Some fluorescence properties of cataractous eye lenses

FLUORESCENCE PROPERTIES LENSES

OF

CATARACTOUS EYE

Aleksander Bal Ler

N. Copernicus Uni vers-i ty'

Grudzi4dzka 5

87-1oO Torurl, PoI and

ABSTRACT

T:le foss of Lransparency of Lhe oculal. l-ens is caused

by Lhe increase of lighL scaLLering as a result of s!:_ucLural changes and by ihe -increased absorpt.ion df the wlsible light due Lo t-he accumulaLion of piqmenls.

Fo:1owi ng pigmen!s

] NTRODUCfi ON

The pholol umi nescence of- Lhe eye lens was fifs!

reporled by Regnauld in 1458 [1]. Since then numerous st-rjdi es of Lhis pheDomenon, _{bolh in uil,o and irr uitro,}

we.re perfor_med _te-lo]- Ift ?)il,o lens fluo.escence can be undergo non-radia.t.iwe and .adiali\/e Clumj.nescence)

prccesses whtch can be moniLol'ed spect-.oscopi cal l y. The paper presenLs resldts conce.ning the e>€iLalion specL.a, dacay Limes and polarj.zatlon of t-he 1e::Lt cul ar fluo.escence. Fluorophore helerogeneily

ma::-if esLs llself ln alf Lhe _{e>qperimen.t.al dat.a- A}

st-riking behaviour of Lhe emj"ssion anisot,ropy as a fu;:clion of Lenperat.ul'e is found, part,1cularly for cc:_llcaL calat'acL Ienses, indicaLino

ut il i zed

for lhe

for Lbe ea.l Y

evaluaLion of

deLecLiol., ol caLaracL t11l and Lhe anLicalaract drug effi cacy

L121

chromophores

Cfluo.ophores), also referred to as fluorogens' h;ve

al.aady been ident-ified t13-151 . AhrsorPlion .in Lhe UV

.egion belo\r 3OO nm is mainly due Lo Lhe aromaLic amino acids Lr'ypLophan, L)4'osine and Pheny.Lalanine Present, in .Ieirs prot-eins 1n t-e.tns of visual caPabilily lhis absorplion is insignificani. Chromophores absorbing in the visible range , however , conlrrbule d.irecLIy Lo the loss of Lhe .Lens Lransparency. A haze caLrsed by

their flrjorescence may also ini-erfe.e wilh vision t6l The idenlif.ied, visible lighl absorbing fluorophores

A nunber of

.i-r),?t-ophan _{deri vaLi ves}

Pioperlies Cincluding raPid nano- _{and Picosecond}

Prccesses). The _--xLreme single Phot-on sensiliviLy of

mcde.n fl-uorescenge i-echniques Logelher wiLh Lhe hJs<iFrir i.\r +roir Eitu non-destr ucti ve

view of Lhe

N'-formylkl.nurenine, L-kynurenine and O-B _-D-ql ucosi de oi 3-hydroxy- L-k),'nurenine, as we.L 1 as 3'3'-biLl,'rosine

anC anlhrani.Iic acid Cl,3-151 . In addii-ion, Lhel.e is a pcssibility of lhe accumufai-ion and relention of n!!ne. ous exLraneolrs tluorescenL comPounds, in paj_Lrcular orugs. such as Psoralens, Phenothj. azi nes, pc.ph),'rins or ai.lopurinol f161 .

Fluorescence nelhods are one of the moderr',

.a3idly developing, Loofs fo. probing Lhe biomofecula. s):sLenls t171 . Since fluorescence is sensit-ive Lo 't-he

P].cp-or!i es of lhe f I uoioPhore' s mol ecul ar envit_onm-anl,

i-hese melhods may be used Lo provide infortnaLion abouL il and in parlicular about its slrucLural and dl/namic

l.ansparency an imporLanl properLy is lLs absorptlon

speclrum 1n lhe vlslb].e range from abcrl 38O nanoneLers

ro

about, 7q9.

_{!?q?l1L-9tli.}

a9._9!13!qr _{_q,i:"ect} measuremenLs of lhe spect-I'at disLribuLion of absorbed light are, owlng Lo slrong light- scaLLeriDg and lhe shape of lhe lens,very d-ifficulL Lo perform. T:)ese dlfficuliies can be avoj-ded eiLhef by using :ne phoLoacouslic

met-hod t3,1al o. by record.ing '-he fluorescence

lnlensily as a funclion of fhe exci_,aLion wavelenglh

Clhe ftud.escence excilation spectrrm) al a fixed emission wavelengt.h tal- Such a spect-rirn i"

"ti" to li'" abso.plion speclrum and may be, in p.tnc.iple, measqred in sitz as well as other fLuo.escence characterislics (i-ncluding ils emission sPecL.um, 3olarizaLion and

decay).

MATERI ALS AND METHODS

A Lolal of 102 lenses obtained as a resulL of inLracapsul.ar cat.al'acL exlractlon from Lhe Ey" DeparLnenL ot lhe Joinl Provincial H3spiLal in Torurl wer_e sludied The age of patienls ran3ed f.oJ11 O lo 87. Int-acL .Lenses were placed ln a special hotder belween

lwo quarlz windovs of O.5 tnm i-hickness conlaining no fluids and measuremenls were pei_formed Cunless oi-her.wise sLaLed) al room Lempefalure about one houa afler t-he operaLion . In some cases lhe fenses were

sT-ored _{for several days al OoC. Le;:s e\-Lracts} were prepared accord-ing Lo Lhe follcwing pfocedure-DecaFsuleled ]enses were dessicaled in Lhe presence of

P-O^ for 4 daF .cu! rnt.o iour parls and pounded. Each par! was Lhen mixed wlLh a solvenL Cl:_is buifer pH 7.5

lhe nd><Lure _{'.ras honogenized fo.} 1O minut-es 1n an ul!:_asonic bath and cen!!'ifuged. Aqueous sofut-ions were

adcjlionally dialysed lo reduce lighL scat-le.ing'

ot-i.e.l^llse inlerfertng wj.Lh absorplion measuretlent-s _' and lo slu<iy lhe effecl of .emoval of ProLeins and oLher

rhaca orno.I ec ul, a. malerial 0n Lheir f.Luorescence

AbsorpLion sPecl.a ware measured using a Specord

uV--rT S specLroPholomeLer fl'otn C. Zeiss'Jena CEasL Ge.:.any) - Fl!lo.escence excilat-ion antl emission sPeclra

v,/ere recorded on a home bu-ifL versalile

comPuler-cor-'-r ol I ed photon-counLing speclrofl uori met,e. equtPPed v/i'-r': Dolarizers. MeasuremenLs ot fluoaescence

ex.:'-at-ion spect,.a required the use of Lhe qurntum

co'.i!ef sofuLion of rhodam-ine B in eLhylene glycot

C3g,'1).-in Lhe reference channei of Lhe insli'umenL. Wilh

lh:s quant-um _count-er lhe highest- accuracy rs obrained

in --ire range of excilalion wavelenglhs tt'rriled t,o 36() -6CC nm. Emission sDeclra \teFe nol correcled for Lhe

specLral sensii-iviLy of lhe deLecling sysLem. Ftu.rescence decay curves were measured wilh a home

burll !ime-cor.e]aLed single photon Danosecond flL3roneLe. using a niliogen-filIed flashlanP. The f i:'-: Le lime-widLhs of Lhe excillng lighL pulse and Lhe ins:.umenLal .esponse Can ove.all halfwidlh ot abou! 2 na::3seconds) .equired t-he use of a deconvolut-ron

'he fl!:=rescance inlensiLy lo an infinitely shorL

€x.:t-ai-ion pul.se Clhe so calIed imPulse response). A

leasl squares filt-inq deconvol.uLion p.ocedure was used

lo .ecover t.he fluorescance decay parameLel.s t1Sl . In aI: exci taLi on-eni ssi on sLudies, fron! face exciLaLion

of the anLerior part of lhe -Lens was aFF.lied. fbe ill'rnrinaLed area, abouL a mmz, was cenL-ared on lhe

di.recLion made an angfe of 45- wl!h' Lhe lens axis and

Lhe fluorescence erlission was observed along Lhis axis. Conslruclion det-ails of ou. inslrurnent-s are avallable

LeL us first. presenl Lhe resulLs obLained for

cat-aracLous lens e\'lracLs. TyPl ca.t absorPlion specLra

ol such exlracls are shovrt iTr-:iFigsiti and

2-AceLon-it.ile e\.!racts absortrLlon in i-he 2OO

-exhi bi led

600 nm range- The

st-rongesi-t-he f] uorescence -of i-he sludied ex-Lracls. In

absorDlion was found for aqueous exl-.ac1-s and app-aars lo be main.Iy due to lhe Presence of Lhe aromatic amino

acids in lhe exLraci-ed sutrslance. The speclra' ot e!hano]ic and cyclohexane e).Lracfs are similar lo each

olher,bul dllfer slrongly in t-heir inlensiLy' shape and posit-ion wiLh resPecl Lo lhe speci-ra of aqueolJs ex'LracLs. It is lnteresLing Lo nole LhaL Lhe aqueous

ex-L.acls spectl'a markedly exLend iDLo t-he .visible range for nucLear ar,d

'rli xed cal-aracLs CFig. 1) ' bul

nol for Lhe sugar calaract- CF.ig.2)

The fluoaescence speciaa were' measured for all df lhe prepared e\-|racls,using Lwo exciLatlon wavelenqLhs:

366 nm and 436 nrA. OnIy lhe aqueous e\-t-racls exhibiled

nreasurable fluol'escence under Lhese condilions and lhe posit-ion and shape oi Lhe enrission band \'/er e iound to

be exciLalion wavel englh-dePendent cFig- 3). The same

figure also shows lhe p.onounced effecl oi dialysis on parlicula. dialysis leads lo a several-fo-ld increase of Lhe emission inLensiL,y and shifLs Lhe maxi tnum of Lhe e)llission exciLed wit-h Lhe 436 nrn ligh! fl'on about- 5OO

nm Lo 52O nm.

All of Lhe following .esu.It-s were obt.aj- ned fol'

whole lnlacL lenses. Flgure 4 sho\'.s examFLes of normalized f.luorescence spect-ra of lhree .lehses. These spect-ra are strongly e),-ci!a!ion rtavefengl-h-dependenl,

revea-Iirig Lhe he|erogeneit-y of f.Luoraphore,s. Flgufe 5

displa]s Lhe same speci-ra, buL wiLh l'elalive inlensllI€.s p.eserved for each excitat'ion wawelenglh.

Figure 6 pl.esenls lypical f.Luorescence exciLalion specL.a {Lhe .aLio or LI_,e Dumber of em-ilLed phoLons Lo Lhe nunber of incide.lt- phoLons as a funclion af Lhe excilalion wavelengLh) of Lranspareni Ca) and cat-aracLous Ct',cf IenEes. These excilaLion specLra

sLrongly depend on t-he wavel, ength used to observe

fluorescence (5OO,55o,600,650 and 7OO nm) which is again arr -indicat.r,rn of t-he hele.ogeneily of t-"he

fluorophare populalion CFartlcularly evldent- in Fig.6 b

and c)-An inleresting flndinq is Lhat 5 oul of 6 su€raf

cal-aracL fenses are charact-erized bv Lhe same sDecLra *hown 1n Fig.6c.

Figure 7 p.esenls fluorescence exciLaLj-on speclr.a

detined as

4J ll-1ry761 ll.s 211 , where I and T- are the parallel

and Lhe Ferpendicular polai_lzalion cor*ponents af Lhe ffuorescence inlensiLy lrt Lhe case of a linearly pofarized excitat-lon ligh!, fo. Lwa Lyp.i ca.L cases ot

cataraclous and t-t.anspafenL Ienses. The enlsslot) anisoi-foFy aFFears Lo be essenLia.Il.y consLanL across t-he exclLaLion specLrum. A sfiEhl increase \cl t-h rising

excilat-ion wawelenglh may p.obabfy be attriL'uled Lo

some Ieakage of the st-rongly scaLt-ered excitalion l-ighl lhrough Lhe evldsslon monochr'omaLor. This effecl is more

F.onounced fo. t-he cat-aracLous Iens.

deDendence of Lhe emission anisot-ropy' oblained on healing CFigs.8,s) and bot-h on heat-ing and cooliDg.

Typicat resulLs oi Lhe fLuorescence decay measuremenLs lor lenses no.6'F and I Csee exciLation speclra in figures 4 and 5) obt-ained using lhe 337 nio

ex.il-aL.ron are shown in TabIe 1. T1-t"y .ePresen! Lhe

inpulse response of fluorescence' which was assumed Lo

be a sum _{of three exDonent-ials: T],"" " ',}

TABLE 1

A:ri i Ludes d Cre]al-rve) and Decay Times

F!uorescence Dec.y for Lenses

"".$"fl ana 5 d:fferenL Endssion Wavelenglhs Xen

\tavel engt.h: 337 nrn. ExcilaLion I ns] t nml I nsl lnsl

6(

o. e4 1.34 o.7 o.a o.7 4.7 o.5 o.24 o. a6 o.17 o.

04

13. 6 o.

05

17. L o.

04

s.4 o.

02

10.3 o.

01

12. 6 q? o- 56 o.55 o.5e 1.8 r-4 o.27 o. 1s o. eo o.

oos

27. a o.

04

10.9 o.

01

e7.7 o.

oo4

86.2 a6 o.70 o.55 o.50 o.6 1.5

o.4t

o.25 o.

o9

7.7 o.

04

14. a o.

os

11.8

The resulLs shown .in Table 1 indicale lhal Lhe ffuorescence decay of a lral.tsParent- Iens no.5 tnay

indeed be .epresenl.ed by Lhree exponenLials wiLh decay lirnes essenlially independenL of lhe emission

\ravelengLh, bul in Lhe case of Lhe lenses no.6 and 7 more componanls are requ.ired i-o adequalely represenr

lhe daLa.

Dl SCUSSI ON

Fluorescence heLerogeneily of cat-aract-ous eye

lenses is maniiesLed in aII ol Lhe resulls Presenled

in t-he foregoing secLion. Dialysis did nol remove.lhe excilaLion wavelengLh dePendence of the aqueous ex'lracLs, \rhich indicales Lhat t-he main faclor in Lhe observed helerogeneiLy is t-he p,'esence ol warious

fluorophores, white Lhe effecl of lbe f.Luorophore si!e

heLerogeneiLy rs ]ess imporLanL. The inlLuence of Lhe macromolecu.lar env.!ronment cabsenL rn dialysed a\-|raclsl nanifesLs it-seff, howeve. _{' in Lhe} quenchinel

of lhe ffuorescence emission in Figure 3. A

dialysis, in Figul'e 3b, indicat-es LhaL Lhe environmenL

ol lhe ftuorophores seleclive]y exciled wil-h Lhe 436 ntn

IiqhL is in non-dialyzed e\-i-ract-s mosl likery hydrophob.i c.

' Fluorescence sPecLl'a shown in Figure { demonslrat'e lhaL Lhe Lluorophore PoPuIaLion oi the Lransparent- neonalaf lens is -.Iess heterogeneous

comFared wiLh mat-ure and s--n-i]e caLa.act- lenses This

concfusion is supporled by t-he decay Lime dala sho\{r1 i-n Table 1.

The lluorescence of lhe ll.anspa:'enL lens 1s of rele!j.vely smal,I int'ensii-y CFig-5). In parLicular'

ccxnpa:ing Lhe inLensiLjes in F.igure 5c one can iudge

!:::.! Lhe concentratlon of f.IuoroPhiores Lhe t:ans?arenL lens, exciLed with Lhe 4O5 nm Irght-' is a:oul lv/o orders of magnilude smaller Lhan in the Lwo

c_-her lenses.fhis conliI'ms earlier findings t15l LhaL l:ght absorption of 'he aging lens rs proerressj vely e!'LenCing inlo .Lonqer wavelengLh' regions of lhe

s:ec L l_ uJlr.

SirdLar .ernarks can be made aEoal lhelruoieltence axcilat-ion speclra in lhe visible range shown in Figu.e e. f:1 addi!ion, bowever, one observes a disLincl

s?,ecl:_a] palle.n foi^ suga. cat-a'.acL Ienses

CFig-6c)-ResulLs of lha emlssion anrsotropy measu'.emenLs canncr be unFqu.iwoca--y InterpreLed. ILs val ue rs 1::tluenced by t-oo many factors involving lhe f: qorcphores and their environmenL' .which are beyoid

canLrol- These include Lhe sl.uctural' _{ordering '!he} r::lransic angles belween abso.pt-j-on and emiss.ion dpo-Les of aluo.ophores, Lherr rol-alional mobiliLy and i:)lernal f.Iexibilily on lhe nanosecond Lime-scale, Lhe e:ect-ronic exciLalion energy transfer t17l and also l:gh! scattering and brrelrr ngence r2Ol of the Iens

The obt-ained t-emperalure dependence of the e:!ission anisolropy is qu-ile challenging CFigs-a,S and 13)- For i-nsLance, lhe incl'ease of Lhe emission

a.iisolropy wilh rising lemperaLure CFj.gs.8 and 10) is inexplicable. one would ralher expect- iLs value lo deci'ease, which is fhe (lsual properLy of the f-uorescence anrsoLr.opy of solulions t17l and is caused

b-,' rapld .andom:zalion ol phoi-ose]ect-ed

,n3fecular o.ient'at-ions due Lo fasLe. roLalional d:ffusion aL elevai-ed lenp-^raLures. A st-rikiner

behavlour of the emission a-isotropy is obserwe,d for co.llcal caLaracL Ienses (Fl9,9). 1l appears lhat, a sorl of phase _transiL1on Lakes pface in Lhese lense,s aL

abouL zO-C. A diflerenl palt-din is Sbsefve(faoa't

auga-shovrn in Fiqure 14. These spec1-r a cor r espond calaract- .Iens CFig- 1O) whose enlssion anisot.opy

dlsp-Lays a m.inimum, z.1so al abouL zOoC, on heating.

This lrdnimum does nol appear on cooling.

In view of lhe above findiners il seems approprtale lo draw lhe at-lenLion ot olher inve.st.igat-ors lo Lhe necessiry of Lhe temperaLure conLrol Cwithin 1oC) in Lhelr sLudies of physical p].ope.Ltes of lhe lens.

The daLa shown in :table 1 were analy.zed in lerms

of lhe fract-ldnal cont-ribuLions f CX€m) - d?., t .x.r. Ci = 1,a,3) of each of t-he t.hnee cornponenLs i-o lhe t'olal fluorescence int-ensiLy. This unique., Lhree component, decomposiLion was posslble only fol.

-Lhe

lr_ansparenl neonalal lens no.6. ResulLs oi such an anall.'sis are shown in Figufe 11- The fracLionaL

conlrib(llions f CX ) were Lhen used Lo resolve Lhe

composlle emission spect,.um of Lhe Iens no.6 CLhe solid fine in Fig.4a) inLo t-hree componenLs- This vas accompfished by simply nulLip-lying the composi !e speclrum by f and Lhe resulLing componenl s'pecLra are presumably _{t.o lhrae diffefent- ffuo.escent species.}

Such a unrque lhree componenl decomposit,ion was

not Dossible for lenses no.it and no.tr which indrcares a larger helerogeneily ot lhe fluorophore populalion

Cmore t.han Lhree speci--s) in opacified Ienses.

Sioce the presented resuf!s raise more questions Lhan _{trrovide furlhel. si-udies of Lhe eye_ .Iens}

fluorescence are needed. ,The author hopes Lhat lhis oplnion wiII be shared by lhe l'eadef.

ACKNOWLEDGE}'€NTS

T::e author is graleful for Lhe cooperation of

L, tsi €;qz:!3\,,sk i , M. D. and lhe personne] ol Lhe Ey.

De?aI.L=e:l'. ol t-he Jolnt- Provincia] Hospit-al in

Torun-Ti? assisLance of :f. Marszalek, A. Prenlka, A. iaai.bcL::r.zek and D. Heweli is also appreciai.ed- The v,c.k r_a-: ca''.ie.d oui under lhe - Fesearch Projecl

RR.

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Figure capt-ions

FI CURE 4

Normalized fluorescence speclra of leises oblained al warious exciLaLion wavelengt.hs: 3Oe ni - doLled line, 334 nm _- broken line, 4O5 nm _{- solid l:ne.}

No.6 - transparenl neonatal rens, no.p - partj.ally opaque .lens f.om an enucleaLed eye, no.$ - senfle

cat-aract- lens.

exLracls for wiLtsl O. O? %

31 _{, .lens no.e}

-spec!.a ob',ai ned

^L fi>(ed : a) Lransparent, Iens, b) and

Absoipl-ion specLl.a of caLa.ac,t-ous fens

cm opLical paLh: a) tris buffer pH 7.5

added, b) elhanol , c) cyclohexane.

Lens no. 1 - nuclear caLaracL, ma-Ie mi xed calaracL, fema.Ie 85.

FI GUPE 2

Same as in figufe 1 fo. sugar cat-a.acl- lenses.

Lens _{no-3 - f.76,} .Lens _{no.4 -}

f.65-..

Influence of diatysis on the fluciescence aqueous axLract. oi Lhe ..Ie!1s no. 1 exciLed'

v/avelenglhs: a) 366.nn and b) 436 Dm.

FI GURE 5

Same as in iigure 4, bul wiLh rela.,ive inLensil.ies

FI GURE 6

F.Iuorescence e)<ci Lali on emission wavel englhs for c) cat-al.act-ous .Lenses.

NiNg

FI GURE 7

Typical flliorescence exciLat-ion sp--c:-.a obt-ained for lhe 55O nm emission and t-he correspc:ding spect.ra ot

TemperaLul.e dependence of Lhe ffuorescence anisc]Lropy

of: four' lenses: a) f.66 and b) f.Aa - m-i xed cat,aract

.IeDses, c) f.61 - almost transpareni- non-cataracLous

-.--fens--cf _{f.68 -} nucl€ar- cai-aiact fens.

Temperat.ure dependence of

for L\..o cor!ical calaract- lenses: a) f.6a, b) the fllrorescence ani m.59:soLropy

dependence of lhe fluoiescence anlsot..opy caLaract- Iens Cf.78): a) on Lhe day of the

b) five days l aler

-FI GURE 10

FT GURE 11

Fr acLi cnal .inLensilies of lhe

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Li feLi cte-r esol ved speclral components of !he UV

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