Macroscopic laser-tissue irritation evaluation
For establishing a reference for skin irritation, healthy skin was exposed to the same laser parameters as the laser-treated areas (Figs. 2 and 3). Areas irradiated with 532 nm wavelength were more severe using the 2 mm spot size beam, that produced intense and localized erythema. Barely perceptible edema increased in severity past 75 mJ pulse energy and became moderate at 120 mJ (Fig. 2S-2). In comparison, the irradiation effect with 4 mm spot size was milder, resulting in a well-defined erythema and edema only at 120 mJ, and the erythema that was developed by the pulse energy of 75 and 120 mJ had a uniform distribution (Fig. 2S-4).
Dermatoscopic evaluation of untattooed skin immediately following treatment with 1064 nm laser therapy.
The laser treatment on healthy skin using the 1064 nm wavelength displayed almost no indications of laser-induced patterns at 30 mJ, but a rapid increase in erythema was observed past 95 mJ with the 2 mm spot size, developing a severe form (Fig. 2S-2). Areas treated with the 4 mm spot size up to 95 mJ pulse energy were comparable to healthy skin. Sporadic petechia formation (< 1 mm) was observed in areas treated with 155 mJ using the 4 mm spot size (Fig. 2S-4).
The degree of tissue irritability was assessed to ascertain how various laser parameters affected different colored tattoos. The erythema and edema for all areas were overall reduced (Fig. 4B) using a 4 mm spot size at a 532 nm wavelength in comparison to the treatment using a 2 mm spot size (p = 0.054) (Fig. 4A). The severity of tattoo removal, as measured by the outcome of treatments utilizing 75 mJ (p = 0.011) and 120 mJ (p = 0.041) energy levels and matching pigment color, was found to be higher when using a 2 mm spot size compared to a 4 mm spot size.
Assessment of tissue irritation immediate post-laser treatment. Laser treatment parameters: (A) wavelength of 532 nm and 2 mm spot size; (B) wavelength of 532 nm and 4 mm spot size; (C) wavelength of 1064 nm and 2 mm spot size; (D) wavelength of 1064 nm and 4 mm spot size.
When utilizing a 2 mm spot size, pulse energies of 75 to 120 mJ, and a 532 nm wavelength, black tattoos exhibited the most pronounced response to laser treatment, resulting in moderate to severe edema (Fig. 4A). The laser-tissue reactions elicited by 532 nm wavelength were found to be the mildest in red and green tattoos, with only moderate edema observed even at the highest pulse energy levels. The analysis of the data presented in Fig. 4B revealed that, apart from black tattoos, all laser-treated areas subjected to irradiation with 120 mJ pulse energy using a 4 mm spot size resulted in comparable or reduced irritation scores when compared to the control skin.
The skin irritation scores of the areas treated with 1064 nm wavelength were significantly higher (p – 0.037) after treatment with 2 mm spot size compared to 4 mm spot size (Fig. 3C, D). The use of a 2 mm spot size at both 30 mJ and 155 mJ pulse energies resulted in a significantly higher irritation score compared to the 4 mm spot size. Specifically, at 30 mJ pulse energy, the 2 mm spot size resulted in a 2.7-fold increase in irritation score, primarily due to the presence of edema. Similarly, at the highest pulse energy level of 155 mJ, the use of a 2 mm spot size led to a 1.9-fold higher irritation score (p = 0.013) compared to the 4 mm spot size, as shown in Fig. 3D.
The overall irritability of the treated areas was found to be highest in black, blue, and yellow tattoos, due to lower edema severity at pulse energies ranging from 30 to 95 mJ with a 2 mm spot size (Fig. 4). Conversely, the treatment of red and green tattoos resulted in the least irritation compared to the other colors, due to a slight reduction in edema formation.
Tattoo clearance evaluation
Different wavelengths of laser light have varying degrees of effectiveness in tattoo removal. The clearance rates demonstrated a positive correlation with pulse energy. When comparing the clearance rates achieved using different wavelengths, it is observed that the 1064 nm wavelength produced lower clearance rates compared to the 532 nm wavelength, with the exception of black tattoo (Fig. 5).
Dermatoscopic view of laser-treated tattoos 30 days post third treatment. For 532 nm wavelength a pulse energy of 75 mJ was used and for 1064 nm – 95 mJ. Tattoo color—(A–E). Spot size (S)—2- and 4-mm. Treatment wavelength (λ)—532/1064 nm.
The highest clearance rate for black tattoos was achieved using a 2 mm spot size at 1064 nm wavelength and 155 mJ pulse energy, resulting in a rate of 76.46% (Fig. 6C). Conversely, the least effective treatment, which only achieved a clearance rate of 16.41%, was observed when using a 4 mm spot size at 532 nm wavelength and 25 mJ pulse energy (Fig. 6B).
Assessment of tattoo clearance 150 days post-treatment. Laser treatment parameters: (A) wavelength of 532 nm and 2 mm spot size; (B) wavelength of 532 nm and 4 mm spot size; (C) wavelength of 1064 nm and 2 mm spot size; (D) wavelength of 1064 nm and 4 mm spot size.
The most effective treatment for removing blue, green, red, and yellow tattoos utilized a beam size of 2 mm, a wavelength of 532 nm, and an energy of 120 mJ (Fig. 6A). After three laser treatments, yellow and green tattoos exhibited the highest clearance rates, achieving 71.87% and 71.69%, respectively. Blue tattoos were the third most effectively treated, with a clearance rate of 65.04%, followed by red color tattoos, with a rate of 48.96%. The lowest clearance for blue, green, red, and yellow color tattoos was observed after treatment utilizing the 4 mm spot size at 1064 nm wavelength and 30 mJ pulse energy, achieving 20.24%, 18.74%, 5.15%, and 10.20% respectively (Fig. 6D).
Microscopic tattoo removal evaluation
The histological characteristics of the epidermis in untreated and laser-treated tattoo samples were evaluated. The epidermis maintained its characteristic architecture after treatment with various laser settings. Furthermore, no extracellular ink particles were detected in the epidermis, even in the accumulations of necrotic tissue that formed in the stratum corneum of the epidermis.
The skin tissue slides stained with hematoxylin and eosin (HE) showed variations in ink color of non-laser treated areas, with black tattoos appearing black, blue tattoos displaying a range from dark blue to black with a bluish border, green tattoos exhibiting a range from dark green to black with a greenish border, red tattoos showing a range from dark red to black with a reddish border, and yellow tattoos presenting a heterogeneous black color with multiple pigment granules (Fig. 7).
Histological analysis of pre-treatment tattoo pigments: control samples 150 days after tattooing. HE-stained sections of pigment clusters in different colored tattoos: (A) black tattoos; (B) blue tattoos; (C) green tattoos; (D) red tattoos; (E) yellow tattoos.
Ink particles were observed in the dermis up to a depth of 1.2 mm, with the highest concentration locating within 100—600 μm from the skin surface (Fig. 8A1–2). The majority was concentrated in clusters distributed in mononuclear infiltration, comprising mainly of macrophages surrounding dilatated blood vessels (Fig. 8). The cytoplasm of phagocytic cells contained large pigment deposits, which showed a high degree of color saturation and homogeneity, except for yellow tattoos.
Histological assessment of black tattoos 150 days post-laser treatment with 532 nm wavelength and 120 mJ pulse energy: (A1) control (untreated) tattoo; (A2) black pigment cluster in untreated tattoo; (B1) laser-treated tattoo (2 mm spot size); (B2) pigment particles in laser-treated tattoo (2 mm spot size); (C1) laser-treated tattoo (4 mm spot size); (C2) pigment particles in laser-treated tattoo (4 mm spot size).
The study showed that 532 nm wavelength at the 4 mm spot size only reached up to 250 μm (Fig. 8B1–2), while the 2 mm spot size was capable of achieving pigment fragmentation up to 400 μm (Fig. 8C1–2). Reduction in overall pigment quantity and the transformation of large intracellular pigment deposits into considerably smaller particles containing granules resulted in a lightening of the treated areas. By increasing the laser pulse energy from 75 to 120 mJ, the fragmentation effect was significantly enhanced, as demonstrated by the tattoos’ clearance (Figs. 5 and 6).
Laser treatment at 532 nm wavelength produced detached melanin‐containing parakeratotic mounds or microscopic epidermal necrotic debris (MEND) that reside in the upper levels of the SC in sub-granular position. The laser beam spot size, pulse energy, and pigment characteristics affected the size variation of MENDs. The treatment with a 2 mm spot size resulted in the formation of MENDs in blue, green, and yellow tattoos, especially past 75 mJ pulse energy. The largest MENDs were observed in yellow tattoos, measuring up to 1.5 mm in diameter. In blue tattoos, the detached necrotic tissue was measured up to 500 μm, and in green tattoos—up to 300 μm. MENDs were not observed in black and red tattoos, and they were not observed in tattoos of any color treated with a 4 mm spot size. Vascular dilatation of superficial capillaries was observed throughout the samples treated with 532 nm wavelength and both spot sizes. Using the 2 mm spot size the dilatation was observed up to 1.5 mm in the dermis and using the 4 mm—up to 500 μm (Fig. 8).
The application of a 1064 nm wavelength for tattoo removal was particularly effective in decreasing the amount of pigment deposits in black tattoos (Fig. 5), especially when a 2 mm spot size was used (Fig. 9B1). The total black pigment amount in the samples treated with both beam sizes were lower than in the untreated areas (Fig. 9A1–2), which positively correlated with pulse energy past 95 mJ using a 4 mm spots size and 30 mJ with 2 mm spot. Treatment up to 95 mJ with the 1064 nm laser using a 4 mm spot size affected mostly the superficial layer of the black pigment clusters up to 300 μm, leaving the lower layers visually comparable to control tattoos (Fig. 9C1–2). Fragmentation of pigments throughout their distribution depth was observed to be successful after irradiation with a 2 mm beam width at all energy levels, and with a 4 mm beam width from 155 mJ pulse energy (Fig. 9B1–2).
Histological assessment of black tattoos 150 days post-laser treatment with 1064 nm wavelength and 155 mJ pulse energy: (A1) control (untreated) tattoo; (A2) black pigment cluster in untreated tattoo; (B1) laser-treated tattoo (2 mm spot size); (B2) pigment particles in laser-treated tattoo (2 mm spot size); (C1) laser-treated tattoo (4 mm spot size); (C2) pigment particles in laser-treated tattoo (4 mm spot size).
Our observations indicate that colored tattoos exhibited fragmentation to some degree throughout the entire range of pigment distribution. In addition, we noted a slight dilatation of superficial capillaries up to 300 μm, along with a significant infiltration of mononuclear cells in all areas containing pigment. MENDs were only observed in histological samples of blue, green, and yellow tattoos treated with 2 mm spot size. The highest MEND formation was observed in blue and yellow tattoos up to 1.5 mm in diameter past 95 mJ, while in green tattoos the detached necrotic tissue measured up to 200 μm.