 nm, fluoresene ws proportionl to the 2.12th power (Fig. 3), onsistent with two-photon effet. High levels of A2E in mie with efetive retinoi yle Mie lking retinoi yle enzymes A4 n Rh8 (Supplementry Fig. 1) show oth efiient trnsport of ll-trns-retinl from the internl iss n reue pity for ll-trns-retinl reution n lerne 18,26,27. Consequently, A4 / ;Rh8 / mie umulte lrge mounts of A2E n other ll-trns-retinl onenstion prouts. Imges otine with wvelengths of exittion light tht inrese from 72 nm to 91 nm were onsistently righter thn those from mie (Fig. 4), with exeptionlly high fluoresene t 91 nm. Spetr with 73-nm exittion showe the emission pek hrteristi for either retinol or retinyl ester together with pek t out 56 nm ttriutle to retinl onenstion prouts (Fig. 4). As expete, the ontriution of retinosomes to Fluoresene (AU) nm, Rpe65 / 73 nm, 91 nm, Rpe65 / Slope: 2.12 Seon power Thir power ROI Lser power (mw) 81 nm 85 nm 91 nm Figure 4 Two-photon exittion of 3-month-ol A4 / ;Rh8 / () intt mouse eye. () Fluoresene intensity s funtion of exittion wvelength, normlize to mximum emission t 72 nm exittion. () Emission spetr from RPE of n mie exite with lser light t 73 nm n 91 nm. Blue rrow points to 58 nm. () Imges of mouse RPE t the inite exittion wvelengths. All imges were otine with 1 mw of lser power; photomultiplier tue gin n offset were kept onstnt. Sle r, 5 μm. () Zoome-in TPM imge of unstine mouse RPE. Green olor ws ritrrily hosen to mke the imge etils more visile. Sle r, 25 μm VOLUME 16 NUMBER 12 DECEMBER 21 nture meiine

4 21 Nture Ameri, In. All rights reserve. All-trns-retinyl ester (pmol per eye) , 1,6 1, weeks Rpe65 / this emission spetrum ws reue. Notly, the right fluoresene, lthough puntte, ws spre throughout ells (Fig. 4,) t ll wvelengths teste (Fig. 4). Syntheti A2E stnr h similr emission spetrum, with the mximum shifte to the longer wvelength when exmine y two-photon exittion (Supplementry Fig. 2), n oservtion similr to previous finings 28. These results n those otine with, Lrt / n Rpe65 / mie (Supplementry Fig. 6) strongly orrelte with levels of ll-trns-retinyl esters n A2E foun in the retins of these mie (Fig. 5). Beuse A2E umultes with ge, imges otine with exittion wvelengths of 73 nm n 91 nm oul e extremely helpful in isriminting etween essentil retinois of the visul yle, suh s retinols n retinyl esters present in retinosomes, n potentilly hrmful retinois, suh s exessive mounts of ll-trns-retinl n its onenstion prouts. Two-photon imging to etet retinl ging To test the ility of TPM to etet retinl ging, we otine fluoresene imges from mie t vrious ges n with multiple exittion wvelengths (Fig. 5). Fluoresene emission erese mrkely with inresing exittion wvelengths in 7-week-ol mie, ut in 23-week-ol mie fluoresene emission t longer wvelengths ws elevte (Fig. 5). Therefore, we systemtilly evlute rtios of fluoresene otine with 91 nm exittion to the fluoresene with 73 nm exittion n plotte these rtios s funtion of ge (Fig. 5). As expete, mie showe n inrese in these rtios s funtion of ge, presumly euse A2E n other ll-trnsretinl onenstion prouts umulte. Moreover, 14-week-ol ontrol A4 / ;Rh8 / mie with signifintly elevte A2E h very high 91 nm / 73 nm fluoresene rtio, wheres 17-week-ol Rpe65 / mie, whih o not proue A2E, h very low rtio 22, even elow tht of young mie. We then teste the level of retinyl months 23-week-ol 7-week-ol Age A2E (mau/eye) 1 8 Rpe65 / 6 4 Lrt / Rpe65 / Lrt / 2 N D N D N D Fluoresene rtio 6 weeks 6 months Age Age (weeks) Figure 5 Age-epenent hnges in fluorophore umultion in mouse eyes. () The two-photon exittion fluoresene intensity s funtion of wvelength in 23-week-ol n 7-week-ol mouse retin. Fluoresene emission vlues were normlize to tht oserve with 72-nm exittion. () Rtios of fluoresene exite with 91-nm light reltive to fluoresene exite with 73-nm light. () Levels of lltrns-retinyl esters in the eyes of,, Rpe65 / n Lrt / mie of ifferent ges (n = 5 for eh group). () Levels of A2E extrte from,, Rpe65 / n Lrt / mouse eyes of ifferent ges (n = 5 for eh group). In n, t re mens ±s.. ND, not etete. esters s funtion of ge n foun tht this retinoi umultes with ge, s previously oserve (Fig. 5) 24. Of note, A2E unne inrese with ge, s well (Fig. 5). In the presene of oth A2E n ll-trns-retinyl esters in n A4 / ;Rh8 / mie, we oserve muh more fluoresene from A2E, euse exittion of A2E seems to e more effiient t the longer wvelengths thn the three-photon exittion of retinyl esters. This ws inite y inrese levels of fluoresene t longer wvelengths. At longer exittion wvelengths, the two-photon emission spetrum of lipofusin in humn RPE overlps the spetrum of mouse RPE (Supplementry Fig. 7). DISCUSSION The ims of this stuy were to optimize multiphoton imging of retinosomes, use multiphoton spetrosopy to istinguish mong retinl fluorophores n exmine the use of noninvsive multiphoton spetrosopy to monitor hnges in the retin use y inherite visul isorers n ging. Our stuy revels tht utofluoresent imges of the retin, speifilly of the RPE, n e otine t two exittion wvelengths, one t ~73 nm n the other t ~91 nm. Exittion with ~73-nm photons lerly imges retinosomes in the two-photon moe, wheres ~91-nm photons primrily exite fluorophores relte to the onenstion of ll-trns-retinl, for exmple, A2E, A2DHP-PE n retinl imers 16. Only uner speil irumstnes n retinosomes e imge with ~91-nm photons, s in Rpe65 / mie, where they re enlrge. In suh ses, nerly simultneous sorption of two n even three photons n tke ple. These finings re onsistent with the mximl sorption of ll-trns-retinyl esters or ll-trns-retinol t 326 nm (Supplementry Fig. 5) n A2E t ~44 nm 16. There re severl ifferenes etween retinosomes n retinoi-onenstion prouts. Retinosomes re lote lose to the plsm memrne n re surroune y ipose ifferentition-relte protein 12, wheres onenstion prouts re spre throughout the ell. Eh hs fluoresene emission spetrum unique to its hemil n ellulr strutures, n ll-trns-retinl onenstion prouts not only umulte with ge ut lso re either highly elevte or sent in mie with eletions of ertin genes enoing enzymes of the visul yle. Conenstion prouts re propose to e generte in photoreeptors n umulte in lysosomes of the RPE, n the istriution of onenstion prouts oserve here (Figs. 2 n 4) fits this esription 13. In the RPE, levels of ll-trns-retinl onenstion prouts inrese with ge 16, euse eh y out 1% of humn s ro outer segments, where these prouts re forme, re she n phgoytose y RPE ells 29. These onjugtes umulte within the RPE euse their rekown is either slow or nonexistent in mmmls. Even though we lerly oserve fluoresent retinosomes in mie when fluoresene ws mesure upon exittion t 73 nm n 91 nm, emission from onenstion prouts ws ominnt upon 91-nm exittion. Thus, it seems tht the emission fluoresene rtio fter exittion t these two wvelengths, tht is, the 91 nm / 73 nm rtio, might e use to monitor the helth of the retin n evlute the effiy of therpeuti gents, t lest in mie. The totl kgroun utofluoresene of the funus exmine y snning lser ophthlmosopy (SLO) t the sme lser power (sensitivity t 1) lso reflete the mounts of A2E in mie (Supplementry Fig. 8 n Fig. 5). However, euse justments in lser power re require for eh iniviul to ompenste for vrious eye onitions suh s trts, iret omprison nture meiine VOLUME 16 NUMBER 12 DECEMBER

5 21 Nture Ameri, In. All rights reserve. of SLO vlues etween iniviuls n time points poses potentil prolems. Moreover, the infrre lser of urrent SLO nnot use two-photon exittion. Therefore, the 91 nm / 73 nm exittion rtio of TPM offers greter vntge in ssessing the helth of the retin. TPM provies severl vntges over single-photon mirosopy in monitoring the humn eye. The humn lens n mulr pigments re highly opque to ultrviolet n lue light, respetively. Therefore, it is espeilly iffiult to eliver exittion light sfely n effiiently to ultrviolet- n lue-soring fluorophores in the mul. Inste of using short-wvelength exittion, TPM tkes vntge of infrre illumintion to exite ultrviolet- n lue-soring fluorophores suh s retinyl esters n A2E, whose ynmis n quntities hnge in the mul of people with pre ge-relte mulr egenertion (AMD). In AMD, inresing RPE fluoresene is one of the erliest hnges oserve 3. A reent lrge-sle prospetive stuy of iniviuls with geogrphi trophy showe tht funus utofluoresene t the mrgins of geogrphi trophy ws the est preitor of geogrphi trophy progression. The unique ility of TPM to simultneously imge intermeites n yprouts of the visul yle in the intt eye enles monitoring formtion of ll-trns-retinl onenstion prouts in vivo y ompring the rtio of fluoresene exite with 73 nm to 91 nm. Moreover, s oserve y us n others, the ll-trns-retinl levels require to proue toxiity in vitro (5 μm) re lso foun in vivo, espeilly fter right light exposure. A lifetime of light exposure hs een propose s one of severl risk ftors for vne AMD 18,31. Other vntges of TPM inlue less light sttering with resultnt eeper penetrtion, intrinsi three-imensionlity n erese risk of photolehing n phototoxiity. TPM is lso growing tehnology with potentil pplitions to oth si siene n linil reserh 32. However, most urrent TPM pplitions use exogenous fluoresent mrkers rther thn enogenous fluorophores, n most other proeures involving lrger orgnisms re invsive. By tking vntge of n niml moel for Leer ongenitl murosis (the Rpe65 / mouse) we quntittively etermine tht three-photon mirosopy n generte fluoresent imge hrteristi of this isese. Three-photon mirosopy requires three photons to e sore per fluoresent event, n the exittion is proportionl to the instntneous intensity of inient lser light (I) to the thir power. If imging onitions re ientil, the rtio of threephoton exite fluoresene to two-photon exite fluoresene is proportionl to δ 3 I / δ 2, where δ 2 is the two-photon exittion ross-setion n δ 3 is the three-photon exittion ross-setion 11. We re not wre of ny t iretly eling with two or three-photon exittion ross setions s funtion of wvelength for ll-trnsretinyl esters, so we use existing t for retinol inste 33. On the sis of severl ssumptions, we lulte whether three-photon exittion of retinyl esters might our t 91 nm. We ssume tht the two- n three-photon fluoresene quntum yiels (η) re the sme, tht the three-photon exittion ross-setion spetrum is offset from the two-photon exittion ross-setion spetrum y ftor of 1 33 (ref. 11), n tht it prllels the two-photon exittion spetrum if the wvelengths re set to 1.5 times the orresponing two-photon proess wvelengths. Then, using t for the wvelength-epenent two-photon fluoresene tion ross setion of retinol (η δ 2 ), we estimte tht t 91 nm, η δ m 4 s per photon, n, tking into ount tht mximum sorption of retinol is t 326 nm (Supplementry Fig. 5), we estimte tht three-photon ross setion extrpoltes to η δ m 6 s 2 per photon 2. Using these numers n the intensity t 91 nm, I91 ( po p ( NA) )/( f p t p h l) = photons per m s where is the spee of light, h is Plnk s onstnt, NA is the numeril perture, p o is the verge inient power, τ p is the pulse urtion, f p is the pulse repetition frequeny n λ is the pulse enter wvelength 34, we lulte tht t 91 nm the rtio δ 3 I / δ 2.3. Following the sme methoology ut now t 73 nm, we lulte I photons per m 2 s, η δ m 4 s per photon, η δ m 6 s 2 per photon 2 n δ 3 I / δ These estimtes suggest tht three-photon exittion of retinyl ester is fesile t 91 nm ut not t 73 nm, supporting our experimentl emonstrtion. Thus the three-photon effet hs een emonstrte, perhps for the first time, in the intt, unfixe fresh eye. In summry, TPM of enogenous fluorophores, inluing retinyl esters, ll-trns-retinol, n A2E, provies powerful wy to monitor the visul yle 15 iretly n ontriute to unerstning the pthology of humn retinl iseses. If we oul visulize n unerstn erly errtions of these pthwys in live humn eyes, we woul e etter le to evise n monitor effetive therpies for lining retinl iseses. Methos Methos n ny ssoite referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Meiine wesite. Aknowlegments We woul like to thnk M. Golzk for help uring the ourse of this stuy n Z. Dong for expert hnling of mie. We lso thnk L.T. Wester n memers of Plzewski lortory for ritil omments on the mnusript. Rpe65 / mie were kin gift from M. Remon (US Ntionl Eye Institute). This reserh ws supporte in prt y grnts EY861, EY9339, EY1988, EY1931, EY2715 n P3 EY11373 from the US Ntionl Institutes of Helth, TECH 9-4 from the Stte of Ohio Deprtment of Development n Thir Frontier Commission, the Europen Life Sientist Orgniztion n the Klus Tshir Fountion. AUTHOR CONTRIBUTIONS G.P. n K.P. oneive of n irete the projet. G.P., T.M., Y.I., W.S., Y.C. n A.M. esigne n onute experiments. G.P. n K.P. prepre the mnusript. Y.I., D.W.P. nlyze the t n eite the mnusript. D.R.W. eite the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre ompeting finnil interests: etils ompny the full-text HTML version of the pper t Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions/. 1. Shenke-Lyln, K., Riemnn, I., Dmour, O., Stok, U.A. & Konig, K. Two-photon mirosopes n in vivo multiphoton tomogrphs powerful ignosti tools for tissue engineering n rug elivery. Av. Drug Deliv. Rev. 58, (26). 2. Zhng, E.Z., Lufer, J.G., Peley, R.B. & Ber, P.C. In vivo high-resolution 3D photoousti imging of superfiil vsulr ntomy. Phys. Me. Biol. 54, (29). 3. Kim, J.S. et l. Imging of trnsient strutures using nnoseon in situ TEM. Siene 321, (28). 4. Shohm, D. et l. Imging ortil ynmis t high sptil n temporl resolution with novel lue voltge-sensitive yes. Neuron 24, (1999). 5. Hell, S.W. Fr-fiel optil nnosopy. Siene 316, (27). 6. DCost, R.S., Wilson, B.C. & Mron, N.E. Optil tehniques for the enosopi etetion of ysplsti oloni lesions. Curr. Opin. Gstroenterol. 21, 7 79 (25) VOLUME 16 NUMBER 12 DECEMBER 21 nture meiine

6 21 Nture Ameri, In. All rights reserve. 7. Evgenov, N.V., Merov, Z., Di, G., Bonner-Weir, S. & Moore, A. In vivo imging of islet trnsplnttion. Nt. Me. 12, (26). 8. Piston, D.W. Imging living ells n tissues y two-photon exittion mirosopy. Trens Cell Biol. 9, (1999). 9. Imnishi, Y., Loowski, K.H. & Koutlos, Y. Two-photon mirosopy: sheing light on the hemistry of vision. Biohemistry 46, (27). 1. Denk, W., Strikler, J.H. & We, W.W. Two-photon lser snning fluoresene mirosopy. Siene 248, (199). 11. Xu, C., Zipfel, W., Sher, J.B., Willims, R.M. & We, W.W. Multiphoton fluoresene exittion: new spetrl winows for iologil nonliner mirosopy. Pro. Ntl. A. Si. USA 93, (1996). 12. Imnishi, Y., Sun, W., Me, T., Me, A. & Plzewski, K. Retinyl ester homeostsis in the ipose ifferentition-relte protein efiient retin. J. Biol. Chem. 283, (28). 13. Imnishi, Y., Btten, M.L., Piston, D.W., Behr, W. & Plzewski, K. Noninvsive two-photon imging revels retinyl ester storge strutures in the eye. J. Cell Biol. 164, (24). 14. Imnishi, Y., Gerke, V. & Plzewski, K. Retinosomes: new insights into intrellulr mnging of hyrophoi sustnes in lipi oies. J. Cell Biol. 166, (24). 15. von Lintig, J., Kiser, P.D., Golzk, M. & Plzewski, K. The iohemil n struturl sis for trns-to-is isomeriztion of retinois in the hemistry of vision. Trens Biohem. Si. 35, 4 41 (21). 16. Sprrow, J.R., Wu, Y., Kim, C.Y. & Zhou, J. Phospholipi meets ll-trns-retinl: the mking of RPE isretinois. J. Lipi Res. 51, (21). 17. Me, A., Me, T., Golzk, M. & Plzewski, K. Retinopthy in mie inue y isrupte ll-trns-retinl lerne. J. Biol. Chem. 283, (28). 18. Me, A. et l. Involvement of ll-trns-retinl in ute light-inue retinopthy of mie. J. Biol. Chem. 284, (29). 19. Zhou, J., Jng, Y.P., Kim, S.R. & Sprrow, J.R. Complement tivtion y photooxition prouts of A2E, lipofusin onstituent of the retinl pigment epithelium. Pro. Ntl. A. Si. USA 13, (26). 2. Zhou, J., Kim, S.R., Westlun, B.S. & Sprrow, J.R. Complement tivtion y isretinoi onstituents of RPE lipofusin. Invest. Ophthlmol. Vis. Si. 5, (29). 21. Sprrow, J.R. et l. Involvement of oxitive mehnisms in lue-light inue mge to A2E-len RPE. Invest. Ophthlmol. Vis. Si. 43, (22). 22. Ktz, M.L. & Remon, T.M. Effet of Rpe65 knokout on umultion of lipofusin fluorophores in the retinl pigment epithelium. Invest. Ophthlmol. Vis. Si. 42, (21). 23. Vn Hooser, J.P. et l. Rpi restortion of visul pigment n funtion with orl retinoi in mouse moel of hilhoo linness. Pro. Ntl. A. Si. USA 97, (2). 24. Vn Hooser, J.P. et l. Reovery of visul funtions in mouse moel of Leer ongenitl murosis. J. Biol. Chem. 277, (22). 25. Drent, R., Bryl, K. & Olszewsk, T. A wter environment fores retinyl plmitte to rete self-orgnize strutures in inry solvents. J. Fluores. 7, (1997). 26. Me, A., Golzk, M., Me, T. & Plzewski, K. Limite roles of Rh8, Rh12, n A4 in ll-trns-retinl lerne in mouse retin. Invest. Ophthlmol. Vis. Si. 5, (29). 27. Me, T., Me, A., Lehy, P., Sperstein, D.A. & Plzewski, K. Effets of longterm ministrtion of 9-is-retinyl ette on visul funtion in mie. Invest. Ophthlmol. Vis. Si. 5, (29). 28. Sprrow, J.R., Prish, C.A., Hshimoto, M. & Nknishi, K. A2E, lipofusin fluorophore, in humn retinl pigmente epithelil ells in ulture. Invest. Ophthlmol. Vis. Si. 4, (1999). 29. Kevny, B.M. & Plzewski, K. Phgoytosis of retinl ro n one photoreeptors. Physiology (Bethes) 25, 8 15 (21). 3. Holz, F.G. et l. Progression of geogrphi trophy n impt of funus utofluoresene ptterns in ge-relte mulr egenertion. Am. J. Ophthlmol. 143, (27). 31. Wielgus, A.R., Chignell, C.F., Ceger, P. & Roerts, J.E. Comprison of A2E ytotoxiity n phototoxiity with ll-trns-retinl in humn retinl pigment epithelil ells. Photohem. Photoiol. 86, (21). 32. So, P.T., Dong, C.Y., Msters, B.R. & Berln, K.M. Two-photon exittion fluoresene mirosopy. Annu. Rev. Biome. Eng. 2, (2). 33. Zipfel, W.R. et l. Live tissue intrinsi emission mirosopy using multiphotonexite ntive fluoresene n seon hrmoni genertion. Pro. Ntl. A. Si. USA 1, (23). 34. Xu, C. & We, W.W. Mesurement of two-photon exittion ross setions of moleulr fluorophores with t from 69 to 15 nm. J. Opt. So. Am. B 13, (1996). 35. Me, A. et l. Role of photoreeptor-speifi retinol ehyrogense in the retinoi yle in vivo. J. Biol. Chem. 28, (25). 36. Btten, M.L. et l. Leithin-retinol yltrnsferse is essentil for umultion of ll-trns-retinyl esters in the eye n in the liver. J. Biol. Chem. 279, (24). 37. Remon, T.M. et l. Rpe65 is neessry for proution of 11-is-vitmin A in the retinl visul yle. Nt. Genet. 2, (1998). 38. Btten, M.L. et l. Phrmologil n raav gene therpy resue of visul funtions in lin mouse moel of Leer ongenitl murosis. PLoS Me. 2, e333 (25). 39. Imnishi, Y. & Plzewski, K. Visuliztion of retinoi storge n trffiking y two-photon mirosopy. Methos Mol. Biol. 652, (21). 4. Miti, S., Sher, J.B., Willims, R.M., Zipfel, W.R. & We, W.W. Mesuring serotonin istriution in live ells with three-photon exittion. Siene 275, (1997). nture meiine VOLUME 16 NUMBER 12 DECEMBER

7 21 Nture Ameri, In. All rights reserve. ONLINE METHODS Mie. Lrt / n Rpe65 / mie on C57BL/6J kgroun were rosse with C57 lino C57BL/6J-Tyr C-2J mie to proue lino Lrt / n Rpe65 / mie. Rpe65 / mie were kin gift from M. Remon; Lrt / mie were generte in house 36. C57BL/6J mie were purhse from Hrln. Alino A4 / ;Rh8 / oule-knokout mie were generte y rossing A4 / ;Rh8 / mie with mixe 129Sv n C57BL/6J kgroun 17,35 with 129/SvJ lino mie (Jkson Lortory). PCR genotyping of these mie ws performe s previously esrie 17, Mie were house n rossre in the Cse Western Reserve University Animl Resoure Center Fility, where they were mintine uner either 12-h light-rk illumintion yle or in omplete rkness. Mnipultions in the rk were performe uner im re light trnsmitte through filter (trnsmittne 56 nm; No. 1 Sfelight; Estmn Kok). Mie were provie free ess to stnr how iet n wter. Prior to experimentl nlyses,they were nesthetize y intrperitonel injetion with 1 μl per g oy weight of 6 mg ml 1 ketmine n.44 mg ml 1 xylzine n then kille y ervil islotion. All mouse proeures were pprove y the Cse Western Reserve University Animl Resoure Center Fility Animl Cre n Use Committee n omplie with the Amerin Veterinry Meil Assoition Guielines on Euthnsi. Mie were gvge with 9-is-retinyl ette oring to previously pulishe protool 38. Retinoi n A2E Anlyses. All experimentl proeures relte to extrtion, erivtiztion n seprtion of retinois from issete mouse eyes were rrie out s previously esrie 17,35. A2E ws synthesize from ll-trnsretinl n ethnolmine n purifie y HPLC 35. Quntifition of A2E fter HPLC ws omplishe y omprison with known onentrtions of pure syntheti A2E stnrs 35. Multiphoton exittion mirosopy. TPM imging ws one with Lei TCS SP2. A Ti:Spphire lser (Coherent Chmeleon XR) elivere <14 fs lser pulses t 9 MHz. The exittion light ws fouse on smples y Plnpohromt 1.25 numeril perture,.1-mm working istne ojetive lens, n fluoresene ws ollete through the sme lens. Fluoresene intensities n ross-setion imges were otine y offline nlyses with Lei LCS Lite softwre, version For imging, Hmmtsu R6357 photomultiplier tue ws use s nonesnne etetor fter filtering light through n-pss Chrom filter, HQ 465/16. Before trns-slerl imging n immeitely fter enuletion, mouse eye ws ple t the enter of glss-ottome 35-mm ish (MtTek), with the sler ontting overslip n hyrte with solution of 9.5 mm soium phosphte, ph 7.4, ontining 137 mm NCl n 2.7 mm KCl. The typil time etween enuletion n t quisition ws less thn 3 min. For flt-mount imging, the orne n lens were first issete out of enulete eyes with spring-loe sissors, n the vitreous n retin were remove with tweezers. After four ril inisions, the RPE with horoi n sler tthe ws li in the enter of glss ottome MTek ish n iretly imge with its pil sie fing the glss. Emission spetr were otine with Lei TCS SP2 spetrlly sensitive etetor in the esnne onfigurtion. Regions of interest were outline with Lei LCS Lite softwre version 4.. Spetr were normlize to the mximum vlue for eh smple. The z-position on the mirosope ws kept onstnt when imging with vrile wvelength exittion, tking into ount tht retinosome size is greter thn 4 μm in the z iretion, s eue from Figure 1. Chrteriztion of fluoresene s funtion of lser power. Imging ws one with 73-nm n 91-nm exittion wvelengths with pixels per frme n 4-Hz line spee. Pixel size ws.367 μm. Lser power ws juste with Lei TCS SP2 eletro-opti moultor. At lest 1 h ws llowe for the lser to stilize n eletro-opti moultor to equilirte with het introue y the infrre lser. Power ws mesure immeitely efore n fter imging in the smple plne with lirte Coherent FielMx-TO lser power meter n PM1 sensor. Lser power ws mesure y using the point leh option with isle snning n lnking. To ensure tht the lser em ws fully pture y the sensor, mesurements use FL PLAN.25 numeri perture ojetive with 17.6-mm working istne. To etermine whether two- or three-photon proess ws ominnt, we first ssume tht the two-photon exite fluoresene (N ex2 ) ws proportionl to the inient lser power (p 2 ) n the two-photon fluoresene exittion ross setion (δ 2 ) 1, N ex2 δ 2 p 2 ; n tht the three-photon exite fluoresene ws proportionl to the p 3 n the three-photon fluoresene exittion ross setion (δ 3 ) 4, N ex3 δ 3 p 3. Then the orer of the proess ws etermine from the slope of the line on the log-log plot. Sttistil nlyses. Dt re expresse s the mens ±s.. Liner regression nlysis of fluoresene s funtion of lser power ws performe with Sigm Plot. The squre orreltion (R 2 ) ws lulte s higher thn.997 for eh se. nture meiine oi:1.138/nm.226