Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy

Re inal cell imaging in myopic chickens using adap i e op ics mul ipho on mic oscopy Juan M. Bueno,1,* Raquel Palacios, Anas asia Giakoumaki,1 Emilio J. Gualda,1 F ank Schae el,2 and Pablo A al1 1Labo a o io de Óp ica, Ins i u o Uni e si a io de In es igación en Óp ica y Nano ísica, Uni e sidad de Mu cia, Campus de Espina do (Ed.34), 30100 Mu cia, Spain 2Sec ion o Neu obiology o he Eye, Oph halmic Resea ch Ins i u e, Calwe s asse 7/1, 72076 Tuebingen, Ge many * [email p o ec ed] Abs ac : Abno mal eye g ow h induced by isual dep i a ion can modi y he s uc u e and densi y o he e inal cells. We ha e used an adap i e op ics mul ipho on mic oscope o image pho o ecep o s (PRs) and ganglion cells (GCs) a di e en e inal loca ions in uns ained e inas o chicken eyes wi h abou 10D o myopia and hei no mal-sigh ed ellow eyes. In all samples, he local a e aged in e -PR dis ance inc eased wi h eccen ici y. No signi ican di e ences in PR densi y we e ound be ween con ol and myopic eyes. GC densi y declined in myopic eyes compa ed o con ol eyes and he in e -cell dis ance inc eased. In no mal eyes, he size o he GC cell bodies inc eased app oxima ely wo- old be ween he a ea cen alis and he pe iphe al e ina. In myopic eyes, his end was p ese ed bu he GC bodies we e la ge a each e inal loca ion, compa ed o con ol eyes. Ob iously, GC mo phology is changing when he e inal a ea is enla ged in myopic eyes. ©2014 Op ical Socie y o Ame ica OCIS codes: (170.3880) Medical and biological imaging; (180.4315) Nonlinea mic oscopy; (110.1080) Ac i e o adap i e op ics; (170.4470) Oph halmology Re e ences and links 1. J. Wallman, J. Tu kel, and J. T ach man, “Ex eme myopia p oduced by modes change in ea ly isual expe ience,” Science 201(4362), 1249–1251 (1978). 2. F. Schae el, A. Glasse , and H. C. Howland, “Accommoda ion, e ac i e e o and eye g ow h in chickens,” Vision Res. 28(5), 639–657 (1988). 3. C. F. Wildsoe and J. D. 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Neu ol. 140(3), 359–397 (1970). #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 665 42. J. K. Bowmake and A. Knowles, “The isual pigmen s and oil d ople s o he chicken e ina,” Vision Res. 17(7), 755–764 (1977). 43. R. O e and D. Moo e, “Spa ial acui y o he chicken,” B ain Res. 211(2), 424–426 (1981). 44. H. M. Johnson, “Visual pa e n-disc imina ion in he e eb a e: II. Compa a i e isual acui y in he dog, he monkey and he chick,” J. Anim. Beha . 4(5), 340–361 (1914). 45. L. R. DeMello, T. M. Fos e , and W. Temple, “Disc imina i e pe o mance o he domes ic hen in a isual acui y ask,” J. Exp. Anal. Beha . 58(1), 147–157 (1992). 46. K. L. Schmid and C. F. Wildsoe , “Assessmen o isual acui y and con as sensi i i y in he chick using an op okine ic nys agmus pa adigm,” Vision Res. 38(17), 2629–2634 (1998). 47. E. Died ich and F. Schae el, “Spa ial esolu ion, con as sensi i i y, and sensi i i y o de ocus o chicken e inal ganglion cells in i o,” Vis. Neu osci. 26(5-6), 467–476 (2009). 48. N. J. Cole a and T. Wa son, “E ec o myopia on isual acui y measu ed wi h lase in e e ence inges,” Vision Res. 46(5), 636–651 (2006). 49. T. Y. Chui, M. K. Yap, H. H. Chan, and L. N. Thibos, “Re inal s e ching limi s pe iphe al isual acui y in myopia,” Vision Res. 45(5), 593–605 (2005). 1. In oduc ion Chickens ha e been ex ensi ely used as animal model in emme opiza ion and myopia s udies. I is well es ablished ha dep i a ion by placing di use s in on o hei eyes al e s he no mal ocula g ow h pa e n wi hin hou s, gene a ing excessi e axial elonga ion and subsequen myopia [1–5]. The g ow h o he pos e io globe is con olled la gely by he e ina which can inhibi o s imula e scle al g ow h, depending on he sign o he de ocus imposed on he e inal image. As a esul , he bes ma ch be ween he ocal plane and he pho o ecep o (PR) plane is achie ed du ing de elopmen [1]. When excessi e g ow h is induced by nega i e lenses o di use s, cho oidal blood low is educed [6], cho oidal hinning occu s [7], and he cone inne segmen s hicken and od ou e segmen s elonga e [8]. The an e io chambe dep h enla ges [9] and he scle al c eep a e inc eases, a leas in mammalian myopia models [10]. The chicken e inal s uc u es ha e been imaged wi h luo escence [11] and b igh ield mic oscopy [12], OCT [13] and mo e ecen ly wi h con ocal scanning lase oph halmoscopy [14], adap i e op ics (AO) undus imaging [15], and AO mul ipho on mic oscopy [16]. Two-pho on exci a ion luo escence (TPEF) [17] has been epo ed o be a mul ipho on ool sui able o imaging biological specimens. Since Imanishi and colleagues epo ed ha bo h he e inal pigmen epi helium (RPE) [18] and he PR ou e segmen s [19] o he mouse e ina p o ided TPEF signal, di e en au ho s ha e explo ed he isualiza ion and dis ibu ion o e inal cells in animal models [20–22] and humans [23–26]. In pa icula , he combina ion o AO and mul ipho on mic oscopy p o ided imp o ed images o e e y e inal laye in non- s ained human and chicken e inas [16,23,27]. AO mul ipho on oph halmoscopy also p o ided images o PRs in li ing p ima e eyes [28]. Al hough chicken e inal cells ha e ecen ly been imaged in i o [25,26], he e a e ew da a on he e ec s o induced myopia on e inal cell spacing, dis ibu ion and densi y in chickens, excep o a his ological s udy on he spacing and a bo iza ion o e inal be a ganglion cells [29]. Mo eo e , PR densi ies ha e no been mapped ou . Non-linea imaging echniques a e minimally in asi e and p o ide in insic axial esolu ion, allowing imaging o he di e en e inal s uc u es wi hou s aining p ocedu es. In his sense, he aim o his wo k has been o use AO-TPEF mic oscopy in e inas o myopic chickens o s udy he spacing, appea ance and dis ibu ion o e inal cells o be compa ed wi h no mal chicken eyes. 2. Me hods 2.1. Adap i e op ics mul ipho on mic oscopy Mul ipho on imaging was pe o med using a esea ch cus om-buil scanning mic oscope, combining a mode-locked Ti:Sapphi e lase (760 nm), a modi ied in e ed mic oscope and a pho on-coun ing uni de ec ion (Fig. 1). Fu he de ails can be ound in [16,27]. The se up also includes an AO module composed o a Ha mann-Shack wa e on senso and a de o mable #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 666 mi o , used o compensa e o he lase beam and mic oscope op ics abe a ions in closed- loop. A DC-mo o con olled he loca ion o imaged plane wi hin he sample along he Z di ec ion. A non-imme sion long-wo king dis ance mic oscope objec i e was used (20x, NA = 0.5). TPEF signal om he sample was collec ed in he backsca e ed di ec ion h ough he same objec i e. A spec al il e was placed in on o he de ec ion uni (PMT) o selec his TPEF signal. All TPEF images shown along his pape we e eco ded wi h he AO module in ope a ion. A he specimen plane, and depending on he analyzed sample, he lase in ensi y anged be ween 2 and 20 mW/cm2. Fig. 1. Schema ic diag am o he AO mul ipho on mic oscope. PMT, pho omul iplie . Image acquisi ion was ca ied ou by means o cus om-w i en LabViewTM so wa e. TPEF mic oscopy images we e eco ded a a a e o 1 image pe second (0.82 mic ons/pixel) a di e en eccen ici ies along he e inal dep h (in s eps o 3 mic ons). Ha ing in o accoun he used wa eleng h and he mic oscope objec i e speci ica ions, he heo e ical la e al and axial esolu ion alues o a mul ipho on con igu a ion a e espec i ely 0.52 and 2.13 mic ons. Al hough TPEF signal clea ly isualizes mos e inal laye s om he ne e ibe laye o he PR ou e segmen [16], in he p esen wo k we a e only in e es ed in e alua ing he spa ial dis ibu ion o PRs and GCs in bo h con ol and myopic e inas. A each e inal dep h-loca ion six indi idual ames we e acqui ed and a e aged o ge he inal image. Image p ocessing was pe o med using bo h Ma LabTM and he public domain image p ocessing so wa e ImageJ. Fo each image he densi y o PRs was manually compu ed by he ope a o [16]. To calcula e he densi y (and a ea) o GCs, each image was i s p ocessed using di e en il e s o imp o e he isibili y o he GCs (Fig. 2(a)). This included b igh ness-con as adjus , backg ound sub ac ion and median il e ing. Then an image con as h eshold and a bina y p ocess was also applied (Fig. 2(b)). Wa e shed segmen a ion was employed o au oma ically sepa a e indi idual GCs (Fig. 2(c)). Cells loca ed on he edges o he image we e no aken in o accoun . Some imes, as a esul o he au oma ic segmen a ion p ocedu e, small and i egula pa icles a e gene a ed as a e ac s. These we e also excluded om he inal compu a ion. Finally he quan i y o GCs wi hin he image and he a ea o each indi idual GC we e calcula ed (Fig. 2(d)). #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 667 Fig. 2. Example o he p ocedu e s eps. (a) TPEF image o GCs; (b) image a e di e en il e ing, h esholding and bina iza ion; (c) wa e shed segmen a ion; (d) inal his og am o GC coun ing and a ea compu a ion. The a ea cen alis o he chicken e ina has been epo ed o be he loca ion wi h maximum cell densi y [15,16]. Fo each e ina i was loca ed in a p e ious ope a ion by using a b igh - ield (linea ) mic oscope. Wi h his echnique, only he PR oil d ople s a e clea ly isible [16]. Two di e en obse e s (co-au ho s) independen ly chose he loca ion ha was always simila o bo h. This was se as he o igin o he eccen ici y scale. To de e mine he e inal eccen ici ies om he linea dis ances on he e inal su ace, he alues o pos e io nodal dis ance (PND) and adius o he ocula globes o schema ic chicken eye models (bo h emme opic and myopic) we e used [30]. Mo eo e , he isual spa ial esolu ion (VR, in cycles/deg) o he chicken e ina o di e en eccen ici ies was calcula ed as [31]: , 180 2 GC PND VR D π =⋅ ⋅ (1) wi h DGC being he a e aged GC in e -dis ance di ec ly compu ed om he Fou ie powe spec um o he GC images [16]. 2.2. Samples Fi e male whi e legho n chicks we e ob ained om a local ha che y in Kilchbe g, Ge many, and aised in g oups unde a 12-h ligh /12-h da k cycle in he animal acili ies o he Ins i u e o Oph halmic Resea ch (Sec ion o Neu obiology o he Eye) in Tuebingen, Ge many. Food and wa e we e eely a ailable. Fo he de elopmen o myopia, chickens wo e di use s o e one eye o 7 days acco ding o a well-es ablished p ocedu e [32]. The ellow eyes had no mal isual expe ience and we e used as con ols o compa ison. A e di use emo al, ocula e ac ion was measu ed using au oma ed in a ed pho o e ac ion [33]. A e one week o di use wea ing h ee ou o i e eyes de eloped myopia o −10.4 ± 2.9 D on a e age. The wo eyes which became sca cely myopic we e excluded om he s udy. As ypically ound, ellow con ol eyes exposed o no mal ision emained nea - emme opic (1.3 ± 1.6 D). The animals (4 week-old) we e sac i iced wi h an o e dose o e hyl e he anes hesia (see u he de ails on [34]) and he ocula globes we e immedia ely excised. A e cu ing he ocula globe along he ho izon al plane behind he scle al ossicles, he i eous was ex ac ed and he e ina de ached om he undus. The e ina (wi hou he RPE) was ixed o e nigh in a mois chambe wi h pa a o maldehyde solu ion (4%, in 0.1M phospha e bu e ). The e inal issue was la -moun ed on a mic oscope slide and co e ed wi h a 130-µm hick co e slip. None o he samples was s ained. A o al o eigh e inas (3 myopic, 5 con ol) we e in ol ed in he p esen s udy. Con ol eyes we e he same as hose used in [16]. Expe imen s we e conduc ed in acco dance wi h he s a emen o he use o Animals in Oph halmic and Vision Resea ch and app o ed by he Uni e si y o Tuebingen Commission o Animal Wel a e. #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 668 3. Resul s 3.1. Visualiza ion and analysis o he PR mosaic Despi e he anspa ency o he e inal issues in he isible pa o he spec um, mul ipho on mic oscopy p o ides images wi h su icien con as ac oss he en i e e ina. As an example, Fig. 3 shows images o he PR mosaic in a con ol and an a e dep i a ion myopia eye. TPEF signals clea ly deno e indi idual cells in bo h images. Since he samples we e no s ained, his signal co esponds o endogenous luo escence. A he PR laye he signal comes mainly om he oil d ople s, hea ily pigmen ed sphe ical o ganelles loca ed be ween he inne and he ou e segmen [35]. Fig. 3. TPEF images o PR laye o con ol (a) and myopic (b) chicken e inas a he same e inal eccen ici y. Se s o PR images o a ious e inal eccen ici ies p e iously epo ed by hese au ho s can be seen in [16]. Scale ba : 50 mic ons. Fig. 4. (a) Dec ease in PR densi y (cells/mm2) wi h e inal eccen ici y o myopic ( ed symbols) and con ol (blue symbols) eyes. Linea eg essions i ed o he da a demons a e a signi ican dec ease in PR densi y wi h e inal eccen ici y. (b) A e aged PR densi y alues as a unc ion o he e inal eccen ici y g ouped in in e als o 20 deg ees. In o de o e alua e he spa ial dis ibu ion o PR in bo h con ol and myopic eyes TPEF images o he co esponding laye we e acqui ed a di e en e inal eccen ici ies. Figu e 4(a) shows he densi y o PRs o all specimens as a unc ion o he e inal eccen ici y in bo h #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 669 myopic ( ed) and con ol (blue) eyes. Blue and ed lines a e he co esponding bes linea i s which showed a signi ican dec ease wi h eccen ici y (R2 = 0.79, p<0.0001 and R2 = 0.74, p<0.001 espec i ely). A pai ed - es showed no signi ican di e ences be ween bo h se s o da a. Fo comple eness, he changes in PR densi y wi h e inal eccen ici y g ouped in in e als o 20 deg ees a e shown in Fig. 4(b). A he cen al e ina he PR densi y o hese pa icula samples was abou 20600 cells/mm2. These alues dec eased o app oxima ely one hal a he pe iphe al egions o he e ina. To u he explo e his spa ial dis ibu ion o PRs, o each TPEF image he local a e age PR in e -dis ance was compu ed om he Fou ie powe spec um [16]. Fo bo h myopic and con ol samples he inc ease in PR in e -dis ance as a unc ion o e inal eccen ici y is depic ed in Fig. 5(a) and he ela ionship be ween his PR in e -dis ance and he densi y o PRs in Fig. 5(b). Fo bo h plo s signi ican linea co ela ions we e ound: R2 = 0.40, p = 0.008 and R2 = 0.36, p = 0.001 o myopic and con ol eyes espec i ely in Fig. 5(a), and R2 = 0.61, p = 0.003 and R2 = 0.56, p<0.001 o esul s in Fig. 5(b). As also shown in Fig. 4, dis ibu ions o myopic and con ol eyes did no di e signi ican ly. Fig. 5. (a) Inc ease in PR in e -dis ance wi h e inal eccen ici y. (b) PR in e -dis ance (mic ons) e sus PR densi y (cells/mm2). Myopic and con ol eyes a e ep esen ed by ed and blue symbols espec i ely. Linea eg essions i ed o he da a demons a e a signi ican inc ease in (a) and a signi ican dec ease in (b) o bo h se s o e inas. 3.2. Visualiza ion and analysis o he GC laye TPEF images o he GC laye in con ol and myopic eyes o wo di e en e inal eccen ici ies a e shown in Fig. 6. Whe eas he cell nucleus emains da k, he bo de s (cy oplasm) p o ides a high non-linea signal, allowing eadily dis inguishing indi idual cells a e e y e inal loca ion. #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 670 Fig. 6. TPEF images o GCs o con ol (a, c) and myopic (b, d) eyes. Images co espond o di e en e inal eccen ici ies, ~15° (a, b) and ~85° (c, d). Scale ba : 50 mic ons. The densi y o GCs was also compu ed o di e en e inal loca ions. Figu e 7 p esen s he esul s. A signi ican dec ease wi h e inal eccen ici y was also ound o bo h myopic (R2 = 0.64, p<0.017) and con ol (R2 = 0.86, p<0.0001) e inas. O e all GC densi y o eyes exhibi ing dep i a ion myopia was 13% lowe han ha o con ols ones, al hough his di e ence did no achie e signi icance. Fig. 7. Change in GC densi y (cells/mm2) as a unc ion o he e inal eccen ici y g ouped in in e als o 20 deg ees, o myopic ( ed symbols) and con ol (blue symbols) e inas. The changes in GC a ea wi h e inal loca ion a e depic ed in Fig. 8. A signi ican inc ease in GC body size wi h e ina eccen ici y was obse ed o myopic (R2 = 0.42, p = 0.058) and con ol e inas (R2 = 0.49, p = 0.003). I is in e es ing o no e ha he size o he GCs inc eased app oxima ely wo- old when compa ing he a ea cen alis and he pe iphe al e ina. Fo each e inal loca ion, he GC size in a e dep i a ion e inas was la ge han in con ol ones (83% on a e age). Mo eo e , his di e ence was s a is ically signi ican (p = 0.01, pai ed - es ). Fig. 8. A e aged GC a ea alues (mic ons2) o di e en e inal eccen icies. Red symbols, myopic; blue symbols, con ol. Lines co espond o he bes linea i s. #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 671 Fo comple eness, Fig. 9 shows he co esponding alues o GC in e -dis ance (pa ame e DGC). As expec ed, o bo h ypes o e inas, he inc ease wi h eccen ici y was signi ican . Mo eo e , o each e inal loca ion DGC was la ge in myopic eyes (24% on a e age), and his di e ence was s a is ically signi ican (p = 0.005, pai ed - es ). Fig. 9. Signi ican linea inc ease o GC in e -dis ance (mic ons) wi h e inal eccen ici y. Myopic eyes ( ed do s): R = 0.77, p = 0.015; con ol eyes (blue do s): R = 0.80, p = 0.0001. F om each TPEF image o GCs, he VR was compu ed as explained in Eq. (1). As expec ed, VR dec eased owa ds he e inal pe iphe y (as depic ed in Fig. 10) o con ol and (op ically co ec ed) myopic eyes. Fo mos e inal loca ions VR appea ed highe in con ol e inas (14% on a e age, 20% a he a ea cen alis) bu his di e ence did no achie e signi icance ei he . Fig. 10. Values o calcula ed GC isual esolu ion (c/deg) in he chick e ina as a unc ion o e inal eccen ici y compu ed using Eq. (1). Labels a e he same as in p e ious igu es. 4. Discussion AO mul ipho on mic oscopy has been used o image e inal laye s con aining PRs and GCs in bo h con ol and myopic chicken eyes. Indi idual cells a e clea ly dis inguished in all specimens. Pa ame e s such as cell densi y and in e -dis ance ha e been analyzed o di e en eccen ici ies in o de o p o ide addi ional in o ma ion on cellula a angemen . Al hough e inal cells ha e been imaged wi h his mic oscopy echnique [20–22,24–27], o ou knowledge his is he i s ime ha TPEF images ha e been used o compa e densi ies and spacial dis ibu ions be ween myopic and emme opic eyes. Resul s he ein ha e shown ha PR densi y dec eased wi h e inal eccen ici y in myopic and con ol eyes. Howe e , no di e ence in PR densi y was de ec ed in myopic eyes compa ed o no mal ellow eyes. Cone in e -dis ance was signi ican ly ela ed o densi y in bo h se s o eyes. GC densi y declined by 13% when myopia was induced (which did no achie e signi icance). This ag ees wi h he inc ease in su ace o he ocula globe o abou 18% o a 10-D myopic chicken eye. Mo eo e , he a e aged spacing among GCs inc eased #203105 - $15.00 USD Recei ed 17 Dec 2013; e ised 30 Jan 2014; accep ed 31 Jan 2014; published 7 Feb 2014 (C) 2014 OSA 1 Ma ch 2014 | Vol. 5, No. 3 | DOI:10.1364/BOE.5.000664 | BIOMEDICAL OPTICS EXPRESS 672