“Te previo caper deste te geal fares of inna pen foroenc. We dented the seca [popes of the wos unio sis sa oe prop nes are ence by he sanding pon esta We non dese line relied messzement of ps Ahcecnce. Such enurenens ne cre ees: Wy common ecte of the crened aly of ine opin 7D) aed teazency-darain (FD) insrment- thm’! However imetenved sods fiat prtein ‘ioscan sve me ehenping by tbe ek of sips poled ah sowoss. Ped inser des ae ot yt eal {hie for eninion of rin Moorescenc,Puleé LEDs fot fhetutoe of proe Muoesence have just ber sh oc he ple whe ae ove ot oanoecod Singl-pbooe eciases of protia Merescence regu ‘eters ihe age fo 200305 Pio ost 2100, he doc ple ight ace forth ange of ‘aslegs was» serene pumged ai duped fe ler piel danioe 66, whch war doable fo ceuin he UV wavelengths. The syetemanly pumped ye ers ear an ever eke pp ee icaly an ago lot NEYAG hse At rset the ave 'y mde ecko er ae bso lee valble bese 1 the wien os of Tsai aes These ls ‘pamamsesy mos lek may ot we on ave ote Ioan Wales srl fo exciatinof t M- rescence canbe oie by teeny pie log ‘ovlegth pat of Tse Ting he ue 840 am yds 28 The fennel from Taupe Iers make acti to genet ch amon, Extuenof mee prow resem can sito be acomplia wih ston raion. Many of ‘he reent ses of tiered nti rot eres cence sed the eguery tle pal pled Tsay elie or syenren ration, Even withthe bt abe omental tic Jeni ere te ane deed’ a fom roti Time-Resolved Protein Fluorescence The iment decays of poten a asuly compen and stlensepend on cburatonwinlngt, The onples Suto oh the presen a mae eypophan st 2 sin protein an the complex dey knees asplyed ty even taal ypuptan ree In pons, Hh ie ‘clan and hip peal meascres ae el {0 solve he malople component i Owe dys ‘One te malé-apmenta ieniy does re ee itemptingostign the ses component oh ini taleyptopin esis aire prone. How ver, even pres wih single Hepa side type ‘yop to or re ety en" Honor, ere ‘uot believe tht he alia ey Unes erst ‘noid rp eas, at Ue Rte deontnie by {ical experiments In soe ee, wrth docty time evry flrs has bean posible ain doy tie ini! tpn redus,This assumes ‘ypealy ri one we of maa pris conning ne ‘papa Toner wane of eplopbat than we re mine wipe proten, ‘Ae ena completion sat yobs selina eal pt dps mlierpeenal on cxpotestl deny. The berogenty oderely weak ‘Mow of te erst fom iypophan cers wi ey toe ter 3.1 9 Tees aos tnd cogent i ecay ume ovat 05 ne Fe See une eon of is oaporet wos so tnown appear otf Pleo fovy of he typrpian decay is du he peer of nloratna zone callodtanes. wich Spy = tnt dst tes, Here here yb onl er, Sal conintate oe comples day Ket of yop Tnerreution of teensy decays tecones mae complied besaue of won proces tat myo teri forthe led ino bh, Energy ast an sce from rexte yplptan, or eresn syoghan tidus emi. Flowing eactan he Sypuphon ison canopy Unedependet spec sis dae 0‘rstedtne dpe mons, Pris my ex i several ‘onformatns. ach of whch plays arent testy ‘cay. Ath sore of compe the posit of nie fect in ealition! cuething, duet eiber ded ences © Be prsence of eaby quenching {Foor te rain Inport al ae phere ‘eae sng ine esolved examen I patie. ‘ciclo dtermine te conrasione ofeach pana: onto te itn ety The we of engined IN Clo eohing thee compen nets Wile Be iterpeation of ine eles peen Duo reset i 0 nye spl, the one rege aa ae operates ora more dead ended of ten sro and fn. In vrai cates it posse to rele he emlsion jer of indo! typoptan ‘ides bse ont tine depeniat drys evar ‘asooreoinion wnelngie The Secs decays ‘he tnatines be nerd items of he aein of he typloptan eaiies in protean Be erent of these reoos wi nary moa esos ie r= ‘sin Coeferatico changes ia prin fie rel ‘changes in De ety cay Oe aed io ‘wih ey groups. The tine dopenden anorey ea ‘ae iasuhly ieee wow he een oe pein Aibiiy ané Be Imercions of » ptio wid ober ‘macemeleae Alo laoreet uae i be ied by tme-esolved mesures, whieh can snes Sennen Shen dynamic proces. AR ay i ‘ow gon it pole canbe aspect a ea “emper. The phosphorescence decay ines we Hea ‘veo eaposore 10 the aes pa swell pes nce of meaty qpetchen In ths caper we pe a Stenew of timesived poe Muorecene wih ‘er tha site be ge bts mn tons Wi INTENSITY DECAYS OF TRYPTOPHAN: ‘THEROTAMER MODEL On dix in meee hn tele inesty ‘ery of proeeshas teen lc ners te emt deny eypopian eK Tn etal Spent ‘olen he tent day oF tryptogtin is kxown oe 8 oul exponential wih ey Umes ear 3. and 5 ot (ise (72 The dominator of Seenpoenal ey of wypophan she preseece of ution! somes (Gg 71). sown the sd hale of eyrapan xt opt aia conformations te, which appear te. ange slowly one ws ese A wypepn Solon fa be pal 3 mine of tee relma ome alle roars. neal ajo soliton, yap [en in te wenn fom in whieh he io op om SESS eran ‘ovo eg tre es fo “he 74 ey Dnt fa ArmA A nd Rela Compnn 2° 9H? Tar ce ae OP Bree mt perro 3B oom ss) mo. fo tol A eisai 5 Bek iexsiry \ yee : / wg fol pee bo BLY i t t t ‘oe 12 eee mcr sey a ene EDT yesh ee toon ew ne om Secowce ae inertia tne is pooaned (NH) and te exon aru i nied 20.5 tn salaiee ie emion of pan esa tnlloencted by an rake proce eg te indole te sd the polly chggedarnoam F40. Recall noes ug Snsie quenching, poe slay by ekcvon eet specie, Reped quae of Indole ede aye, ses. niazbin,wrne 0, oebonine, sine, ines, pepsi bots, ciuo- ‘artim, aod alzwon saver! Tplepion tobe quenched byte amino sei ide ci, actng Pltamie spain, rte ero poops on Be lanai and wate ose, ard iti ‘Guoctng bythe smunais som seen fo he Aepentece fh uypophan quanti yd np gue 172)-Arthe pt rincrased for 81010 tani ep ‘eer doin oe neal orn aah qa Died and ean eine serase eprsinately ree {ois Taree qa yl iene cos because eel aio group des ot quech ends ‘Sot. The aenchng let of rene arco sep Cnmpopan gral a scar for Reba of O- Pharconiiag pops Typoptan argues bg the amin gps ich as Someta or eseanes ‘ymoptan deter Qa prevent conte of he amano [Boup wih nd ring ot hw he pend [tees in quart yl erren pt 8 nd 10. Compe ton te sino pop ios crown eer esas nese hole in arscence ens Bease he te ‘ou cn o longer omen cana with nl ng tamination of he pltdependere estes of epogton Indcaes ta he intensity dees fer tow pH 3 nd shove pT cease pnts blow 9 dhe ineamoleclar gueactng ot ile Wy te eta ‘crhonl ponp. wie sowes a an lecbenacepoe. A ‘igh nace echedy Nyy groups, ch ay (edu in cana ucehng by OM or by excise {rennin of be pros on he doe open ous. “ow des quendung by te ano oap ex be iexponenta decay of pegs a pT? The questing proces is though 19 be mast efiint in ne of the ruers, adh poser aly he boner Os desy ‘ime (pare 17. le). The amt exption probe ‘iy someutac more comples.Fornstanc, more an ee calor ae pose oe cose he ose = maton of te ndoleing" Ala ow ts gench- in is onaly companied by want eles Oat ype suber comes nthe mens deny, Beaee of ie ‘Simply needed by he ast ad cab! ops, cxperment a eqn performed on wae P0- fan ances (ge 173) lr he news! eypOpINN Sralogut NATA the anno oup i bene ad ca tovyl group coveed o an amide, octane sie Aejvnivr we ao wed (NATYA) These fas of the tine wt ini he ene foond wen he io ‘ie we conned ina plypepie cn. "Ta rence ter robb he oan son forthe ml expoertal nny f wpa ats) DH. However may be ete (sor hat abo con {rite fo the comer cecaye of epg ad pts (ne of thee factor econ by pepe onde The sryptornan Trp W ara ines. nents BATA ant Noe Sram aaqn ied of NATA 9 wat 0.14 an he gant eld of inet ndole AM ner 00.38. The wet {mor ik of NATA iu oe ee quent ono beh the amie goes on NATA Fire 78, ahs nie hen taping hat he se ops thon of NATA oot ea ins moli-eponetl dey a ‘cur In rupapan. The ee of queehieg by aes epee on dst. pay. anche bw ‘hn sorouning inden wie row. The Churge dtm aroun the plas x8 cher trae or devas shape separation ao guechitg nde Hence» wt sts come te com et inesy das of reins 7a, WHE RESOLVED INTENSITY DECAYS OF “TRYPTOPHAN ANO TYROSINE ‘Conover net ines cecay of ypophan persis ‘3 for many asad oe ay wander hy the podem ‘he Vn ofthe aalbl inne ad mats of ‘hl rai. Many of the vine cys of "ppaghn nee ese ing lamps wit ane ‘Sd ute we The low ines nd epion a of the Mage red nda jo aan dete Shon cnn, ut ato eb ese i ey me td anplie, Lang dts toon tines wee ted 0 un he mint of pote sours mended 9 eve th {sca component, Daring ke he Dahan pret ld cange am eeu ac Adina now owe tt he 5d ee deay components diy fee emsson ses, Sone ofthe exer mest fem tiowed nl ong.raelentheson soe 560 am, ere enson rm he Os omen "Te ety im rsing the tu ines dase semponents ilsaed by We ncnsy decay of fron pH 7 igre 174). The bg sec wns vais ump hed OC dye ln th was Feeney ds tied 298 am, which provid ult aout? ps woe The deter wt a mrctunel pla (MCP) FAT dle. ‘he dow were fo the single nd Soy-cagonenisl Key Eeewinry any, 5 18033 and 3258, 8 _E . Grcoie ans dee roth ee “ Tae: of await! 2 Hae vb sine tos) fr 4 Tesi ty ny sp (Sta ute ewe teat Shin aera mo sehr reaps of he component ey ‘ia Th ied curves fr Bth eagle ad Soo ‘toons! models aoe itllytapeimpete onthe mente a Devauoe ote dt rma singles ‘renal ae bry vie te deen Fg 178. Tove prl andthe decreas in yl rm 2620 123 fort ingle on dobeenponet fi epee 1h, Bren wher TESCE iruenin int {iia etl he two des ins of typophon “The multexpnena dey of BORMAN can aes teen teed aig te egery dome mis Fue 175) Theda were oan wing sone Be ae ign source, 8 MCP PMT dewioeand te Purmone cote, trtbd (Chapter St The deistions fon the enleag esta dt len noms ene pe. The SM deren in or te colegio 5891009. comeing eens fore nani np reat deay of pian FREQUENCY hss) Pern ay en pain (gs wo vee 172.1. Decay Associated Emision Spectra of ‘Typtophan Suppose sale dpays 9 m-eapoenil de0y. 268 Ute decay en a lees ein wavleng ‘henley deny stem deseo 1. = Wi) = Zar) apioey 022) ‘where ae he wavelet ependent r-epoen Taran te yesh egeion ame Be ey tne te independent of wae Te mien Spezou ds cach component Cnt aed is tiny Lap, 10) wy 1 his epresion 12 6 he se se emison er: tram Te pote a, opera he aera ane ‘heady satiety is properonl hs pro, The im inthe deromscio i open the at mee fy athe wane Thee spect) af clle "cated gen (DAS) bent hey repent the ems ‘Son pecan of the component emg with ete, r0stne FLUORESCENCE INTENSITY ais a0 as 000 WAVELENGTH (nm fees yee Sms noe wpe sion wanltgth are se const he erison see assceimeé wih each decry dims To excl the dec cited pects on haf eal ht he son eon nao ) eh spies woe tease ines Propane th pret, Ph coniben stor ‘eeytime componente ead sete lower than be mise smelt (of the sherdy-me compen athe ery dey nthe ee of OPS ‘heton cmponen ents ony abo 4 oe ‘niin of pops eal pH pure 17.6 ras ‘oe om te tho components erred ea 335 mae curt = sighlyshoer wale th ot overall ‘ison, is ble eit of We emisin fe cnsgent with ‘he eget eet fs ery posi charge he pla "Use of indle. Lite contain fm By ste pec, comersio of" 0", ora le han 3 172.2 tteralty Decas of Neutral Teypeophan Derivatives Inpricin th amine std abeylsouptotiypphan ae evened imo neal rouge by the eemtion ef pepe Sande The coercing effet of he eae grou 90 longer pee, News yap taloges pias ny simpler decay net ha yplophan sl The on onmonly wed anlgue is ace Lrywoporamn ‘tien ho evenly he cane scare 23 epee ‘ube to poi Figure 173 The ny deeSeer : L met06) 3 ‘ Bot |e Eesorcemencme ss [ATA pps 17.7 een single exponen 2" Comparable feginty-domain data for NATA ao feveled 4 dominant sngieenpnens decey Pgse 178), There sme deri! the ube rem so at the deny of NATA may gly 2 wk ‘Sroed somponent, However, fe all pacieal purposes [ATA cpanel deny ie Tle 71). 2.3. Incensty Decay OCT rosne and les Neutral Derivatives Tyrosine eam ko dap complex day bine. tu is roeris egy otha fey oa. Frese 109} 30 “400 360 "5000 FREQUENCY (He) Rial epee re fan 0 Cap 8 lanai ita decay of tose and ts neal an lope ace -Lsprnnamige (NATTA) ae show Fig 179 Toe olf pays wsighoexpoen dey whereas NATYIA dspays 9 Deexponenit dsp TOBE 17 The molec in of he le ‘rpoanal day of NATYA i ee bt may ee foihe presence of round ate rotamer.” The erie bn or beng! ous may quench ihe phen usion by an ls arter mechani * = Cone [iy ea ifort arable he erst decays of pheriaaninae=© l ependetietmes Rave nt Be teen “einen deny of rosie in ome is eal # single cape, bt flere reac be absioed & Gite omenaton wavlngh (Fre 1710), THe inersydeeiye were reateed wing ?77-am ein Frome heen ped sep lae and mira fel pla PMT deter Porte pak ron esion a Soa te nnsy cay en ge ego "AS the hseaton wavelength ntestes he ple bce uve. Showing he prewi fwsitonl components Te cone pene with day tte of 098 ei ue YF AIH Yoms ug te scat Meine. Th compe. ovo pon nena he rap ete pier i yey 8 amen ay ety wt cea ue tne seng apa ra Seachem sna Soe hor ine a ong Maen esinton fhe yo: eoup occurs etn pK, dese hat nthe “hved te Te assignment ths compares! one Tyrosine isppored By Oe ens dees of Onna ‘yr gue 1710 lower pan, which 60 ash {his camponen: The DAS fo the to main component > ‘he tyfom y ae stown Fg 71, Theos trioion ene at 10a i sed with he 3096 emoonen Te 038-6 component i de 16 NE ave emision centr fer 360 which eS se to sine 7 INTENSITY AND ANISOTROPY DECATS We Wow conser the stensiy dens of upptphan ‘eee in pons. Sse NATA delays singly {hee we cam expt epetnpophin res 0 py sgl tone decays Meee the a {Suey a ah singing pro. ep venta wes oun to ply 3 el ey ine “hse repo wa cote yma eee Mae set stomp tha ow single uypiogtan prone iis omaunea oobe or tpeaponi eye (Tae 172 an of ore mul-rpapan rt maria gly mal epoca decay abe ‘Wher ae the genera Teac of poet ensy ey? The satya teensy dra ae of fron state Ths Ins dest and ewe ay Fine re moe ie or use pit ha for dea toed pois The eas ten for dena pins can te ped noo tat, with eye er 15 3 1D 5, whe inter dpa arg eision wae Tenth Ti deny i enanedip Rte prc, ad mam fie show hime a log 267. ply ov Ue tedsseo he nsin Sopra be ypiophan reidce sem 6 pay sar fete. Te gs Tine of expesedryptphen eats ave Ben peslng esau exjorae in water essed 0 ret in sont Mies: now know har Fee ds can gach tnaphon crssion® Hence. he shone tings ‘hele 172 Inc ect Sage Fopohan Pong“ate 173. tea Dees of Mab Toe Fes ured resiuer may te tease te bared Inptophin resis have more conn wih pepe onde ha the posed reies However, te ile colton Between the meen times emison maxima ofp teins ste Tipu 1612). pela a cont che tmopore groups leeds a yeah Po. thon render ave olen nacached by reanace Cy tat reslag Ie stonnsrecond sey times (Tbe Muiveponeil ces eee sping fo mais :oaphen ping. However. he bp fl pone (Burnes, tps cane tember ee til dcays in sng rnptophn pron ses cer Give hal NATA ops gle des ie sors PtSi Peinploghn rede na sepk imaue poten exon. et shat ln sigl ea) re. One posible Fol muleexponeml eye iste eves 0 elle procis conformations Snot ney srimoaid res Cintas quenters keene open tah ier onferratons ca tu Glee eye fo nate Protein. However, muliexporeal decays ean be ‘ore eben when epi ar thugs fet ‘gl conformation Ar howe ihe owing won ning of » pin can change © sngleeponetl ‘ey not mai-esyoort ey, whch sx ote ‘onset wah tpn of ii conermaon fo * fndor ol pete It appes ta malexponet {acy of ingle ypapan pron an sul np om yma proses cringing ie exces ie ‘ime Tse dame rete can Ide ety ‘atc of qpeene spe eso, anor som ery vse, 13.1, Siagl-Exponental Incensy and ‘Aiotropy Decay of Ribonuclease T, Inte previous con te am hat mom singles prota dspny malexpoeial dons. However. ee [eso known exepons—apoarrn ad nbonucles , From peril one Mot bones 6 ol oni epg, Ronis Tuas phat ices 3 Snell rest dunt some condos ie a) enpeexpoenin dey, RNawe Ty cos of 2 Single pope ch of 14 so was gue 112) ‘als, he or hey bnes me ove, nda slsypoohan ree 3 pion 9. This typloghin ‘eds is near ache se. The emision ai ‘ear 323 om soe that eae Bane ne rote guise and 91 ego to fhe sino pte ‘Quecching sede hve conned ths thse at si accesible to quence In he ages pe = Ribonuclease met dy 2 single exponential ap SS (pr 1713, 19) cy becomes dole exponen 7. single: ‘npc cy isceneniet bcs hae po fom mnie can be expected 0 rea i mare cogs {sey oes esr 10 dete a sngeespet {ecay becoming a mtverpneea Sony thn 1 (de change nt elicenfonenl dey rose z “ee oa Maw E02 ase Bost | ° I 0 8 ie aa TIME (ns) Siete eke nn supine 173.2. Anwoxin¥A Callum Sensitive Single Tryptophan Protein Rcowlene T, proved an exampt of snleayp Pbangratcin th ped singe-expocnilintersity and {apy decays gue 1713. bate). For most lem, cron wih sgl trypophan eau, he icy snd anaopy deca ve more compe One example fmnesin Vwi posses single poptan reside (Gina 171), Annes a 6 of Danolopos fo tent bind et eres aap ‘rune The ey ava trou o be dierent th rd itou bends Ts emiion mtn “ymuphan eden eau Adon 38 nn gore 1715). Inte shece of clam the tee decay ily en enporental Figue 1716) Adon of oun es (eth ni ess fo Become mote lke epee sem Ti dart chang ncaa sugges the pe sve ofa mb guenching rp nth clcu-efem, {Thee srces of sexi cco ih i ‘ogeesion heer the Upon residue Mo. 283) Irom he pen ne presence of se gt 78 "At wt deteried m Cape he mi expeeei ‘roel canbe od at any te cea he (he acl decay hat some eer unetona form. When eamining ibe of mulreseneat cay poree ‘eit obama inne vison fhe dene om fhe mumencal vibes, The forme of comple fers espe canbe Velie om te Weim dns$ ‘yh | : sco & 2 10 msn Weng (nm) a Parc non cn fp aa ‘aeoucir amas: Ren spilt oem Anan Came Sen (ov a0 Bas 1300" 16007000 channel (0012 one Figure 17.17 tow the ieee of sein sn sence a esc of alum. Thee pas bows Inte othe nist day fr each decay tire The Teer axe lose a omen wih asin {uc felon canbe undeod to Be dy cor oven rw 0.2 nr see he ene eran. Bing ‘telus Vrs Ie aerate decay time compotent at 4. “Teen depent hinge nny lap expose isexpated oa eoicrapy xt Argel ors race engsed est, he asitopy deca to esos tore apie psec of ncn Fre 1718) Ore {Span tel ovale ase sco dsibtions. iniiscn hrbtonf selina mes. nck ben raleam te aisevopy Sey of meni V ety ae {© otal rosin difsion vhs eorelon time wet ‘tcecmeion nes ee Hp 12a 1 Fg 1719) Taken gether hese suis comin ah he Annexin V 0.7805 ant 3200 0220s 1.8506 air) of 10 10 anisornory TIME Last SSiewapnenfein Ou lon ae ‘sc arr chang inde by Ca woh ee Alplcenen oe 187 roma ane evronmet oan ‘xp evel in which te side chun Weed ithe sues pe Fut 171) eapaen hate Scere tty ip Sy eM 173.3. Aeisotony Decay of «Protein with “Two Typtophane ‘ine topo retccan be ngs el bye po ‘sno dpiaysegmal malin nsegenen of overall ‘ote! dition Ina mal eypoptan pron rs, hohe ay be igi or mobi The pe of eka ‘ssn erste eptotan mutans fe TA ton ‘Tre prota fom Eco aed in ame fp at peu so we wl allt he posphoy ant pr tener ste fom esis 19 0 168 Resa wo 8 ae nt ‘aL and pray ao sohton Wil iype PIP dosnt ‘ont any penta residue Singlengiphin tas See pened wih seskdues a postions Sand 21 whieh help. Timezeses misc ees a se ta fo 0.08 annerin ¥ one sapne Aniactrony {eos fe shows i Fig 1220. Aries longer a 2m ‘wc mutant pay og coat 6) ney nen 0 “This coetion ime ager tan expects fr « oi wha molecular weigh ear 71 De Table 103) wed ‘ros te de othe ene chai, The muon show very AierenReinior Mines Below 3 ae eer the FW ‘sant aly shan cemlation ins (four (me Te how carton tine dae to of eee I-18 Proaphary Wanaler Proten 220 01s 8 [| oseine oscars 2 raw ine Ins)Tle 174 Annoy Osa of NAA Sle sso ma SF 2 ee which are independent fhe corp! domi of he po- ‘ei These res show how opm eid ‘nlcaions within te same pre can have ees moon od ier ancy day ‘Tia, PROTEIN UNFOLDING EXPOSESTHE “TRYPTOPHAN RESIDUETO WATER ‘he spec popes of poten are epeders wpe tir vento sacar. Thi dependence cap Be sea by esturng te esin of ted eared protons ‘The eit of desaeation ae ay 10 eon fr rn cles, (gue 17.12 when dpa gl exponent) nny ad eiso4opy decay nthe ave onfermain (Pigue 713), Emin pera of boas TRO ‘))ae own nia 1721. Av exciton wavelegh of 285 nov wa chen fo aid scion of the sie ‘eid. or he epee emo asm ia 535 om, which nde he ine group Ima orgs Feuoee scence mtensiry Tena een nom TET sion. bough no sent ia Figure 17.2 ther ave repo he prota f weak wba sac ‘he emieson spec wae een fore ee Pres ot be anole Nigh emperare ob the dion of enatrnis such rea or guanine Ideas, Tee endian elt ea cama shin ‘he emipon seca af Re ger wale a the presence of 0M puantine hyarchonde or °C. "he emiion specu of RN Ts fo oger wave lenge ome carers of plop ei ht ie tly epomdto mater Figo 1721) Thee as how ht pele sua dees the emsion maxima of pono, comorely, th rn foling em be ied By changes inthe nay remain spec of pone. 17.41 Conformational Heterogeneity Can Result 1 Complex Intonly and Aniatropy Decay Freqvency.domain mesienens ware aid to sy he Incraty deny rte and dead raze T go 1723, Inthe nine te apt 5 the eqn dn dares a Siie-etgenentin ty with * = 97 ms ‘When the potent denature By hea the iy dec ecomet aon eogecos. ‘Prisin ung awe the anzatny dec ‘axa Ty, The fegueey-domarn da for Base T a5°C techie ot singe corsa ine gr 1723. Irie temperate need i oe a dena ‘tee eam nora nthe ih fequncy ee pase pean wet in the module anton. Mee ‘fects it Fagen dominant ae hrs FREQUENCY Wt ore 1738 Pegsy eno eT weal Scale ec ett ae FRESUENCY (2) Pepa tewpysanen rime esi api motions ofthe esi addin t slow ovr tations iftsion Alo Tegieey he md led entropies ae eile the eds ape. Ach fegeecin the mele aso ppc ‘Be fonsimentsnaraopy (rp Te FD essere ‘con be ed enue We Umedepeece rip, {ecaye me Inthe nano tee mane ey vs tinge capone, Wheeled by ber mde Iyeachone or hgh ler, We eieoy ce) plays componen dro seen mon of Crpionrin reas ene, eid components In the sisopy decay canbe erste oe anon col pei, ror ypiopnin rit os sce pons Prensa 2 vary of nnavopy dcr Mest pro ees sho st componensiethe dees tees? ‘ypc ary Grae ae tow n Figure 1728 The singe pop i he lune pin sbows 9 ip TY cis sgn otal roe! ino he pro 04 amarine protein .45ns yn? ne oa ‘AnsoTRoPY al Jou Zoo z Ta 57 % 8 i cermin eee ean ‘rons a he epiopan rede ndependen: of ever ‘inion! gifasoe Anaterexate te Pops ‘ede ibe rope hich soe shane rors ‘over tn isin any ees he ped com eee ne snarpy Sen rae be mesa, [Sabres for soompi comps pron hit se ost 785 of the atapy fom de WP rmtion. Te Sart centton ve aber npr “he andanes rm S000 pe. wih aonb cermin i ot he ie reson he ramet ‘he shor colton ine spresimaels eel a heed for SATA i so fo sl pple Tae 1.) These share comie ines pels ensa 8 So on nes i. evi of he slate “Te pcos compen ake niin ets of prea piy son chav sheen Me sion of het apy isp for sl une ted pees ham pop eta bared po sin mati, Using o «ptm rss the trait he Et tore The maple ol he det somata ie e b mtyidperen fe e Culex rng rom dle. mypopban he eee Invern ane” Tne kin ere Ue in ropes sac yh ural canines ge ‘8S forall pede. prea Be 175.1, Elects of Artocition Reactions on Anisotropy Decay: Melt. he singleespopon pete mein proves a goed hamlet bow x prin ars dey depts oe sesame be Dh reset Wh 1S aan, Danny i price Ihe lesa pan 02 Eo? 2M NaCl zo a OM Nati aaa) 5 10 TIME tos em ns tem ante EF sor dee aad etion af sepa et, ‘nor di rita hae een epoed y 3 fe labore Typos for he ne on Fecmer ae shun a Fe 1726. Theatr as ‘ex ioleapnesal noth sy wth 3-op frnescinciersne The seep decay nw 2 TM NIC! woe ielitinfnne homtier The 2nd 85 arte eve, which nuns ho ci Ss ii es oie nese te pes oe mn ren he Hele va" Th Ding tin tic changer sao de (ig 1720, which now sree 2 {© vtain much nenaon afer 1D he ie th ‘ore shoul sl rote whe Ut Hie Ie Thedaain igi 12 ihc ily see The money te vem etn Fender sty 3th eon atlegts Tow sparen near aon can fe store i ne rion othe toma, he ore ime in et esses Jost wh shia op tn ae wn Boman pS 0 “ Fics) wa “oe io \ce z soli / go eat # 00 so S| ol. : 200) 380 ‘00 WAVELENGTH (nm? 3 ri gt yt 1741, Decay Rescinted Spectra of Banate In Sea 198.2 we deere oe esi propetin of msi The wipe poh coin I npn ins leper shang in te ta wa nb ‘eed eeehingo W3 by Ie ie a nnd Iie rue HIS. Tmeswsled msnens ed fie treed mutagenesis were ae 0 dente ie the nea) ate The wn aeons limes of W71 ond 33 ae ey independ of “The rt iene 0 WU anger dramas trom 19a pS Sto Sean 89 Te te IGHIS. he aces 4 he ine prot fom, ‘These eke coal Oe oacains hed ng th ream ha coined 0 propia eds (est 63s 1742, Otuiide Oxldoeductate DibA ‘Themis of pupa dus in prin ret lyase by oe pop in pri Sach kes ec Rene he tig oxen kA Im # oo Tre wilispe OWA conse tao pepe ride elise xb, W124 wi fro be seal on Pgrces ierpeine of the fly ste she poi, The enn fin Gotan af W126 vn DXA wes snd sain ket mugen, The moan roe AF see conse eben» pein th sand oy ‘NioKe The eign ofthe quenting wos sed ne 1766 by mang anno ides ce to 19 Siece amd a ks nbe pile gucthr phon. omine “Sond opie 127 were rence sh The meek inten ess of te OMA rzann woes mesa fern Watley neeDs A W76F INTENSITY (au) Dsba W7EF/NIZ7A 707A FLUORESCENCE 1.03 g.13 2 300380 = «40080 WAVELENGTH [m} ste sed cael the decay-asscated sper (Fe 1729) Ate emally es wih igleseypophen pig tetany decays were mahi cxpnentil The DAS show thar Qe an NI2T we reson fr quenhing of 1126, Forth poce cnn hse reas, W76E. he lps compnart inthe DAS fora ey tine of. 6 Fore potcnlastng ceaues NIZ7 etd QM4 (Fe 17.2 aber panel he DAS ar dominated by acompon mpenat to nti tht be ind Wes ware ot tpt to 3 glemhed or ungonohed ree. In The (ches mae! pen (Fgare 1229 tp) ahd nthe nguenced musnt praeine oHom) te oh reidae dle et de day ns. Te eee of (Qo and NIP were demined by ceesdenng. wich 1743, tmmunophiin FRBPS9-: Quenching of “Tryptophan Fluorescence by Phenylalanine Immonophiin FRBPSP2 provides anier example of feshing of rypophin Sr = tewhy mina ie hain to his cse the each is phenyl, wens et wnlyconieed# gueacter.tmmanapies are ‘aeopors for imunvspprestn drugs ech as year {penn The imps orn FRRPSP conn 0 \yophan enias a ptsios 39 and 89 (Figue1720), In penal would be ica io vp te coins te mo typhi eae ng a ojo FPS. An oalogas pein FABPIZ 638 ave tt cone lane jst WH. The fen of tes Ho pets markedly ren, ith W39 chown mch ower gah toms yn (0425 some 19 for FKBPS9), Treen FRPI2 shows ore pines decay han PRBPS9 ‘ih pipton sie gue 1731), eee po: (ee with tw eyptonan ress, oe of whi igh Foororn the etn bon aay show om ina dey ie se 6 16 ns. Foc te single-vyptopon Dron he erty ay Soins by ere weak Ty eriktng rete, which dplye« ate Reese Imeaty dees win + domiman decay ee earO2 me ee na ep 10° tot counts 0300 10001500 CHANNELS (@02498/1) w99" woe 13 FRaPso-t Trp $9 ares) z FRAP te 4 Trees) 072 243 52606 Lt AM lh o 1 to LIFETIME (ns) fig 113. loner pn), Thee reseed an net: elseif seeing 2 WHS Apply OS Ee Phan esi ueneed de 10 eb phere eide. Bese noun 0 decrease te Geo} es of Indl" Aowgh wot pened one would expect dea ‘inf the retin to decrete heen gute tonmalve ie Mresene of W99, which nod spon (he cote at 29 sepomil fo guenching wae, “Two neractingTryptophane Stearate mesgencsc aed euens domain mere ens etude dhe two ephan Fes ihe wie wane / Trp Repressor FLUORESCENCE 300-325-380 378 400 WAVELENGTH (nen) prea etn ante ype Aryplgtan spree Ecol The wily poe teineayennencl Sime nd onan me og st [oson 199 in eae en The W29F mu con {ans only eypteatan19(WI9) and he WF mt Caine ony tpplpten 99 (W99) raeban pet he ‘ee peeis shw aie pti wih W19 as a her {istum yin hepa wi 99, ever (Spe pen wis wo uygophan ees (pe 17.30, Frequent dee ofthe ee ee see grin show tha eth apne 8 tio tees) deep igure 1739). The lope sray ie shown {heron wth Wc Ge aged bye er pase tla later feqincce. The mean dey tine of WIS ‘even longer ha at of te wipe prem Tes ‘escnice hr W9 ts 5a gence ne Wi PE The sess in sae the emision fro. ech88 PASE ANGLE Io) at wen en ones ny 9 omer inesay dca Tale 175). Bod te pe tin Ande snpletypophan mutants ply Seubenpo- tenia decay The ceca nes ove (0 aD thee pe tin. Al, erations bree eran ears ings at te nteeny decay of he wipe pein ot eat of adn he in decay fe tn snp: {ryplotan mats. The no the mlype pron irate sim of he omsson of te wo Sele peop 1745. ThermophileB-Gicosiease: ‘A Mats Tryptophan Protaln nthe receding section we au sabe rine wth» lied numberof plop esis, AS he wuber oF ‘ypophanresives ices, Beco ingicta © rele fhe india ee, Eee i ingleeypophan ‘rants canbe made i unaly the pec ‘remain sable fer abstain of ge miter oF typo phan esiues Ia snch eae eae he lorie ea ‘neti ite disnbton o he wipe pe. 0 “ale 15 fey Daa he Ty Ree tate Sopletnpapue Mae a8 opuLanon reo oe nef i at forces wexrene wemopbile going a 87°C. a8 vce 9 24DDa testa conins 6 yp. tan reid 17 per abun (gue 1734 Te Pty oie remains ave and sew wove 85°C. Ii Spaces atempl wo rssle the india emisin Spec and eels impraca popu singe rp ‘han mins One ca snes ese he ea) {rye of he pein which in i enue wor measured angie Frequency omar eo The decays ight Iaterogeneos ad were ere in ers bine ftne dubaton (Fig 1715). As ibe temperate Ineresed he wo componels nieprorssiey ewan 1744. Haan Protaine Display Urea nena Foe many yous was aud that heme pias were ronfucrecen Thi war» resoable srpon Ber Thal eines Set stern ed of he ese reap ‘peso en Fortra) fry ome 08 (B-Giycoscase we os ‘otros oz! A 08 ese oe oa rr LIFETIME (ns) Iai transfer eae 2-34 A. Since hi snc gr be 2A rds of a myorloba mole, RET ieapecte be nel complete ‘esc of rypiophan emis rom bens prong ‘sexpeently lea Suppore RET quenching is 99% fice sooth oH rote Slane x 1 npr a he spoptin some wore pei ‘The sipal Fob puny wil be proximate eo © "ht rom Ne Reme poe Fre isene fet 0 owe i he obseved weak emi Is Be (9 Ramer ‘ns 1 impure, ot te apoptins. fo spi oF ese ‘cakes sme poop were ster ndeveting ee prokin misc fro he seas dt" Subsequent Timeseoled experiment confined tha ene pros fncipay weak mnie Tureen and te pe Sc ines foun in hee pres can be coat with het “Ancoample of ntsc Marescenc roma ee r= lenis proved by seta sstea eo (SUP == “Tigo coniais 326 aio akan eglesypo- peo ede, W117 (gue 17 36) THe mney ery oF IWII7 shih etrogeneos sowing tea fox ey ines (gate 1737) The ine bon eves Fromme ens dey iafoaive nowy at 75 ‘fhe ads nensty ve dco 3p component “Tiere estes eee in aso emer sen fresco kn if ene ngs [time conponents ede ote ee pri tor wi? ore 36 Soe of wl han ee Impure, epopttein or uns conformation: of te ame prc tJ on alow fia ener ane "The emion of here rent a hen ied sing he Peqpeny-donain mead Represare 10°F socom F rool rrendase ie Fay o 3 68 8 1 a5 TIME (na) Tass 3 4,30.97 $? 030ns 2 R88 E 20% sa o oor oh veoFREQUENCY (Mis) em 8 reno nS a The fendi ee oh pad dey Times Limplime ey conpeni wee 86 Fan foe mvs, Tese ng ivedcrporete esp 1 be eco ofnmaieal somes he prin wich tbe Teme cmhe plone one sa em trator ean ceo The sessed a ean {sel fonoton ud coin hte ears We nie sen ny eames of eating the emision From ind’ seypophan ce Ia lin popb eins, rie ve alee we eres ec Fhe revi dyed he var ie tal emo “rrcengtn Hower tn ifinst f alesplopn fo ge tonm\, 390° i at00a\ 300m °o © e 1216 TIME (neh there all nie clue Howes the ts tecme sues doprden sn ein waren Tingle In pte at 89°C. Tae cnn ve wee ‘8 the emu ie i an he ey 9390 Fyn which on be genet ef hemi “Thisaewce de event ton unde es ede which gringo» siecle comps 0 the deca tine Chere ney dy a3 “oe mens dey a ek watelengh cin Bea yee byte nki-eporenta dl hs ba sith emer were numedrne dependent of wae eg nd hes aan sed 0 depen ‘ensengih Ths ihe une sumptions te sm ‘mony wed len alin wsengedpenie ot {to rain. Te wanker depen Seca ll ea Colao ote day ened pei gr 7.19) {The sas terete in wena Te DAS fer sre dex ines show sre xc ems cima oe angles fe reuinew soe Hae ten Fovindlen inert wed cept ain otis specie a the DAS pre 17 gv he milening mpsion of dist emiing ses. TRe tse esse cur oberg sash imeem Sion spesta gee F70. hoto) The TRES sow 8 fea iso Seer wale aed pcb rie = mre retoaieceenpuon ot de yes. Te Sependet cr esusign cnet in DAS a3 ‘ees vig ening wees. Topeharetn ns wenn ase dae apg fa pec acinar {ioc bt ee ay ak be pon ere ation fees nt crer When wonclegthdepener itr de trier 0 ue DAS oe TRES Fe a union pr team fete oe DAS an 0 ara he DAS ‘Ste inoiual tspuphan fends Tas asceine fan to says cones. Sa. ea wep. Fran won thre zeny Ho aie he AS th WAVELENGTH (nmi 300340380 «20 pas Inaole in sone Glycerol 20°C ald) TAA 35 30 25 WAVENUMBER (kK) rule enorme Hower is imporaotorenen tert be DAS and TRES se epee fhe sno fhe pote Mos need a ol ad une 9 ating been ductete DAS o comin vaere fr me depend lason of iptetan resides pera bat npc. One hares oF ‘pus reteaion bap inves of te mean den ine shining bees wave enh Sine ve ces fens and ter me ines ace Figure 1612 oe ‘ould be eqs mers ofa py ers boxe ime with nceating waveegh Fw ny est eh. wish ie shot ‘if apc eta, sor uagesypmn cies {tsp betes ac neue nah ening wile ‘Av onanism saeco of exc pas see mp ered osm ne tls ehmisieay pct er. However eco Tnbetvcen hater eninen wclonpis there mecca dew quseng By peoude Dd “hei nner he te om of DAS secre rlan Tred y aie of eye ‘ie pin NP." which cnn asleep rel Ther owed in the centro om ‘sched wth mje clin he asece of mee eves, MBP ic heh to be my asad, Tie sles decaye re clkeed sou he emilon see Hanh The DAS sem py lip conformation and Timeresohed da asl doe indicate meter the {eager dar to rele cmt gis acon: |p chmengpomaasce. Mos oper on prin Nore eee welt tar he DAS of TRES for poset ening epee nrrtain of the ce ‘Ue favre cndins ont sate seogeny or special natin can be ditingvihe by Geto of ‘xpi erwin henge te Te Yoel se of ee ep ‘Sift ding ese Metin and cl oe eevee billy enced nd rand es hopin) ‘equine prexponctal factor can be sen ow in erates under vee thal ry imi te nena of paces Fig 7a show time drain For tryplepan oy tier OE) in micelles fan rchaed deen On the shr-waeeaghs side 15 ‘nin ample eal esse Tepes ore negate ome lngnneep 17S sie he ems Sion whieh proven pec elton as ote to wens the tine-depentert petal site mea) ‘slr an ib nlp onan ries it fo nisin Tete fiom pons presente 585 ceo pe preeapoema xt Fmt en it's protany fe nr wane that melisepiaWAVELENGTH (amt @ soo 4038020 oye NH BOM toe = den 5 des = 205nm 8 | o| = | 2550 g a = Op 3 a75nm = 0 NT y 3290 285 2 oot or 0 WAVENUMBER (4K) UFETIME (st re act ge ee ein ede, ipl confrmatins, nd petal non a Prosphorecence esl cbeeé a ow temperate scorn he cml sence of guocher = Tse endiion ae nsdedbecaise hosporsconce Metin ypc long—iliecondh Io scented ths {ueched ty lw cometary geno inp Clow temgoraurs ae ako needed erase eae of sonra sey be empl wih he po gh ence cision res Other, fhe quam eld of pophorecene wil beer eal MEketecince mere ae Rede fig 1740 donee Nets an par cence type ina lo stow tempera Po fhorscece ssa linger mnt an Ore Gene, ed porpuresiee spc eal) me Secu The pngporwce i owe tes eee (prtom to be Renate. A tpn pire her estan oy chen dette fe poe tuevcnes peter at yer Kir npc pce ee i ie 720 NATA ioe pte (0D 019K) the pporcenee ttpent tote langle sof he Rercenee ‘Sere Win gud deteson th esos oted: The wnclgh wooo oe pogo Sone vere ip Ta en Pe ie a 300 386 soo SO =SOO Fear 4 oan ower pgm specin 17.8 are mori ean» bands er $ fm pc wedi eoring Nsrecence et. I ‘he dry ine were measured o 80 an 50am he = ‘mension wold shew a ilseond decay ie be Inthe 190 reons tee epee om cbserton f eypioghan pongo fom pte om tee persee™ some case i te peer of oygen. "swat crgig poet ng eines fr spore: cence Some oe ay tps ce ner do pe or of otagen wih meme selon ia" NORMALIZED INTENSITY [WAVELENGTH (om) or ese yfRAs ashen E gz Pew ators i uy a a of inal confi von seeped hat oe (res display posporecence in slain at 00m erature" Oberon of wf ghorphrescenee ‘ous the comple exlaron of oxygen, tse {Shove been pores wih x) gen orctnisions op 0 sou Bn “he phosphorescence deca ies of pris al 0m ‘ener hn be sri lone Cire 1748). Ia ontas to Meorescace decay te decay of pooper ence scr 0 be mote Me sige exponents. Bales "popbae with ston emission maeength tend 9 Bane longer phosphorescence cy times han esos reds (ae 176)" Toe wary snglesponeildeeays of povshoecence sn hele ng a peepee ec es suggest he possi f releg the emis ‘on of ever ypephane in» malo Pen “Te higlysrctire emeson of sptopan pogo rescence eam allow he reson of t0 opm ‘ides ingle pen Ts possibilty if wm foe issepeuyptepan aniReesor A” Jw \/ - snore : war zfwrk 4 : 2 lv an, 8 to o___s00 © | sor wi Buena int * § ope va 5 380 400480560 580 3 wow WAVELENGTH (nm) of ‘ea Loma aera rae as wan (Y263) 4 om a) a) 5 10 15 (gare 12.46). The wibiype pron shows cwo ap peaks na 0 om and each of Ie ingle nypep me {an tow single yes earths waselopth Ea Pose pests tur doa iia ropa roe ‘Matileposphrsconce pals tae born obsened for ba pris with wo woop ees * Se ‘iret mazes ba een sea ily amino Side chins tha queschtrplophon plospbareceree pee 1729 shows the phosphorescence emsion specs ot lyeraehyae-potpaedetytropense (PD fom Beis srarhermphan. Te wleaype ote co tao uyplgn resi: WEE apd WSID Te en on prema! he WHEF moti sllowr argnnent oe peaks nea 0 nm o eh ey rsd The ve Fane shows the ghosprecere decay at °C. The fata contining W310 shove wry son ppb fence cyte This sho decay ee ido rose ‘sien poion 209 1Y280. Reptcement of hi = Sine with aie (V) esas 8 Ss eres he ea ine of W310 7a, PERSPECTIVES ON PROTEIN FLUORESCENCE ue wndertanding of rte Muerte as incre Sway ote pot fe year Te ay a8 “TIME (mel Dicuss Sacre nde WH ‘moa a foe Age: Povheeme try So loge suber of pon snetres sows enlation be een scr eles the prtens an Bee sec ropes. Gone rn ae begining Io emer. ac at (eben ado uns ae most Bly 1 gotchsypoptan neg ower rom shoo ong waselengh npn abana forever pres. Emin masa canbe ome wi capoe een ae rom te po tence. Tis mproved anders of pot 0 feoene wl lw fe eo sorcr merece ues. ties swt pti incon ad prin nero wih nace ming tne wget coming. ITs » CASE Apt Ao Snead Rr th fin Paces cy of pie Sogn Lope om tie U.N open oe ‘aoe ep ni curt lng 2 imme ance pom woe dn aoe Fests PG 0. Reig 97 Farce ‘nan ean congne!Ehe BA ss oe open an ys. Bh Bhs Ae Sry. onk ompan R98 Se se ment Sy oe ‘tpn opp renin whe te Fromenceung saint eat ‘en pie cea CMRF Sot Qantas! emsmee ss es oa se nan Fee fain Ta DC ening OR 198 Speco Mr ‘ergo ns npn eo te sie oye Ran an ety Fan. {ita Cah Rog nd 92 yin Narener nt ponte bon pe rae Inigo meen meray Bora Sp i eR ta tisoy nc ee acm Sir war Yona $8 gs Wen [Sih Patna wee in pei eve sie rn ‘even cm on ion? ep inns snp inpepun bt in tat anes ed nee sen eee ‘ei cy nnn mee ‘ema en ap 785558 frye tna os pac cat i ie ae Crean ton pi 508 INA et ete ar fel nce Soh chine Ch 1S. an os ps ma ‘eset ane pence Aa A ci en CA. 197 gmp i etn he tpg nate ae a gn ey ise of mse Gin ge nn Pn Siew ssc oy kc Screed ere ee atu nace need ee ee ‘ramen a Be TEES SEIS Rate of Rao 6 mw ‘opm pesmi tp ee. on ona RX bg 198 Can eine ‘come tection a tere poe ‘roman nessa i endineonon Sera ba ney ag ‘Scanner pent p/h ‘Sere oa oe Hore Goan Rao F si Sneek pied serton Seeman ars sey Soy op ‘put nares mpm se ets 5 Tampon Yogi AK Me Fsepein manson ecto mest i Bg Kany a2 19 Prac eeinged ‘ecient pl ye Bt oe ie AO. Sel en cia iter ee eee Econ oh Remc peg ie bs Fe isnt Gamo A tronngpta ag eran FD Ue Se Sevan dinners ac um Saeed SRS don Ota Peel ste oen respon uae frm ao ees oye. ‘hese emp st ee Ea Fetnn tna np eae ase om m tne her mei span cpa \neuatty inated enn er mee Fone nd or gas feemerrini ns Senta ae fee Ree Green & Poe Grn akk ‘pope ama ofngina manne is hf ‘anew ay fete pment or Ml sate “rag me. Sey Seed oeeligi van sme mtn Bon “ped cect Se Reames (soya tuen a onde a Micon MG 3 arte A 9 Ssh tain inh ae Pater tn WC 1 Pee ey mine seein ttn roi Bee Cou sit c8 22 Tyga nepaeies ape fee ten pin meu rae Bop 1 [Ee Iman 99 Teme end fe phe ‘Not asneo yer a acetone CIR en) ss enh mega pan settee geno rece at oe ‘Sr Bersmnsms a i tetpere to ane a ep tne 9 Toppa Ponte ren ‘gus semis ae tnpetee ees Rept tc pu pogceene np He Paes ‘romance sts Sete vt a mc By Ca Sent ein Teese sane etn Man OB Ein $4 1817. Ope Taimongwue pte perenne ee [Meso Sipingne tempest smug erm P17. Rear gion Pas Ue eis ae Iain of Rae Tin sna aon, A pet ale fr phen et. depend fet RIDA, Codon of avs Anew Spc Te atone er ba The ny Se he monty Saye ae poe in Tale 78. term ofe mem eine nacht Colne dvs seated spe aa ine le 12 ety Dstt Et ie ener Aon 0 eae6 Now cond you contin fas msiamen of DAS wexchypepanedo? PIS, Colaoion fe Topham Hee Dor Fg Yok moe he en ace yen pr fue. Tae demi coonet ithe pean Trea ey Sp, whch eran fe 7% fe mon, Atume th he congnents e 6 ‘murs poi. so habe ee fey ip 9 Calste te ippahanso hee Lee sng he nap of 1999 0" Moor he rer de of 35.1 fore =, “Tecra eines = 2. Aue gua rec ine of 13. eisai Senna {te reo chapel is bok me ered ne emi fon eng fm oneyhton excinion (1) By TPE ‘semean ft an ects Moor ba eschad e xc sae by abseion of sige poten. We ow eonsiee {werphton (PE) and de oon (3) exciton. The teow 2PE ines the Corpor has reached he iad ste by arorption of wo potent. We wl ty Consider sures absorption of wo or mov pts. "Weil pot nae seu bern here ei ‘el defied nemetiate ste Uni 199 ovkiphin secwscopy we consort wo bean exe phermege at as ed pint henicl phys ad ep specooeopy. Taper ‘orbance of excinion regues NpY peak powes 10 incase te probability th two pons we ewlanoes 1y ava for aban, Beets of te tent of ‘0 photos with be Morpher. he set rules for gh bern ai poeple aren om hae Cor ote poto sposopy Bese fhe ier son ‘let, trghoonspctacopy cap bested a 2h 10 Shuey ihe ete symmetry of orgie che ‘nophores Mapholon eipenmens equ complex ‘ees a ih opal powers Id wet Sar psi to se maligne econ (MFE) iy ops! mene) Teeaue te high per woul daage te blogic se Pet Sapeingl: MPE ow dl an in freee Picreenpy. Mutipttce micscopy (MPM pes Decne fie foarte properties of anion Sop (Fesaphi) ser and he nclopnens of ber ceig Incescopes Moiphten excaton usualy dang tng to bapa! smplo shin 9 oe in exon. Mubipteoe microscopy ma ngosaced 199" and sow detest aging = Multiphoton Excitation and Microscopy Tei, INTRODUCTION To MULTIPHOTON The phenomenon of litte exsation canbe dees ina labios gra Figur 161) For one poen 3b ‘opin a single gston eles he Moorophore o ‘ced se Degoding upon he sbsctin Spec ad he exciton welegh te Quorpbore may eect do Sipher iron tea the stor evento he, te Inespesive of te etiam wanlengh the Mropore Dr echd the eid ne by burn of nl RO ‘on, Amst all fap eat from the loves! ergy ive te lead se, ‘MRE is accompli sg loge-eaelengt xi Sin toavo he mach tonge gle phon asorpon of the Mroghore (gee IS 50a 03 pots ae ede fo each he Same erry level deo ove lon stoartion This agra can ge he gression 3a ‘rope aso the pons sequently. Howeve MPE 'S de to simataneor eben of male photons, Shieh i why wo mermatine stas we shown a Fie IS. Bigs ilumaanoninensies must asd for MPE Dectue Re ame Pts mst ert si malnecily seth earphone MPE fea tonnes proces. The ‘Sea of 276 end IPE ae proportional oe eaky ‘ated othe second rhe power. respectively To all "uoreptos examined wih MPE have ilaed te sme ‘emsson spc andesimes ney were excited by ‘one pote sroin. Sie he weston mile for oie ‘lion we eee fr IPE. 2PE and 3. me fo ‘eptors my te plod i eet ees wate wih “irene odes of eaten, Howes, te Muresan tem om the tame exci ral. Independent of oe