Ultrastructure: Effects of Melanin Pigtt1ent on Target Specificity Using a Pulsed Dye Laser (577 ntt1)

Arthur K. F. Tong, M .B., B.S. , Oon T. Tan, M.D., James Boll , M.Eng., John A. Parrish, M .D., and George F. Murphy, M.D. Dcp:utmmt of Dermatology, Boston U ni versity School of Medicin c (OTT), Boston, Dcpartmcnt of Pathology, Brigha l11 Jnd Worncn's Hospital (G FM), Boston, and Dcpartment of I ermatology, Massachusctts Gencra l Hospital (AKFT, JB, JAP), Boston, Massachusetts, U.S.A.

It h as been shown recently that brief pulses of 577 nm doses. We conclude that epidermal mela ni n and vascul ar radiation fro m the tunable dye laser are absorbed sel ectively hem oglobin are competing sites for 577 nm laser absorp• by oxyhemoglobin. T his absorption is associated with highl y ti on and damage, and that the target specifi city of the 577 specifi c damage to superficial vascul ar pl exus blood vessels nm tunable dye laser is therefore inAuenced by variations in those with li ghtly pigm ented (type I- II) skin . in epidermal pi gmentation. T hi s finding is rele va nt to the To determine w hether pigmentary differences in the clinical applica ti on of the tunable dye laser in the ablative overlyin g epidermis inAuence this target specifi city, w e trea tment of vascul ar lesions. exposed both type I (fair) and type V (dark) normal human We also fo und on ultrastru cture that the presence of elec• skin to varyin g radiant exposure doses over 1. 5-fLS pulse tron-lucent circular structures of approximatel y 800 A in durations from the tunable dye laser at a wavelength of577 di am eter were observed onl y at and above cl in ica l threshold nn~. U sing ultras tructural techniques, we found in type I doses in those with type I skin and at the highes t dose of skin that even clinica l subthres ho ld laser exposures ca used 2.75 JI cm 2 in type V skin. I t has been proposed that these reproducible alterations of erythrocytes and adj acent der• structures might be hea t-fixed m olds of water vapor. Both mal vascular endothelium w ithout comparable damage to this and ul tras tructural changes o f epidermal basa l cells the overl yin g epidermis. In contras t, degenerated epider• demonstrate m echanisms responsible for alteration of ti ss ue mal basal cel ls represented the predomin ant form of cellular after exposure to 577 nm, w hi ch arc di scussed. J fillies/ d aITlage aft er laser exposure of type V skin at comparabl e D erll/a/o l 88:747-752, 1987

lthough lasers have been in medica l use fo r about 20 and morc w id espread ti ss ue injury results. Therefore, in order to years as ca utery and cutting devices, the multitude produce highl y selective thermal damage to pigmented tissue tar• of facto rs that determine the o utcome of laser-tissue gets such as bl ood vessels or melanin-containing cell s, very shorr interactions are still being defin ed. In general, a com• and intense pulses at selectively absorbed wavelengths must be bin ati on of intera cti ons occurring at the ti me of laser ad ministered. Aexposure determine the sites, nature, and extent of immediate It is now possible to select laser parameters w hich res ul t in tissu e injury, w hi ch then initiates repair processes. Absorption of exqui sitely selective thermal damage to vessels or to pigmcnt cell s lase[' cnergy in sufficient quantity at given ti ss ue sites, such as [1 -3 J. Y cll ow (577 nm) pulsed dye lasers have recently been used blood vessels and pi gmented cell s, succeeds in heatin g these and to ca use hi g hl y specific injury to the cutaneous mi crovascul ature. o the[' nearby structures to denaturing or vaporizing temperatures. C linica l applica tions of such selective vascul ar injury include treat• The extent to which thermal damage is precisel y locali zed to sites ment of port-wine les ions [4,5J, telangiectases, and other vascular of selecti ve absorption is largely determined by w hether the laser abnormalities and tumors. The 577 nm wavelength is strongly exposure is sufficiently short to prevent thermal diffusion fr om absorbed by oxyhemoglo bin, but there is also absorption by epi• these sites . If diffusion occurs, surrounding ti ss ues are also heated, dermal melanin at this wavelength [6]. The overl yin g epidermal pi gment layer, therefore, represents a potentia l barrier thro ugh w hi ch 577 nm rad iati on must pass to reach the underl ying m i• Manusc ript received September 2, 1986; accepted for publi ca tion De• crovascul ature. This is evidenced by an in crease in the radiant cember 3 1, 1986. cxposure dose necessary to ca use vascul ar damage w ith in creasing T h is stud y was supported in part by the Arthur O. and Gullan M . mela nin pigment [6]. Thus, it is possible that individuals with Wellman Fo undation and by Mary Kay Dermatology Foundation Fel• darker skin are less sensitive to the selective vascul ar damage lowship awarded to O. T. Tan, M .D. ca used by 577 nm pul ses . Reprint requests to: Oon T. T '1I1 , M . D ., Department of Dermatology, In ord er to characteri ze the cffects of epidermal melanin pig• Boston Unive rsity School of Medi cin e, 80 East Concord Street, Boston, Massachuse tts 0211 8-2394. m entati on on sel ective vas cular inj ury, we exposed skin ofhul11 3n A b brevia tions: subjects of differing constitutive pigmentation to a pulsed tunable RUC: red bl ood cell (s) dye laser at 577 nm and studied the morphologic and ultrastruc• T: threshold dose tural effects produced over a range of radi ant exposures.

0022-202X/87/S03.S0 Copyri ght © 1987 by The Society for In ves ti gative Dermatology, In c.

747 748 TONG ET AL THE JOURNAL OF INV ESTIGATIVE DERMATOLOGY

MATERIALS AND METHODS Buttock skin of 4 healthy human male adult volunteers (3 with skin type I and 1 with skin type V) was exposed to varying radiant exposure doses of laser radiation from a Candela Model #LFDL 1 tunable dye laser operating at 577 nm usin g a rhodamine 575 dye at a pulse duration of 1.5 f.L s. The laser was fo cused usin g a pl anoconvex lens, into a quartz optical fib er. Laser pulse energies were meas ured usin g a Scientech laser energy meter calibrated to ± 10% accuracy. Radiant exposure dose was ca lculated based on the diameter of the circular fi eld of irradi ati on. Separate 3 mm• diameter sites were exposed to sin gle laser pulses at flu ences rang• 2 2 2 in g from 0.5 Jlcm to 2.75 Jlcm , in 0.25 J/c m in crements. C linica l (morphologic) thres hold was defin ed as the presence of nonblanchable purpura filling the entire exposed site of 3 mm diameter, appearin g within 10 min of laser exposure. The energy required to produce this effect was defin ed as the threshold dose (T). Four 3-mm punch biopsies were taken from each individual at predetermined radiant exposure doses after infiltration of the skin with 1 % Xylocaine without epinephrine. All biopsies were ob• tained within 10 min of laser irradiation and split for li ght mi• croscopy (routine H & E staining) and electron microscopy. The tissue was processed for transmission electron microscopy as pre• viously described by Murphy et al [7] . Ultrathin sections were Figure 1. Superfi cial capi ll ary venule of type I skin after exposure to 2 cut w ith a diamond knife on a Porter Blum MT-2 ultra• subthreshold dose (0.5 J/cm ) of laser irradiation at 577 nm. N ote the microtome, counterstained w ith uranyl acetate and lea d citrate, marked swelling of endotheli al cells (a sterisks) producing narrowing of and examined with a JEOL JSM 100S electron microscope. the vascular lumenal diameter (L) . Focal gaps (a rrow) between adjacent endothelial cells (EC) and retraction of the endothelial cell s (arrowhead) RESULTS away from pericytes (P) can also be seen. There was a direct relationship between laser energy required to produce the morphologic effect of clinica l purpura and skin pig• mentation (Table .I); a mean dose of 1 J/c m 2 (range, 0.75-1.25 melan ocytes were also profoundl y altered and rough endoplas mic 2 J/c m ) was required to produce purpura in those with type I skin, reti culum within keratinocytes appeared dilated and mitochon• whereas it was not possible to produce purpura in the skin type drial swelling was apparent (Table II ). Similar ultrastructural changes V subject, even at the maximum ava il able laser exposure of 2. 75 were also seen in Langerhans ce ll s. In spite of these changes, 2 J/c m . Li ght microscopic and ultrastructural examinati on ofbiop• melanosomes, the basement membrane zone, and nuclei still ap- sies, taken from control (non-laser exposed) sites and processed under identi ca l conditions appea red normal, with intact vessels and a normal-appea ring epidermis [8]. Morphologic analyses of laser-exposed sites are given below. Table II. Compari son of Epidermal Alteration Between Skin Types I and V at Similar Laser Energies Type I Skin Although only minimal erythema was clinica ll y Laser Energy detected aft er exposure of type I skin to a subthres hold dose of 2 2 0 /cm ) Skin Type I Skin Type V 0.5 J/c m , ultrastructural examination of the epidermis at this energy showed the presence of small cytoplas mic vacuoles in basa l 1. 0 Cytoplasmic vacuoles Degeneration of keratinocytes, although organell es including the nuclei, mito• in layers, basa l keratinocytes and chondria, endoplas mic reticulum, and basement membrane zone keratinocytes. melanocytes . remained intact. Venular vessels of the superfi cial vascular plexus Dilated rough Cytoplas mic vacuo les and capillary loops, on the other hand, showed marked endo• endoplas mic in basal theli al ce ll swelling, narrowed vascular lumen, retraction of endo• reticulum. keratinocytes. thelial cell s from peri cytes, and enlargement and dilatation of Mitochondria l Dilated endoplasmic swelling. Intact reticulum. Intact mitochondria and endoplasmic reticulum, respectively (Fig 1). melanosome, nuclei, mclanosomes , Erythrocytes were not observed within the lumen of these vessels. and basement desmosomes, and As the laser energy was in creased to purpura threshold dose membrane zone. Langerhans cell s. 2 (mea n oft J/cm ), cytoplas mic vacuoles became more prominent 1.25 Degeneration of Focal necrosis of basa I in basa l keratinocytes , w hich involved several layers o f ce ll s, par• keratinocytes with kcratinocytes with ti cul arl y those immediately adjacent to melanocytes. In addition, pyknotic nuclei. foca l cleft through Cyto plasmic vacuoles basa l layer. in basa l 2-3 layers of Edematous keratinocytes . keratinocytes. Table I. Purpura Thres hold Fluence in Skin Type I and Skin Dilated endoplasmic Type V Subjects reticulum and mitochondria. Threshold 0 /c m2) N ormal granular and cornea l layers. Skin Type I N o rmal A 0.75 melanosomes and B 1. 0 basement membrane C 1.25 1.50 N ecros is of C left formation more Skin Type V kcra tinocytes, normal w idespread D > 2.75 mclanosomes. VOL. 88, NO.6 JUNE 1987 T ARGET SPECIFIC ITY W IT H PULSED D YE L ASER 749

pear e d unaltered. At threshold exposure (T) (over a range of Table III. Comparison of Vascular Alterati o ns After 2 0. 75- 1.25 )lcm ) , the degree of laser-induced vascular damage Exposure of Skin T ype I and V to 577 nm Laser Irradiatio n appe ared to be related to the radiant exposure dose delivered. Laser Energy Endo theli al cell s appea red necro ti c at all 3 doses but were most 2 2 0 /cm ) Skin Type I Skin T ype V sev e r ely damaged at the hig hest dose of 1.25 )lcm . In addition, endo thelial cell s at the hig her Auen ce contained Aoccul ent materi al 1. 0 " Molding" of red bl ood Sub-base ment and myelin fi g ures (Fi g 2). In additio n, focal ga ps were al so ob• ce lls (RB C) within lnenlbranc zone serve d between endothelial cell juncti o ns. Abno rmall y shaped bl ood vess el s. Dilated edema with agglutinated red blood cells (RBC ), which w ere mo rc electron• endoplasmi c fo rm ati on of de nse than no rmal erythrocytes, were present w ithin the vessel reti culum (ER) and vacuoles/cle fts. No mitochondria in RB C presen t. IUITle n. These altered RBC contain ed o void and spheri ca l elec• endothelial ce ll s and Pykn oti c nu cle i in tron-lucent vesicles approximately 800 A in diameter w hich were peri cy tes . Swellin g of end otheli al and dis tributed throug hout their cytoplas m (Fi g 2) (T able Ill). Similar fi bro bl as ts (ve il cell s). dilatati on of ER and struc tures have previo usly been described by N aka gaw a et al [9] Perivascul ar edema. mitochondri a. afte r exposure of no rmal skin to 577 nm irradiati on at purpura Retrac tion of threshold Au ences. end oth el ial ce ll s from All these ultras tructural changes beca me progressively mo re pcricytcs. Sli ght 2 cys ti c dil atation of sev e re at su prathres hold doses (> 1. 25 J1 c m ): many keratinocytes w ith pykno ti c nuclei were present, mitocho ndria and rough en• fi broblast. Peri vascul ar cdema. doplasmic reti culum appeared mo re dilated, and cyto plasmic vac• 1. 25 As above, prcsence of No el ectron-lucent uo les were o bserved in at least 3 layers of basal keratinocytes di storted RB C in circular areas within (T a ble II). Similarl y, vascular alterati o ns resembling those de• superfi cial vascul ar RB C. Extravasated scribed fo r thres hold Au ences were also m o re severe and wide• pl exus with electron• RBC. Foca l necrosis spread (Fi g 3). lu cent oval stru ctures of cndo th elia l cell s. with 800 A diameter. Retraction of Type V Skin In contras t, it was no t possible to induce clinica l Extravasated RB . end oth eli al cell s from purpura in the subject investi gated w ith type V skin even at the Degenera tio n of peri cytes. Marked m aximum laser exposure dose o f 2.75 )lcm 2 Despite this, de• endothelial cel ls with perivascular edema gen e rati ve changes w ere apparent o n ultras tructure in epidermal loss of interce llular with cysti c dil atati on ker a tinocytes even at a subthreshold dose o f 1. 0 )lcm 2 (Table II ). ti ght jun ction. Sub• of fi broblasts (ve il These alterati ons w ere primaril y confined to the basal cell layer base ment mcmbranc ccll s) . Focal nccrosis and consisted o f nuclear pyknosis, cyto pl as mic vacuo li zatio n o f zo nc vacuoles. of mast ce ll s. ba s a l keratinocytes and m elanocytes, as well as dilatation o f the Peri vasc ular edema with separati on of endoplas mic reticulum and mitocho ndria. Melanosomes, Lang• co lla gen bundles. erha n s cells, and des m osomes appea red no rmal. At higher Au• 2 Degenerati on of en ces (but still at clinical subthreshold purpura dose ofl. 25 j/cm ) , fibro bl as ts (ve il cell s) th ese changes w ere m o re prono unced and a focal cleft was ob• with cys ti c dil ata tion serve d thro ugh the basa l cell layer (Fig 4) w hi ch was accompanied of cytopl as m. Intact by focal necrosis o f basal keratinocytes. A suprabasal vesicle was mas t cells and ncrvcs. p r esent at the hig hest dose o f 1. 75 ) IC Ill 2 and bo th inter- and 1.50 Electron-lu cent circul ar No el ectron-lu cent intracellular edem a w ere also o bserved. In spite o f such m arked areas in RBC. areas in RB C. Sub• Mark ed sub-base ment base ment membrane mcm brane zo ne zo ne cleft. vacuoles . Normal Degenerated mast mas t cells and nerves. cell s with intact granules.

degenerati ve changes, melanoso m es appeared no rmal even at the 2 highest dose of 2. 75 J/cm . [n no ne of the exposure doses delivered to the ty pe V subject was it possible to induce clinical purpura although the laser-ex• 2 posed site appea red edem atous after ra diant exposure of2. 75 )lcm . In spite o f the absence o f clinical purpura, changes were present in the capillary venules and superfi cial venular plexus vessels after 2 exposure to 1.0 )lcm , which consisted of vacuolizatio n of the endothelial cells and sw elling of the mitocho ndria. Unlike type I skin at exactl y similar doses, no RBC w ere observed within the vessel lumen (Table Ill). These changes became more pronounced 2 at hig her exposures (1.25 and 1.50)lcm ) and it w as o nly at these doses that abno rmal RBC w ere seen within the vessel lumen . These RBC appea red distorted and abnormall y electron-dense, w hi ch were similar in density to those seen at thresho ld doses in type I skin biopsies . Extravasation of RBC w as also o bserved in 1 specimen. D ermal edema, evidenced by wides pread spaces be• tween coll agen bundles, w as also present. In addition , w ell-de• fin ed foci of Ao cculent m aterial w ere seen in the dermis lying Figure 2. Hi gh-power view demonstratin g presence of myelin fi gures between collagen bundles. Within some of these foci, membra• (a rro w) and vesicles (asterisks) containin g fl occul ent material within endo• nous structures resembling dilated endo plas mic reti cululll were thelia l cells (EC) . Al so, note the swollen mitochondria (a,.,.owh eads) R = observed, suggestive of degenerative changes in fibroblasts (Fig red blood cell s. 5) (Table Ill). 750 TONG ET AL T H E JOUI1NAL OF INVESTIGATIVE DERM ATOLOGY

Fi g ure 3. Superfic ial ca pill ary venule of type I skin after exposure to Figure 5. T ype V skin after 1.50 ) lcll1 2 exposure. Note the am ount of 2 su prathreshold dose (1.25 ) lcm ) o f laser irradiatio n at 577 n111 . N ote th e dermal edcma prescnt as evidenced by w ide spaces between coll agen presence of altered red blood cell s w hich contain ovoid and spheri cal bundles (as terisks). Degenerati ve changes in fibro bl as ts w ith cytopl as mic el ectro n- lu cent structures throughout the cy toplas m (as terisk). CC = vacuo li za tion (,,) arc also evid ent. EC = endotheli al cel l. endoth eli al cel l.

At th e hi ghest ex posure dose (2.75 J/c l11 "), di sruption o f endo• m ore pro no un ccd at this hi g her Al.l ence. In spite o fthcse changes, theli al ce ll juncti o li and bascment membranes was o bservcd (Fi g dermal lymphatics, nerves, m as t ce ll s, and coll agen fibers ap• 6). Red bl ood cell s exhibited ovoid and spheri ca l electron-lucent peared no rmal. areas w ithin their cytoplas m ill additio n to enhanced electro n DISCU SS IO N density and distortion of the ce ll conto ur (Fig 6). At 2.75 J/cm", some damage was no ted in the extravascular dermal co mpo nents In th is study we ha ve dem onstrated, o n ultrastructure, detailed in type V skin. D ermal fib roblasts and m acro phages in peri vas• evidence of injury o f two competin g cutaneous chro lll o pho res, cul ar loci showed degenerative changes consisti ng of cytoplasm ic namely melanin and oxyhemoglobin, aft er expos ure to 577 \1111 vacuoliza ti o n and mitochondrial enlargement. I ermal edema was laser irrad iation . It has previo usly becn shown 161a nd now CO I1 -

Figure 4. T ype V skin after cxposure to 2.75 ) Icm". N o te the prescnce Figure 6. T ype V skin after exposure to 2.75 ]lCln2 N ote disruptio n o f o f a cl eft th rough the basal layer kera tin ocytes (asterisks). Ill set, He I11n ants endo thelial cel l j unctio ns (arro w/r cads), and disto rted red blood cells w ith o f th e keratinocytes (K) w ith in tact hemidesm osomes attached to the o void and spheri cal electron-Iu ccnt arcas w ithin their cytoplasm (1'). R = dermis (a rrowh eads). Itl sct magnifica ti on X 2833. red blood cell s; EC = endo thelia l ce ll s. VOL. 88, NO.6 JUNE 1987 TARGET SPECIFI CITY W IT H PULSED DYE LASER 751 .... •z: ences in melanosomal damage between nanosecond-domain ruby u 1.4 and excimer laser pulses, and the present study w ith microsecond• .... ' • : . Rb domain 577 nm laser pulses, ca n be largel y cx plained on the basis ... . .' -' Hb0 (H O) of wavelength and pulse durati on. The intrinsic thermal rel axa ti on ~ 1.2 2 2 : ~ -' .. timc for mclanosomes is es timatcd to bc about 50 ns 13,13]. Se• "6 1.0 lecti vely absorbed lase r pulses less than 50 ns, such as the excimer ...... and ruby lasers cited herc, in ves t their thermal cnergy in mela• X nosomes at a rate much fas ter than the melanosomes ca n cool. ..= 0.8 ...:z: The res ult is extremely confined thermal excitati on and damage u 13]. In contras t, the 1.5 fL S pulse-w idth of th e 577 nm dye laser 0 .6 ... used in this study is considerabl y longer than th e intrinsic thermal C> rel axa ti on time of meianosomes. A more uniform hea ting of the u 0 .4 :z: pi g mented cell volume with less focal melanosomal damage might, C> therefore, be expected. The limitati on of exposure dose to 2.75 .... 2 u 0.2 J/c m , however, may have been in sufficient to elucidate melan• :!:: osomal fr agmentati on . ....X ... 0 Another interes ting findin g relates to RBC damage. It has pre• a: 300 400 500 600 700 100 ~ viously bee n reported by N akagawa et al r91th at 577 nm radiati on ~ WAVELENGTH, Nil produced changes in the electron density ofRBC. Of even grea ter interest was the presence of circul ar electron-lucent structures Figure 7. Absorption spectrull1 of oxyhemoglobin (Hb0 2) and melanin scattered within the cytoplasm of these RBC. Similar stru ctures in s kin showin g competin g absorptio n by these cndogcno us chrol11 o• have been observed in some of our skin biopsies after ex posure pha r es at 577 nm. to 577 nm la se r radiati on. However, these structures were present onl y in skin exposed to and above purpura thres hold doses in type I and at the highes t flu ence in type V biopsies. This is the first finne d by this study that morc laser energy was required at this time that the presence of clinical purpura has been correlated w ith wa v e length to produce the clinical threshold of purpura as skin very definite ultrastructural alterations in RBC after exposure to pig IT1.entation in creased. In fact, this morphologic endpoint was 577 n m irradiation. The mechanisms res ponsible for these ob• not clinically detected in the skin type V subj ect inves ti ga ted in servati ons suggest that these ele ctron-lucent circul ar structures this study even after exposure of the skin to 2. 75 J/c m2 In con• might be hea t-fixed molds of water vapor. In spite of this clinical tras t , a mean dose of 1 J/cm 2 was sufficient to produce clinica ll y and ultras tructural correlati on at purpura thres hold doses, vas• d e t e ctable purpura in our skin type I subjects. The reason for this cular alterations were evident even at subthreshold doses w here is clearl y related to competin g absorption of laser energy by epi• the ski n appeared clinica ll y normal. These ea rl y chan ges of vas• ci e rIT1.a l melanin overl ying the dermal vasculature (Fig 7). Ultra• cular endothelial swelling appea red to be similar in both type I struc tural details presented in this study started with the presence and type V skin although they might have been sli ghtl y more o f d egenerated keratinocytes and melanocytes in type V skin at marked in type I skin . However, a difference was noted between flu e n ces well below those required to produce clinica l purpura the two skin types w hen the la ser energy was in creased to hi gher ev e n when the skin appeared clinica ll y normal, to the fo rmation doses when vascul ar specifi c damage beca me more evident and of cle ft s through the basal keratinocytes at hi gher flu ences . These confined to within the vasculature in the type I biopsies . ch a n ges were in marked contras t to minimal epidermal changes In contras t, marked peri vascular edema, cystic di latati on of seen over the same dose range in type I skin consisting of dil atati on fibroblas ts w ith retrac ti on of endotheli al cell s from peri cytes, and o f e ndoplasmic reti culum and mitochondria as well as the presence degenerati on of mast ce ll s were the typi cal findin gs at hi gher of c y toplasmic vacuoles within basal keratinocytes . It is evident flu ences in the 1 la ser-exposed type V skin subject in ves ti ga ted. froIT1. these data that there is absorption of 577 nm radiation by These vascular changes in conjunction with those epidermal m e l a nin, and as one might expect, in darker skin types , epidermal changes described above further support our vi ew that RBC are ci a Inage becomes relatively more prominent and vascular damage the targe chro mophore in type I and melanin in type V skin. rel a tively less specifi c. T hi s study demonstrates that, at some level Fro m th ese data, we conclude that skin type must be taken into of e pidermal pigmentation, probably between skin types IV and consideration at 577 nm. It is obvious that highl y specifi c vascul ar V, selective vascular effects are no longer possible without con• injury ca n be achieved at 577 nm in skin type I subjects but this curre nt damage to the epidermis. Interestingly, selecti ve vascular sel ectivity quickly diminishes as epidermal pig mentati on in• effects also occured, even in skin type V, at exposures insufficient creases. to cause clinjcall y detectable purpura. The data in this study do not s upport the role for thermal diffusion from dermal vascul ar targets as a major ca use for damage to the overl yin g epidermis: RE FE REN CES it is clea r that epidermal and dermal absorption and damage va ry sep a rately and largel y inversely. Therefore, it is apparent that I. Anderson RR, ParrishjA: Microvasculature ca n be selecti vely dam• vascular selectivity is no longer achieved at 577 nl11 in darker skin aged usin g d yc lasers: a basic thcory and ex perimental evid ence in human skin . Lasers Surg Mcd 1:263-276, 1983 (type V) and that the treatment of vascular lesions using this wa v e length will result in concurrent epidermal damage. In ter• 2. Greenwaldj , Rosen S, Anderson RR, HarristT, Macfa rland F, N oe esting ly, in spite of the severity of the epidermal changes noted j , Pa rrish jA: Comparati ve histologic studies of the tunable dye (a t 577 nm) laser and argon la se r: the specific vascular effects of he r e, melanosomes always appeared morphologically no rmal and the d ye la se r. J In vest Dermarol 77:305-310, 198 1 intact. This is in contrast to the shattered appea rance of me• 3. Anderson RR, Parris h JA: Selecti ve photothcrmolysis: precise mi• la nosomes in human skin exposed to single nanosecond-domain crosurgery by selecti ve absorptio n of pulsed radi ati on. Science 220: 351 nm pulses, using an excimer las er at a wavelength specifi ca ll y 524-527. 1983 chosen to make melanin the major target chromophore [7 ,10]. 4. Mo rell i JG, T an O T, Ga rden J, Margoli s 11. , Seki Y, Boll J. Carney Sin1.ilar melanosome-specific alterati ons have also been reported JM, Anderson RR, Furul11 oto H , Parrish JA : T unable dye la ser for nanosecond-domain 694 nm Q-switched ruby laser pulses in (577 nm): trea tment of po rtw ine stains. Lase rs Surg M ed 1986 6: re tina l pigment epi thelium [11] . The 694 nm wavelength is also 94-99, 1986 selectively absorbed by melanin, mainly because of a lack of com• 5. Tan OT, Carney M, M argoli s 11. , Seki Y, Bo ll J , Anderson RR, pe ting chr01l10phores at this wavelength [12] (F ig 7) . The differ- Parrish jA: Histologic res ponses of portwin e stains trea ted by 752 TONG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

argon, ca rbon dioxide, and tunable dye lasers: a preliminary re• skin after exposure to a pulsed laser. J Invest Dermatol 84:396- port. Arch Dermatol 122:1016-1022, 1986 400, 1985 6. Tan OT, Kerschmann R, Parrish JA: The effect of epidermal pig• 10. Garcia R, Tan OT, Flynn E, Szabo G, ParrishJA: Effects of ultra• mentation on selective vascular effects of pulsed dye laser. Lasers violet excimer laser radiation on human melanocyte culture, Pro• Surg Med 4:365-374, 1986 ceedings of the XlIth International Pigment Conference, Giessen, Federal Republic of Germany. Tokyo, Univ of Tokyo Press, 1985, 7. Murphy GF, Shephard RS, Paul BS, M enkes A, Anderson RR, pp 569-576 Parrish JA: Organelle-specific injury to melanin containing cells 11. Ham WT Jr, Williams RC, Mueller HA, Guerry D III, Clarke AM, in human skin by pulsed laser irradiation. Lab Invest 49:680-685, Geeracts WJ: Effects of laser radiation on the mammalian eye. 1983 Trans NY Acad Sci, Series II 28:517-526, 1966 8. Braverman 1M, Yen A: Ultrastructure of the human dermal micro• 12. Anderson RR, Parrish JA: The opti cs of human .skin. J In vest Der• circulation II: the capillary loops of the dermal papillae. J Inves t matol 77:13-19,1981 Dermatol 68:44-52, 1977 13 . Hayes JR, Wolbarsht ML: Thermal model for retinal ·damage induced 9. Nakagawa H, Tan OT, ParrishJA: Ultrastructural changes in human by pulsed lasers. Aerospace Medicine 39:474-480, 1968