Comparison of Responses of Tattoos to Picosecond and Nanosecond Q-Switched Neodymium:YAG Lasers

STUDY Comparison of Responses of Tattoos to Picosecond and Nanosecond Q-Switched Neodymium:YAG Lasers

CDR E. Victor Ross, USN; George Naseef, MD; Charles Lin, PhD; Michael Kelly, MS; Norm Michaud, MS; Thomas J. Flotte, MD; Jill Raythen; R. Rox Anderson, MD

Objective: To test the hypothesis that picosecond la- ated with the study (and blinded to the treatment type) ser pulses are more effective than nanosecond domain evaluated photographs to assess tattoo lightening. For- pulses in clearing of tattoos. malin-fixed specimens were examined for qualitative epi- dermal and dermal changes as well as depth of pigment Design: Intratattoo comparison trial of 2 laser treat- alteration. Electron micrographs were examined for par- ment modalities. ticle electron density and size changes (in vivo and in vitro). The gross in vitro optical density changes were Setting: A large interdisciplinary biomedical laser labo- measured. ratory on the campus of a tertiary medical center. Results: In 12 of 16 tattoos, there was significant light- Patients: Consecutive patients with black tattoos were ening in the picosecond-treated areas compared with enrolled; all 16 patients completed the study. those treated with nanosecond pulses. Mean depth of pigment alteration was greater for picosecond pulses, Intervention: We treated designated parts of the same but the difference was not significant. In vivo biopsy tattoo with 35-picosecond and 10-nanosecond pulses from specimens showed similar electron-lucent changes for 2 neodymium:YAG lasers. Patients received a total of 4 both pulse durations. In vitro results were similar for treatments at 4-week intervals. All laser pulse param- both pulse durations, showing increases in particle sizes eters were held constant except pulse duration. Radia- and decreased electron density as well as gross ink tion exposure was 0.65 J/cm2 at the skin surface. Biop- lightening. sies were performed for routine microscopic and electron microscopic analysis at the initial treatment session and Conclusions: Picosecond pulses are more efficient than 4 weeks after the final treatment in 8 consenting pa- nanosecond pulses in clearing black tattoos. Black tat- tients. Also, ink samples were irradiated in vitro. toos clear principally by laser-induced changes in the intrinsic optical properties of the ink. Main Outcome Measures: In vivo, on the comple- tion of treatment, a panel of dermatologists not associ- Arch Dermatol. 1998;134:167-171

-SWITCHED LASERS have pulses.5 Assuming a thermal or mechanical revolutionized the treat- mechanism for tattoo removal, subnanosec- ment of tattoos. By restrict- ond pulses theoretically should be more ef- ing pulse duration, ink par- fective in tattoo treatment. To test the hy- ticles reach very high tem- pothesis that picosecond pulses are more ef- Q peratures1 with relative ficient than nanosecond pulses in the sparing of adjacent normal skin. This signifi- treatment of tattoos, we compared tattoo cantly decreases the scarring that often re- clearing after treatment with 2 neodymium: sults after nonselective tattoo removal meth- YAG laser systems in which all laser param- ods, such as dermabrasion or treatment with eters were held constant except pulse dura- a carbon dioxide laser. The Q-switched neo- tion. Using both pulse widths, we also ex- dymium:YAGlaserhasbeenshowntobepar- amined immediate particle responses to ticularly effective in the treatment of black pulsed laser irradiation in vitro. From the Department of tattoos.2,3 Neodymium:YAG lasers in clini- Dermatology, Wellman cal use are capable of delivering 10-nano- RESULTS Laboratories of Photomedicine, second pulses. The most common pigment Harvard Medical School, particle, carbon black in india ink, has been Of the 16 tattoos, 12 were judged to clear bet- Massachusetts General 4 Hospital, Boston, Mass. Dr Ross showntobeabout40nmindiameter. These ter with picosecond pulses based on the re- is now with the Department of particleshavethermalrelaxationtimesofless sponse of black ink regions (Figure 1 shows Dermatology, Naval Medical than10nanoseconds;therefore,anargument a representative result). In the remainder of Center, San Diego, Calif. can be made for using subnanosecond tattoos, there was only slight or no clearing

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 PATIENTS, MATERIALS, AND conventionally used in clinical practice (fluence of 8.0 J/cm2 and 2.5-mm spot size). Both laser units used an articulated METHODS arm and focusing handpiece lens assembly for beam deliv- ery. Care was taken to avoid gross overlapping of adjacent Sixteen patients with cosmetic tattoos participated after be- exposure sites. For all treatment sites, the patients under- ing informed of the nature of the protocol and giving con- went 4 treatments in the described manner at 3- to 4-week sent. The protocol was approved by the Subcommittee for intervals. All pulse energies were verified with an energy me- Human Subjects of the Massachusetts General Hospital, Bos- ter (Model 365, Scientech, Boulder, Colo) prior to each treat- ton. Patients were enrolled on a consecutive basis as long as ment. The beam profiles and spot sizes were determined by their tattoos were partly black and previously untreated. The imaging the beam with a charge-coupled device camera restriction to black tattoos allowed for a reasonable chance (Model TM-34KC, Pulnix, Sunnydale, Calif) as follows. A for pigment lightening, given the response of black tattoos black surface was placed at the object distance in front of to 1064-nm radiation and the low fluences used in the study the articulated arm. The surface was brought into the focus (vide supra), and easier correlation between clinical re- of the camera so that the laser spot was in the field of view. sponses and in vitro black pigment alterations induced by la- The video signal was input into a frame grabber (CX100, Im- ser. Of the 16 tattoos, 15 were created by a professional and age Nation Corp, Beaverton, Ore) interfaced with a per- 1 was done by an amateur. Eleven of the tattoos were mul- sonal computer. After capturing the image, the beam size and ticolored, most of these containing black, red, and green pig- profile were determined with the aid of imaging software (NIH ments. The remaining 5 clinically showed only black pig- Image, National Institutes of Health, Bethesda, Md). mentation. Tattoos were divided into 3 parts, the first 2 parts Photographs were taken before each treatment. All pho- being the treatment sites for comparison between pulse du- tographs were taken with the same 35-mm camera (AE1, rations. These sites were designated either as the left or right Canon USA, Lake Success, NY) under similar lighting con- symmetrical parts of a representative portion of the whole tat- ditions. The same film type (Ektachrome 100, Kodak, Roch- too. Part 1 was treated with a mode-locked Q-switched neo- ester, NY) and processing method were used for all pho- dymium:YAG laser (Model YG501, Quantel Technologies, tographs. Local anesthesia (2% lidocaine with epinephrine) Santa Clara, Calif) delivering 35-picosecond pulses. Spot size, was used in patients requesting it. In 10 consenting pa- fluence, and repetition rate were 1.4 mm, 0.65 J/cm2, and 10 tients, 2-mm punch biopsy specimens were obtained be- Hz, respectively. The beam profile was gaussian. Part 2 was fore and immediately after the first treatment, and at 30 days treated with a Q-switched neodymium:YAG laser (Model after the final treatment. Samples were taken from tattoo NY82-10, Continuum, Santa Clara, Calif). Laser parameters areas that appeared clinically black. Specimens were fixed were identical to part 1 except for pulse duration, which was in 10% buffered formalin, processed in paraffin, and stained 10 nanoseconds, and the beam profile, which was multimo- with hematoxylin and eosin. Also, 1-µm sections were cut dal. The remainder of the tattoo was treated with the same and stained with buffered 0.5% toluidine blue O. These sec- laser values as those for part 2 except with parameters tions and their unstained counterparts were examined for

with either pulse duration (Table). Within individual tat- irregularly shaped “clumps” that ranged from 1 to 5 µm in toos, black tattoo regions responded best in all cases. Over- diameter. In some specimens, occasional distinct red and all, clearance of black ink was better after picosecond treat- green granules (1-2 µm in diameter) were identified. Inter- ment (PϽ.002). Although green tattoo regions lightened in tattoo ink depth varied considerably and ranged from 250 somepatients,red,purple,blue,orange,andyellowpigments to 1700 µm deep to the stratum corneum. Intratattoo ink did not clear regardless of number of treatments or pulse du- depths, however, were more consistent, with biopsy speci- ration, and overall, nonblack tattoo regions responded simi- mens showing no more than ±200 µm depth variability from larly for both pulse durations (PϾ.20). Although not for- different sections of the same specimens. Immediately af- mally assessed by the panel of raters, we found that conven- ter treatment, nonblack particles appeared unaltered in the tional high-energy nanosecond pulses produced clearing biopsy specimens; however, in all cases there was a smudg- comparable with that of the lower-energy picosecond pulses. ing of black particles, noted as a transition from black gran- During treatment, picosecond pulses produced more intense ules to amorphous light brown bodies with a lacy appear- immediate whitening than low-energy nanosecond pulses. ance. In general, a line could be drawn demarcating the depth Also, in most cases, picosecond pulse sites showed plasma of transition of the particles. This depth ranged from a mean formation and slight postoperative pinpoint bleeding not ob- (±SEM) of 670±96 µm (n=10) for picosecond pulses to served after nanosecond pulses of the same fluence (Figure 590±107µmfornanosecondpulses.Otherimmediatechanges 1). Edema was noted after both nanosecond and picosec- included the formation of suprabasilar clefts and vacuoles ond pulses. Hypopigmentation was noted in 1 tattoo in the in the dermal interstitium in proximity to pigmentation. Al- picosecond treated portion. No scarring was noted except tered pigment and cellular debris lined some of these der- in parts of 2 tattoos treated with conventional high-fluence mal vacuoles. These immediate histological changes ranged nanosecond pulses. These areas showed persistent erythema frommostpronouncedafterconventionalhigh-fluencenano- and induration. No other adverse effects were observed. second pulses to least pronounced after low-fluence nano- Routine light microscopic examination showed pre- second pulses. No significant fibrosis was noted immediately treatment pigment in all cases. The concentration of gran- following treatment or 1 month after the last treatment. ules roughly correlated to the clinical darkness of the tat- Electron microscopic analysis revealed pretreatment too. Also, the clinical color corresponded to the microscopic particles that were electron dense and ranged from 10 to particle color. The predominant granules appeared as black, 100 nm in diameter (mean, 40 nm). (We define “particle”

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 determination of the depth of altered pigment following portions of the tattoos. For every tattoo, the modal score was treatment. Biopsy specimens were examined by a derma- recorded for each pulse duration. A Wilcoxon matched- topathologist blinded to the pulse duration. Transmission pair signed-rank test was used to analyze the data. Hypopig- electron microscopy was performed in specimens from 4 mentation, hyperpigmentation, textural changes, and scar- patients. Specimens were fixed in 4% glutaraldehyde in 0.1- ring were graded as absent, trace present, or present. mol/L cacodylate buffer, postfixed in 2% osmic acid in buffer, dehydrated, and embedded in epoxy resin (Epon). Thin sec- IN VITRO INK SUSPENSION IRRADIATION tions were stained with saturated uranyl acetate and Sato lead stain and examined with a transmission electron mi- We performed 2 in vitro experiments. In the first, we ir- croscope (Philips CM10, Philips Elect ron Optics, Amster- radiated india ink suspensions composed of stock tattoo dam, the Netherlands). ink for medical tattooing (Sanford-Farber, Lewisburg, Tenn). The stock ink suspension was diluted with distilled water TATTOO GRADING SYSTEM to obtain a usable optical density for testing. These suspensions were put into plastic Petri dishes (60ϫ15 mm, A panel of 8 dermatologists and nursing staff familiar with Fisher Scientific, Pittsburgh, Pa) under which a black piece laser treatment of tattoos but not familiar with the study of paper was placed (Zap-it laser alignment paper, Ken- simultaneously and independently evaluated each tattoo tek, Pittsfield, NH) to prevent backscatter. The suspen- from pretreatment and posttreatment photographs. The sions were irradiated with picosecond and nanosecond posttreatment scores were based on the photographs taken pulses with the same parameters as in the human tattoos. 1 month after the fourth and final laser treatment session. The handpiece tip was placed at the surface of the suspen- Before evaluating the experimental set of data, a short se- sion and the laser beam was moved so that the entire sur- ries of slides from patients not in this study were shown as face was irradiated uniformly. Portions of the suspensions a training set. Using this set, consensus was reached among were submitted for transmission electron microscopy af- the evaluators regarding the grading system for tattoo ink ter in vitro irradiation. Drops of the specimens were placed darkening, as follows: on a carbon and formvar-coated grid, allowed to dry, and examined unstained with an electron microscope. Grade (Response) % Clearing The remainder of the irradiated suspensions were 0-1 (None) 0-9 placed in plastic cuvettes (1ϫ1ϫ3 cm, Fisher Scientific), 2-3 (Poor) 10-34 and the optical densities were measured before and follow- 4-7 (Fair) 35-69 ing irradiation by recording the absorption of a helium neon 8-9 (Good) 70-90 laser beam by the suspensions. This was quantified by mea- 10 (Excellent) Ͼ90 suring the signal drop across the suspension with a pho- Raters were instructed to give separate scores based on todiode attached to an oscilloscope (Model 9420, LeCroy, improvement percentage in black portions and nonblack Spring Valley, NY).

A B C

Figure 1. A black tattoo in which the “W” and “A” were treated with picosecond pulses of a neodymium:YAG laser. A, Pretreatment; B, 5 minutes after treatment; and C, 1 month after the final treatment. as the smallest identifiable structure on electron micros- The in vitro optical density decreased after irradia- copy. This is distinguished from “granule,” which we de- tion of ink in the cuvettes. The suspensions were visibly fine as the smallest structure observed on routine light mi- lighter after irradiation, and the optical density of the sus- croscopy [usually 0.5-4.0 µm in diameter].) 6,7 Particles pensions decreased from 0.1 to 0.06 after both nanosec- resided predominantly in fibroblasts. Immediately after ir- ond and picosecond pulses. radiation, many of these particles appeared unchanged; how- Electron micrographs of the in vitro irradiated india ever, a fraction (approximately 30%) showed a lamellated ink suspension showed a similar mixture of electron-dense electron-lucent appearance (Figure 2). Marked debris was and electron-lucent particles as noted in vivo, with the ex- noted in the cytoplasm of pigment-laden cells. Specimens ception that after in vitro irradiation, many particles were obtained 1 month after the final treatment showed a per- much larger than before treatment (Figure 3). Particle di- sistence of these lamellated particles. These electron mi- ameters ranged from baseline size (40 nm) up to 300 nm. croscopic findings were similar for both picosecond and It was unclear from review of the electron micrographs nanosecond exposures. whether single particles had enlarged or if many particles

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Tattoo No.

Treatment 1 2345678910111213141516 Picosecond pulse 9 10 3 8.5 10 1 8 0 9 9 9 5 8 9 9 0 Nanosecond pulse 7.5 0 2 0 7.5 1.5 5 0 3 6.5 7 2.5 1 2 3 2

A B A B

Figure 2. Electron micrograph showing pretreatment electron-dense Figure 3. Electron micrographs showing in vitro suspension. A, particles (arrows) in cytoplasm of fibroblast (A), and posttreatment mixture Pretreatment particles of relatively uniform shape and size. B, Posttreatment of electron-dense particles and electron-lucent particles with lamellations enlarged “bubblelike” particles mixed with apparently unaltered particles (arrows) (B) (original magnification ϫ62 100); bars represent 0.2 µm. (original magnification ϫ62 400); bars represent 0.2 µm. had coalesced to form larger, more electron-lucent lamel- mal collagen, pulse durations should be equal to or less lated particles. The qualitative nature of the changes was than the thermal relaxation time, defined as the time that independent of pulse duration; however, a greater propor- it takes for the central temperature in a structure to de- tionofparticleswerealteredinthepicosecond-treatedsample. crease by 50%.9 If the pulse duration is less than the thermal relaxation time of the absorbing particle, then heat COMMENT is confined without significant thermal diffusion during the laser pulse. For the 40-nm particles in our study, as- Our results show that picosecond laser pulses are more ef- suming they behave thermally as independent entities, ficient in clearing cosmetic tattoos than nanosecond do- thermal relaxation time is roughly 1 nanosecond, so that main pulses. By holding other laser parameters constant, only the picosecond pulses were thermally confined. pulse duration was shown to be a significant factor in tat- Thus, higher peak temperatures should result from pi- too removal. With picosecond pulses, we were able to clear cosecond pulses, and this may explain the differences in tat- some tattoos with fluences of less magnitude than typical too clearing. We calculated the predicted peak temperatures nanosecond domain pulses used in clinical practice. at the center of a particle for the 2 power densities (2.1ϫ1010 The mechanisms for tattoo removal by pulsed laser ra- W/cm2 for picosecond vs 7.5ϫ107 W/cm2 for nanosecond diation are poorly understood. It has been shown that ul- pulses) by solving the general heat equation for an arbitrary trashort laser pulses can selectively disrupt cells contain- sphere of radius by numerical methods.10 It was found that ing tattoo pigments,6,7 releasing ink into the dermis, some for a 40-nm-diameter sphere (the size of a typical tattoo par- of which is removed by the vascular and lymphatic systems. ticle),thermalconfinementisachievedwitha35-picosecond Fragmentation of ink particles is an intuitively attractive pulse duration (the pulse duration of our picosecond sys- mechanism. The resulting smaller particles should be more tem), so that the energy deposited in the 40-nm sphere stays easily phagocytosed and packaged; moreover, the smaller largely within this diameter at the end of the laser pulse. particle diameters, as they approach the wavelength of vis- On the other hand, if the same amount of energy is deliv- ible light, should result in more intrinsic dermal light scat- ered in a 10-nanosecond pulse, then significant heat trans- tering, making the particles less visible from the skin sur- fer takes place in the surrounding medium during the la- face. Some tattoos are clinically resistant to all laser thera- ser pulse, and the peak temperature attained by the particle piesdespitethepredictedhighparticletemperaturesachieved is less than 3% of the peak temperature achieved with the through selective photothermolysis. Reasons cited for fail- picosecond pulse. The result of the theoretical calculation ure of some tattoos to clear include the absorption spectrum thus supports the argument that picosecond laser pulses ofthepigment,thedepthofpigment,andthestructuralprop- should be more effective at altering the tattoo particles than erties of the ink. Also, some inks may remain in the dermis nanosecond laser pulses. after being rephagocytosed by resident cells.5,8 The lack of qualitative electron and light micro- Regardless of the mechanism for tattoo clearing by scopic differences between the 2 pulse durations sup- laser, the initial event is absorption of the incident beam ports a similar mechanism (thermal) for the laser- by the ink. To achieve the maximum temperature in- induced particle changes. That picosecond pulses resulted crease in the ink particles while sparing the adjacent der- in a greater microscopic depth of altered pigment is also

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 consistent with our thermal argument, since a smaller plasma-generated shock waves in distilled water are able subsurface energy density would critically increase the to propagate hundreds of micrometers below the surface. particle temperature. Accordingly, because more par- The associated shock peak pressures were attenuated by ticles are altered with picosecond pulses, gross tattoo clear- only 50% at 300 µm,12 suggesting that plasma-generated ing is enhanced with the same surface fluence. shock waves might contribute to fibroblast cell death and/or The differences in the beam profiles of the 2 lasers intrinsic ink particle changes. Moreover, the plasma, in cre- should also be considered in interpreting the results. The ating a pinpoint surface defect, might elicit greater dermal nanosecond profile, whose shape was more like that of a inflammation and facilitate transepidermal elimination. top hat, produced a more uniform surface fluence. Still, Results of our study suggest that intrinsic optical prop- the peak energy density (at the center of the gaussian pro- erty changes, rather than particle fragmentation, are respon- file) of the picosecond laser was only about 20% greater sible for india ink tattoo clearing, and that these temperature- than the peak of the multimode (nanosecond) profile. It induced changes occur with lower fluence thresholds with is unlikely that this small difference in local energy den- shorter laser pulses. Further studies with more powerful pi- sity within the spot significantly influenced our findings. cosecond lasers are necessary to demonstrate whether pi- High temperatures have been shown to induce our ob- cosecondpulsesarecapableofclearingresistanttattoos.Also, servedelectronmicroscopicchangesinotherstudies.4,6 Chen because many tattoos contain nonblack inks, studies should and Diebold4 report enlarged electron-lucent particles af- examine ultrastructural changes before and after laser treat- ter in vitro radiation of carbon black with a pulsed neodymi- ment with chemically dissimilar pigments. um:YAG laser. They found that laser irradiation of the suspension caused it to become grossly transparent, and their description of “new particles with a shell-like structure” was Accepted for publication July 29, 1997. consistent with our own findings. In explaining the mecha- This work was supported by the Department of De- nism for these changes, they suggest that the gradual reduc- fense Medical Free-Electron Laser (MFEL) program under tioninabsorbance(blackness)ofthesuspensionswascaused contract N00014-94-1-0927. by sufficient particle heating to initiate chemical reactions The views expressed in this article are those of the au- with the surrounding water. They found hydrogen and car- thors and do not reflect the official policy or position of the bon monoxide gas above the irradiated sample and note that US Department of the Navy, Department of Defense, or the this reaction might be responsible for the gradual loss of US government. carbon in the suspension. This chemical reaction has been Presented at the 16th annual meeting of the American described as an endothermic steam-carbon reaction.4 Society for Laser Medicine and Surgery, Orlando, Fla, April In addition to temperature increases, irradiation of an 17, 1996. absorber by short laser pulses causes rapid thermal expan- We thank William Farinelli for his technical assis- sion, which propagates as a stress wave. Inertial confine- tance. ment is achieved when the laser pulse is delivered within Reprints: CDR E. Victor Ross, USN, Department of the time when pressure can be relieved from the absorb- Clinical Research, Naval Medical Center, 34800 Bob Wil- ing particle. For india ink particles, assuming they behave son Dr, San Diego, CA 92134-5000. inertially as independent structures, this is approximately 25 picoseconds, slightly less than the 35-picosecond pulses in our experiment, so that stress wave differences prob- REFERENCES ably did not play a significant role in tattoo clearing. 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