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Corrected: Publisher Correction

OPEN Pattern analysis of 532- and 1,064-

nm picosecond-domain laser-
induced immediate tissue reactions
Received: 31 July 2018
Accepted: 22 February 2019 in ex vivo pigmented micropig skin
Published online: 12 March 2019
Hee Chul Lee1, James Childs2, Hye Jin Chung3, Jinyoung Park1, Jumi Hong1 & Sung Bin Cho   4,5

Optical pulses from picosecond lasers can be delivered to the skin as single, flat-top beams or
fractionated beams using a beam splitter or microlens array (MLA). In this study, picosecond
neodymium:yttrium aluminum garnet laser treatment using a single flat-top beam and an MLA-type
beam at the wavelengths of 532 nm and 1,064 nm were delivered on ex vivo genotype-regulated,
pigmented micropig skin. Skin specimens were obtained immediately after treatment and
microscopically analyzed. Single flat-top beam treatment at a wavelength of 532 nm and a fluence
of 0.05-J/cm2 reduced melanin pigments in epidermal keratinocytes and melanocytes, compared to
untreated controls. Additionally, 0.1 J/cm2- and 1.3 J/cm2-fluenced laser treatment at 532 nm elicited
noticeable vacuolation of keratinocytes and melanocytes within all epidermal layers. Single flat-top
beam picosecond laser treatment at a wavelength of 1,064 nm and a fluence of 0.18 J/cm2 also reduced
melanin pigments in keratinocytes and melanocytes. Treatment at 1,064-nm and fluences of 1.4 J/cm2
and 2.8 J/cm2 generated increasing degrees of vacuolated keratinocytes and melanocytes. Meanwhile,
532- and 1,064-nm MLA-type, picosecond laser treatment elicited fractionated zones of laser-induced
micro-vacuolization in the epidermis and dermis. Therein, the sizes and degrees of tissue reactions
differed according to wavelength, fluence, and distance between the microlens and skin.

Picosecond-domain lasers target chromophores by irradiating the skin at higher peak power and an extremely
shorter pulse duration, compared to nanosecond-domain lasers1. Laser treatment therewith provides more
effective energy transfer to target chromophores of smaller size than nanosecond-domain laser treatment2,3.
Furthermore, to generate permanent zones of tissue breakdown, picosecond laser treatment elicits expansion and
collapse of great magnitude that is initiated by the irradiation of pigment chromophores in target tissue3. Thereby,
compared to nanosecond laser treatments, picosecond laser treatment better disintegrates pigment particles into
smaller particles that are effectively dispersed into peripheral areas3.
Optical pulses from picosecond lasers can be delivered to the skin as single, flat-top beams or fractionated
laser beams4,5. For the latter, diffractive beam splitters or microlens arrays (MLAs) have been adopted to gen-
erate multiple focused beamlets4. Therewith, high-intensity, micro-injury zones of laser-induced breakdown
are generated in the epidermis or dermis, with limited photothermal injury to the surrounding tissue4–6. These
laser-induced micro-injury zones have been suggested to stimulate new collagen, mucin, and elastin produc-
tion in the dermis during the wound repair process to improve the appearance of wrinkles and atrophic scars5,7.
A previous multiphoton microscopy study provided laser scanning microscopic images of in vivo human skin
treated by 532-nm and 1,064-nm picosecond neodymium:yttrium-aluminum-garnet (Nd:YAG) lasers using a
holographic diffractive beam splitter, and suggested pigment chromophores as the main absorber for initiating
the generation of laser-induced tissue breakdown4.
In this observational descriptive study, we evaluated the patterns of immediate tissue reactions induced by
532- and 1,064-nm picosecond laser treatment in ex vivo genotype-regulated, pigmented micropig skin. To do

1
R&D Center, Lutronic Corporation, Goyang, Korea. 2Global Center, Lutronic Corporation, Billerica, MA, USA.
3
Department of Dermatology, Boston University School of Medicine, Boston, MA, USA. 4Department of Dermatology
and Cutaneous Biology Research Center, International St. Mary’s Hospital, Catholic Kwandong University College of
Medicine, Incheon, Korea. 5Yonsei Seran Dermatology and Laser Clinic, Seoul, Korea. Correspondence and requests
for materials should be addressed to S.C. (email: [email protected])

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Figure 1.  Tissue reactions after 532-nm single flat-top beam picosecond-domain laser treatment.
(a) Untreated ex vivo pigmented micropig skin presented darkly and homogeneously pigmented keratinocytes
and melanocytes in the epidermis and collagen fibers, fibroblasts, and dermal microvascular component in
the upper dermis. Single pulses of single flat beam, 532-nm picosecond laser treatment at a spot size of 5.3 mm
and at the fluences of (b) 0.05 J/cm2, (c) 0.1 J/cm2, and (d) 1.3 J/cm2. Inlets depict close-up views of the basilar
keratinocytes, melanocytes, and basement membrane. (d) Asterisks indicate giant epidermal vacuoles. H&E
stain, original magnification x200.

so, two- and three-dimensional characterization of focused areas, which were generated by delivering MLA-type
beams from a picosecond laser, were evaluated. Then, picosecond-domain laser treatments at the wavelengths of
532 and 1,064 nm were performed on the ex vivo tissue samples of a pigmented micropig model using a single
flat-top beam and an MLA-type beam. Specimens were histologically evaluated following hematoxylin and eosin
staining. Furthermore, we also compared the patterns of tissue reactions between nanosecond and picosecond
Nd:YAG lasers using a single flat-top beam.

Results
Flat-top beam 532-nm picosecond laser-induced tissue reactions.  The untreated ex vivo pigmented
micropig skin in our study exhibited the characteristic layers of the epidermis, including the stratum corneum in
a basket-weave pattern, the stratum spinosum with pigmented, polyhedral keratinocytes, the stratum basalis with
darkly and homogeneously pigmented keratinocytes and melanocytes, and an intact basement membrane (BM)
(Fig. 1a). The upper dermis was composed of homogenous collagen fibers, fibroblasts, and dermal microvascular
components (DMVCs). Immediately after a single pulse of picosecond laser treatment at 532 nm, a 5.3-mm spot
size, and a 0.05-J/cm2 fluence, melanin pigments in the basal layer were slightly decreased, compared to untreated
micropig skin (Fig. 1b). Most of the epidermal keratinocytes presented perinuclear vacuolization. The integrity
and thickness of the BM were intact, and DMVCs were mildly dilated.
The 532-nm 0.1 J/cm2-fluenced picosecond laser treatment elicited marked vacuolation of keratinocytes and
melanocytes throughout all epidermal layers, and melanin pigments in the epidermis were reduced (Fig. 1c).
Under high power magnification, vacuoles seemed to have pushed nuclei in sporadic directions and appeared
with signet ring-like morphology. Numerous round zones of laser-induced tissue injury were found in collagen
bundles at the maximum depth of 274.2 ± 39.0 µm and around dilated DMVCs. Meanwhile, 1.3 J/cm2-fluenced
laser treatment generated more signet ring-like keratinocytes with extensive vacuolization, and the nuclei thereof
were more indented (Fig. 1d), compared to the other experimental settings (Fig. 1b,c). Vacuolar changes in the
basal layer were remarkably extensive, and giant elliptical vacuoles with a size of 32.5 ± 1.6 µm × 15.7 ± 2.0 µm
were also found in the epidermis (Fig. 1d). The maximum depth of round cavities of laser-induced tissue reaction
was estimated at 371.8 ± 30.0 µm in the dermis (Fig. 1d). The maximum depth of tissue reactions was significantly
correlated with the power density (R = 0.765; P < 0.001).

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Figure 2.  Tissue reactions after 1,064-nm single flat-top beam picosecond-domain laser treatment. Single
pulses of single flat-top beam, 1,064-nm picosecond laser treatments at a spot size of 6 mm and the fluences
of (a,b) 0.18 J/cm2, (c,d)1.4 J/cm2, and (e,f) 2.8 J/cm2. (a,c,e) Inlets depict close-up views of the basilar
keratinocytes, melanocytes, and basement membrane. (b) Inlet, close-up view of melanin-containing cell with
small, dark-staining nucleus in the dermis. (d) Asterisk indicates giant epidermal vacuole. H&E stain, original
magnification (a,c,e) x200, (b,d,f) x400.

Flat-top beam 1,064-nm picosecond laser-induced tissue reactions.  Single-pulse treatment at

1,064 nm, a 6-mm spot size, and a fluence of 0.18 J/cm2 elicited decreased melanin pigments in the basal layer
(Fig. 2a), compared to untreated micropig skin. Most of the epidermal keratinocytes exhibited slight perinuclear
vacuolization. BM integrity was intact, although clusters of melanin granules or melanin-containing cells were
occasionally found in the dermis (Fig. 2b). Meanwhile, 1.4 J/cm2-fluence treatment elicited vacuoles of varying
sizes in keratinocytes and melanocytes that pushed nuclei in sporadic directions (Fig. 2c). The overall amount of
epidermal melanin pigments was markedly reduced; in particular, the melanin particles in the basilar epidermis
were homogeneously disintegrated (Fig. 2c,d). Round cavities of laser-induced tissue injury were also found in
collagen bundles and around dilated DMVCs at the maximum depth of 212.8 ± 48.7 µm (Fig. 2d).
Skin treated at a fluence of 2.8 J/cm2 exhibited signet ring-like keratinocytes with extensive vacuolization of
varying size and indented nuclei, compared to the other experimental settings (Fig. 2e). Vacuolar changes in the
basilar epidermis were extensive and appeared to involve the BM zone; few or no melanin particles were found. A
large oval vacuole (size of 40.0 ± 6.1 µm x 48.9 ± 5.5 µm) was identified in the epidermis (Fig. 2e). DMVCs were
mostly dilated and exhibited numerous round cavities of laser-induced tissue injury in collagen bundles (max-
imum depth, 264.1 ± 44.9 µm), compared to treatment at 1.4 J/cm2 (Fig. 2f). Although the depth of vacuolated
reactions was slightly deeper at the fluence of 2.8 J/cm2, compared to 1.4 J/cm2, treatment with 2.8-J/cm2 fluence
generated more extensive tissue reactions in the more superficial parts of the skin (Fig. 2e,f). Furthermore, the

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Figure 3.  Nanosecond-domain Nd:YAG laser-induced tissue reactions. Single pulses of single flat-top beam,
532-nm nanosecond laser treatment at a spot size of 5.3 mm and the fluences of (a) 0.07 J/cm2, (b) 0.1 J/cm2,
(c) 1.3 J/cm2, and (d) 1.8 J/cm2. Additionally, single pulses of single flat-top beam 1,064-nm picosecond laser
treatment at a spot size of 6 mm and the fluences of (e) 0.36 J/cm2, (f) 1.4 J/cm2, (g) 2.8 J/cm2, and (h) 4.0 J/cm2.
Inlets depict close-up views of the basilar keratinocytes, melanocytes, and basement membrane. (d,h) Asterisks
indicate giant epidermal vacuoles. H&E stain, original magnification x200.

maximum depth of tissue reaction was significantly correlated with the power density (R = 0.961; P < 0.001).
Nonetheless, the differences in the maximum depth of tissue reaction and the size of epidermal giant vacuoles
between the wavelengths of 532 nm and 1,064 nm were insignificant (P > 0.05).

Nanosecond-domain Nd:YAG laser-induced tissue reactions.  Immediately after single respective

pulses of 532-nm nanosecond laser treatment at the fluences of 0.07 J/cm2, 0.1 J/cm2, and 1.3 J/cm2, all skin sam-
ples exhibited various degrees of perinuclear vacuolar changes and decreased melanin pigments in the epidermis,
without remarkable vacuolar tissue reactions in the dermis (Fig. 3a,c). Giant epidermal vacuoles and marked
dermal vacuolar tissue reactions were found at the fluence of 1.8 J/cm2 (Fig. 3d). However, dermal tissue reactions
were less extensive that those attained with 0.1-J/cm2 picosecond laser treatment.
A single pulse of 1,064-nm nanosecond laser treatment at the fluence of 0.36 J/cm2 generated mild perinuclear
vacuolar changes and decreased melanin pigments, without remarkable vacuolar tissue reactions in the dermis
(Fig. 3e). While varying degrees of epidermal vacuoles and mild dermal vacuolar changes were found in the
skin treated at the fluences of 1.4 J/cm2 and 2.8 J/cm2 (Fig. 3f,g), giant epidermal vacuoles and marked dermal
vacuolar tissue reactions were found at the fluence setting of 4.0 J/cm2 (Fig. 3h). However, dermal tissue reactions
generated with 4.0-J/cm2 nanosecond laser treatment were less extensive than those obtained with 1.4-J/cm2
picosecond laser treatment.

Microlens array-type 532-nm picosecond-domain laser-induced tissue reactions.  Immediately

after picosecond laser treatment at a wavelength of 532 nm using an MLA-type handpiece, the laser fluence of
0.04 J/cm2, and the distance step of 1 over one pass generated minimal perinuclear vacuolization in epidermal
cells with mildly dilated DMVCs (Fig. 4a). When increasing the distance step setting to 2 or 3, a slightly greater
number of vacuolated keratinocytes were seen in the higher layers of the epidermis, compared to step 1 (Fig. 4b,c).
Skin treated at a fluence of 0.5 J/cm2 exhibited fractionated zones of tissue injury that were composed of
signet ring-like keratinocytes and melanocytes, along with dilated DMVCs (Fig. 4d). The maximum depth of
laser-induced dilated DMVCs therein was estimated at 187.0 ± 25.7 µm. The 1.0 J/cm2-fluenced treatment also
demonstrated fractionated, but deeper and wider, zones of laser-induced tissue injury, compared to the fluence
of 0.5 J/cm2 (Fig. 4g). The maximum depth of laser-induced dilated DMVCs was estimated at 269.2 ± 58.8 µm.
Therein, the maximum depth of tissue reaction was significantly correlated with the power density (R = 0.989;
P < 0.001). Moreover, the distance steps of 2 and 3 at 0.5 J/cm2- and 1.0 J/cm2-fluenced settings generated wider
zones of epidermal reactions, compared to step 1 (Fig. 4e,f,h,i). Delivering an additional two passes at each setting
markedly accentuated laser-induced tissue reactions in wider areas of the epidermis and dermis, compared to
single pulse treatment (data not shown).

Microlens array-type 1,064-nm picosecond-domain laser-induced tissue reaction.  A single pass

of MLA-type 1,064-nm treatment at a fluence of 0.13 J/cm2 and at distance step 1 generated mild perinuclear vac-
uolization in epidermal cells, with decreased melanin pigments (Fig. 5a). More remarkable tissue reactions were
found in the upper part of the epidermis by regulating distance step from 1 to 2; step 3 exhibited similar histologic
features with those at step 1 (Fig. 5b,c). Furthermore, a fluence of 0.5 J/cm2 generated similar patterns and degrees
of laser-induced tissue reactions in both the epidermis and dermis, compared to treatment at 0.13 J/cm2 (Fig. 5d–f).
Nonetheless, DMVCs at 0.13 J/cm2- and 0.5 J/cm2-fluence settings were unremarkable or only mildly dilated.
Laser treatment with all distance step settings at 1.9 J/cm2 generated fractionated areas of vacuolated, signet
ring-like epidermal cells and surrounding areas of mildly to moderately vacuolated cells, with notable decreases
in melanin pigments (Fig. 5g–i). Remarkable vacuolar changes in DMVCs were found, although they were limited

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Figure 4.  Tissue reactions after 532-nm microlens array (MLA)-type picosecond-domain laser treatment.
Single pulses of MLA-type, 532-nm picosecond laser treatments at a spot size of 6 mm and fluences of (a–c)
0.04 J/cm2, (d–f) 0.5 J/cm2, and (g–i) 1.0 J/cm2. The distance settings between microlens and the surface of
the skin were (a,d,g) step 1 (31 mm), (b,e,h) step 2 (33 mm), and (c,f,i) step 3 (48 mm). H&E stain, original
magnification x200.

to only the uppermost papillary dermis (estimated depth, 144.7 ± 25.8 µm). Therein, the maximum depth of tis-
sue reaction was significantly correlated with the power density (R = 0.993; P < 0.001), and the maximum depth
of tissue reaction was significantly deeper in the wavelength of 532 nm, compared to that of 1,064 nm (P = 0.006).
Also, differences in the size of epidermal giant vacuoles between the wavelengths of 532 nm and 1,064 nm were
significant (P > 0.05). Delivering two additional passes, MLA fractionated laser-induced tissue reactions were
accentuated without excessive tissue injury (e.g., coagulation necrosis and disintegration of the skin integrity)
(data not shown).
A linear mixed model analysis was additionally performed to evaluate the effects of beam modes and wave-
lengths on the maximum depth of laser-induced microscopic tissue reaction. In doing so, statistically significant
differences in the maximum depth of tissue reaction were found between flat-top beam and MLA-type beams
(P = 0.002) and between the wavelengths of 532 nm and 1,064 nm (P = 0.003). No significant interaction between
beam modes and wavelengths (beam mode x wavelength), however, were noted in regards to the maximum depth
of tissue reaction (P > 0.05).

Horizontal sections of MLA-type laser-induced tissue reactions.  Horizontal sections of pigmented

micropig skin were obtained to evaluate histologic changes in the epidermal rete ridges and peri-rete ridge dermal
tissue after delivering picosecond laser treatment at a 1,064-nm wavelength, 10-mm spot size, 1.0-J/cm2 fluence,
and the distance steps of 1 and 3 over a single pass. At distance step 1, markedly vacuolated, signet ring-like kerat-
inocytes were found along the rete ridges (Fig. 6a). Additionally, numerous vacuoles with or without nuclei were
identified in the papillary dermis (Fig. 6a). The basal epidermis exhibited decreased pigment components with
numerous tiny vacuolar changes and the protrusion of vacuolated epidermal cells, including melanocytes, into
the papillary dermis (Fig. 6b). The histologic features in another area implicated the exit of epidermal vacuolated
cells by laser pulses from the rete ridges into the peri-rete ridge papillary dermis (Fig. 6c). At distance step 3, the
amount of pigment components decreased remarkably, compared to distance step 1 (Fig. 6d).

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Figure 5.  Tissue reactions after 1,064-nm MLA-type picosecond-domain laser treatment. Single pulses of
MLA-type 1,064-nm picosecond laser treatments at a spot size of 7 mm and fluences of (a–c) 0.13 J/cm2, (d–f)
0.5 J/cm2, and (g–i) 1.9 J/cm2. The distance settings between the microlens and the surface of the skin were
(a,d,g) step 1, (b,e,h) step 2, and (c,f,i) step 3. H&E stain, original magnification x200.

Discussion
In the present study, we microscopically analyzed the patterns of immediate tissue reactions generated by 532-
and 1,064-nm picosecond laser treatment ex vivo in genotype-regulated, pigmented micropig skin. Delivery of
high energy at a picosecond-pulse duration using particular lens array optics generates intraepidermal vacuoles
resulting from the absorption of laser energy by melanin pigments5,8. Accordingly, the use of genotype-regulated,
pigmented micropig skin would accentuate picosecond laser-induced tissue reactions, even with the delivery of a
single pulse of picosecond-domain laser energy at relatively low energy settings.
A previous study demonstrated that more and larger laser-induced vacuoles are generated in skin with a
higher melanin index when using higher energy settings8. In our study, tissue reactions after 532- or 1,064-nm,
single flat-top beam, picosecond-domain laser treatment also exhibited similar vacuolization of irradiated com-
ponents that contained chromophores of melanin and hemoglobin in the epidermis and dermis. We found that
the degree of vacuolization was greater with higher fluence settings and a 532-nm wavelength, presumably due to
greater absorption by both hemoglobin and/or melanin, despite higher scatter loss, at this wavelength compared
to 1,064 nm.
Theoretically, laser-induced tissue breakdown is initiated by the production of free electrons during the pico-
second laser pulse by one of several possible mechanisms. The free electron density increases during the laser
pulse to form a plasma by repetitive free electron generation5. This plasma is optically thick, virtually color blind,
and very efficient at absorbing energy in the remaining portion of the laser pulse. The plasma energy is then trans-
ferred to the tissue via electron-molecule collisions, and the plasma quenches after the laser pulse. The energy
transferred from the plasma to the tissue generates an increase in tissue and water temperature quickly enough to
create cavitation bubbles with large pressure gradients that generate vacuoles5.
The mechanisms for the generation of free “seed” electrons that are needed to begin the laser-induced tissue
breakdown is proposed to be multiphoton absorption or thermionic emission9. The irradiance threshold for
generating seed electrons via multiphoton absorption in water is ~1013 W/cm2 and is weakly dependent on the
absorption properties of the target tissue9. Meanwhile, thermionic emission has a lower irradiance threshold and
is much more dependent on the absorption properties of the target tissue9. The advantages of lowering the irradia-
tion threshold to generate laser-induced breakdown have been suggested to include reducing the risk of collateral
damage and increasing the penetration depth of the laser energy9. In our study, the power density for the single
flat-top beam and MLA-type microbeams was less than 1013 W/cm2 and elicited remarkable histologic features of

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Figure 6.  Horizontal sections of the ex vivo pigmented micropig skin. (a) A single pulse of MLA-type 1,064-
nm picosecond laser treatment at the fluence of 1.0 J/cm2 generated vacuolated, signet ring-like epidermal
cells along the rete ridges and numerous dermal components with vacuolar changes with or without nuclei.
(b) Reduced pigment components in the basal epidermis and protrusion of vacuolated epidermal cells into the
papillary dermis. (c) Exit of epidermal vacuolated cells from the rete ridges into the peri-rete ridge papillary
dermis. (d) Widely and homogeneously distributed vacuolated epidermal cells with remarkably decreased
pigment components. (a–c) Distance step 1 and (d) step 3. H&E stain, (a) original magnification x200, (b–d)
x400.

laser-induced cavitation in the epidermis and dermis in our ex vivo pigmented micropig model. Nonetheless, fur-
ther studies are needed to investigate the precise initiation mechanism of breakdown in our experimental model.
Given our observation of vacuoles together with chromophores in this study, we deemed that the initiation
of the electron plasma is not a color-blind process. The mechanisms that govern this basis (i.e., power density,
wavelength, and pigment absorption coefficient) determine how early in the laser pulse the plasma is initiated and
therefore how much of the remaining energy is available for plasma generation. Accordingly, the location, size,
and density of vacuoles should scale with the laser fluence and pigment concentration and inversely with pigment
depth (to account for beam energy loss due to tissue scatter). However, it should be noted that highly absorbing
plasma effectively blocks deeper laser beam penetration.
Fractional picosecond-domain lasers have been used for treating atrophic acne scars and wrinkles6,7,10,11.
Delivery of high-energy picosecond laser using fractionated optics produces high fluence areas, which are sur-
rounded by low fluence background areas5. The injury induced by the generation of intraepidermal laser-induced
breakdown theoretically stimulates the production of cytokines, chemokines, and growth factors from kerati-
nocytes for dermal remodeling7,12,13. In the present study, ex vivo micropig skin exhibited fractionated zones of
laser-induced tissue reactions in the epidermis and dermis. Therein, higher fluence settings created micro-injury
zones with greater tissue reactions and higher percent coverage at both 532- and 1,064-nm wavelengths.
Moreover, longer distance settings between the microlens and skin regulated the depth of tissue reaction in the
epidermis and dermis.
In conclusion, our microscopic findings of laser-induced tissue reactions using ex vivo pigmented micropig
skin demonstrated that 532- and 1,064-nm picosecond-domain Nd:YAG laser treatment generates vacuolated tis-
sue reactions in epidermal and dermal cells. MLA-type fractionated beam delivery generated fractionated zones
of vacuolated tissue reactions. Furthermore, the sizes (area) and degrees of picosecond laser-induced tissue reac-
tions could be regulated according to the wavelength, fluence, beam type, and distance between microlens and
skin. Although further clinical research using in vivo human skin is needed to confirm our findings, we believe
that our histologic investigation may help with predicting picosecond-domain laser-induced tissue reactions in
dark pigmented skin lesions.

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Figure 7.  Two- and three-dimensional characterization of the MLA-type beam. The beam profiles in (a)
two- and (b) three-dimensional images obtained by a complementary metal-oxide-semiconductor camera and
neutral optical filters using DataRay LCMvD23 software (DataRay Inc., Redding, CA, USA). Picosecond laser
treatment was performed using an MLA-type handpiece at a spot size of 10 mm, a fluence of 0.1 J/cm2, and a
distance setting of step 1.

Wavelength Spot size Fluence Power density

(nm) (mm) (J/cm2) (W/cm2)
0.05 1.11 × 108
532 5.3 0.1 2.22 × 108
1.3 2.89 × 109
0.18 4.00 × 108
1,064 6 1.4 3.11 × 109
2.8 6.22 × 109

Table 1.  Settings for picosecond-domain neodymium:yttrium-aluminum-garnet (Nd:YAG) laser treatment

using a single flat-top beam on the ex vivo pigmented micropig skin.

Methods
Laser devices.  A picosecond-domain 532- and 1,064-nm Nd:YAG laser device (PICOPLUS; Lutronic Corp.,
Goyang, Korea) with a pulse duration of 450 psec was used in this study. The pulse width of this device was con-
stant, regardless of output fluence, by adopting a master oscillator power amplifier configuration14. With appro-
priate optics, the laser energy can be delivered to target tissue as a single flat-top beam or an MLA-type beam
according to therapeutic purposes (Fig. 7). The distances between the microlens and the surface of the skin can
be regulated at 31 mm (step 1, microbeam size of 150 µm), 33 mm (step 2, microbeam size of 160 µm), and 48 mm
(step 3, microbeam size of 300 µm). Furthermore, a nanosecond-domain 532- and 1,064-nm Nd:YAG laser device
(SPECTRA XT; Lutronic Corp.) at a pulse duration of 5 nsec was used for the additional comparison experiments.

Preparation of ex vivo pigmented micropig skin and laser treatment.  All experimental protocols were
approved by the ethics committee of the Catholic Kwandong University Institutional Animal Care and Use Committee,
and the methods were carried out in accordance with the approved guidelines. Fresh back skin tissue was obtained
®
from a Yucatan, wild-type, black female micropig (8-month old, weighing 16 kg; MK Micropig ; Medi Kinetics Co.,
Ltd., Seoul, Korea), with a genotype of i/i (KIT *0101), E+/E+ (MC1R*1); the pig was sacrificed in a humane manner
according to standard protocols. A total of four skin samples of 10 cm × 10 cm in size was prepared, and the skin was
subsequently marked with black ink to outline 1-cm2 grids for each experimental setting (a total of 144 grids). Each grid
was placed at least 0.5 cm from the others to minimize laser-induced photothermal and photoacoustic effects on the
other areas. The temperature of ex vivo micropig skin was maintained between 34–36 °C on a heat plate.
Picosecond Nd:YAG laser treatments at the wavelength of 532 nm were performed separately on each grid at
treatment settings with a spot size of 5.3 mm and laser fluences of 0.05 J/cm2, 0.1 J/cm2, and 1.3 J/cm2 over a single
pass using a single flat-top beam handpiece (Table 1). Picosecond laser treatments at the wavelength of 1,064 nm
were then delivered at treatment settings with a spot size of 6 mm and laser fluences of 0.18 J/cm2, 1.4 J/cm2, and
2.8 J/cm2 over a single pass using a single flat-top beam handpiece. Meanwhile, 532-nm, single flat-top, nanosec-
ond Nd:YAG laser treatments were additionally performed at treatment settings with a spot size of 5.3 mm and
laser fluences of 0.07 J/cm2, 0.1 J/cm2, 1.3 J/cm2, 1.8 J/cm2 over a single pass using a single flat-top beam handpiece.
Additionally, 1,064-nm, single flat-top, nanosecond laser treatments were delivered with a 6-mm spot size and
laser fluences of 0.36 J/cm2, 1.4 J/cm2, 2.8 J/cm2, and 4.0 J/cm2 over a single pass.

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Power density of microbeam (W/cm2)

Wavelength Spot size Average Average power
(nm) (mm) fluence (J/cm2) density (W/cm2) Step 1 Step 2 Step 3
0.04 8.89 × 107 4.45 × 109 3.91 × 109 1.11 × 109
532 6 0.5 1.11 × 10 9
5.56 × 1010
4.89 × 1010
1.39 × 1010
1.0 2.22 × 10 9
1.11 × 1011
9.78 × 1010
2.78 × 1010
0.13 2.89 × 10 8
1.06 × 1010
9.33 × 109
2.65 × 109
1,064 7 0.5 1.11 × 109 4.08 × 1010 3.59 × 1010 1.02 × 1010
1.9 4.22 × 1010 1.55 × 1011 1.36 × 1011 3.88 × 1010
1,064 10 1.0 2.22 × 109 4.00 × 1010 NA 1.00 × 1010

Table 2.  Settings for picosecond-domain Nd:YAG laser treatment using an MLA-type beam on the ex vivo
pigmented micropig skin. NA, not applicable.

Using an MLA-type handpiece, picosecond laser treatments at the wavelength of 532 nm were performed with
a spot size of 6 mm and laser fluences of 0.04 J/cm2, 0.5 J/cm2, and 1.0 J/cm2 over a single and three passes and the
distance steps of 1, 2, and 3 (Table 2). MLA-type laser treatments at the wavelength of 1,064 nm were delivered at
treatment settings with a spot size of 7 mm and laser fluences of 0.13 J/cm2, 0.5 J/cm2, and 1.9 J/cm2 over one and
three passes and the distance steps of 1, 2, and 3. To obtain horizontal micropig skin sections, MLA-type laser
treatments were additionally delivered at a 1,064-nm wavelength, 10-mm spot size, and 1.0-J/cm2 fluence over a
single pass and the distance steps of 1 and 3. All experiments were performed in triplicate.

Histological examination.  Micropig tissue samples for each treatment setting were obtained 30 min after
treatment, collecting the epidermis, dermis, and subcutaneous fat. The tissue samples were fixed in 10% buffered
formalin and embedded in paraffin. Then, approximately 20 to 30 serial tissue sections, which were cut along the
longitudinal plane at a thickness of 5 μm for each condition, were prepared and stained with hematoxylin and
eosin. Additionally, 5-µm thick horizontal micropig skin sections were serially obtained and stained. The maxi-
mum depths of laser-induced tissue reactions were measured from the uppermost layer of the epidermis, except
for the stratum corneum, to the deepest parts of laser-induced vacuolar tissue reactions in the dermis using Image
J software (Version 1.48; National Institutes of Health, Bethesda, MD, USA).

Statistical analysis.  Values are presented as a mean ± standard deviation unless otherwise noted.
Independent two-sample t test, chi-squared test, Fisher’s exact test, and Pearson correlation analysis were per-
formed by parametric criteria using SAS software (Version 9.2; SAS Institute, Inc., Cay, NC, USA). Additionally,
the results were analyzed via linear mixed models with Bonferroni post hoc analysis. Differences with P values of
less than 0.05 were considered statistically significant.

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Acknowledgements
We would like to thank Anthony Thomas Milliken, ELS, at Editing Synthase (https://editingsynthase.com) for
his help with the editing of this manuscript and Hye Sun Lee, PhD (Biostatistics Collaboration Unit, Yonsei
University College of Medicine, Seoul, Korea) for her help with the statistical analyses. This study was supported
by research funding from Lutronic Corporation. The funding company had no role in the study design, data
collection, data analysis, manuscript preparation, or publication. The authors have indicated no significant
conflicts of interests in relation to commercial supporters.

Scientific Reports | (2019) 9:4186 | https://doi.org/10.1038/s41598-019-41021-7 9

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Author Contributions
H.L., J.C., J.P., J.H., and S.B.C. conceived the method. J.P., J.H., and S.B.C. performed experiments. J.C., H.J.C.,
J.P., and S.B.C. analyzed the data. H.L., J.C., H.J.C., and S.B.C. wrote the manuscript. H.L., J.C., and S.B.C. guided
experiments and data analysis.

Additional Information
Competing Interests: The authors declare no competing interests.
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