A femtosecond laser at 1,030 nm (pulse width: 200 fs, repetition rate: 1 MHz) was coupled into a microscope system to perform micromachining on live mice (Figure 1A). The laser beam was expanded to match the back aperture of the objective (×20, N.A. = 0.65) and then focused by it onto the mouse skin at around 50–100 μm deep. The laser focus was around 1 μm in diameter. The micromachining was accomplished by the scanning of the galvo-mirror that could be predefined by any region on the skin of mice. In this study, a rectangular area of 5 mm × 0.64 mm at the mouse back was scanned line by line by the laser. Each line was 0.64 mm, defined by 640 points. Each point was exposed to the laser for 200 µs. The area was finally scanned by 5,000 lines as a trial. The laser power was used at 0.2 W–1 W at the skin surface. After experiments, the mice were sacrificed and the skin tissue were dissected for analysis.

For in vivo experiments, 6- to 9-weeks-old Kunming mice were anesthetized with 4% chloral hydrate. Before experiments, the hair was removed by hair removal cream (Veet Hair Removal Cream, Reckitt Benckiser Ltd.). After the surgical level of anesthesia was reached, the skin was treated by the femtosecond laser. After that, the skin tissues were removed and fixed in 4% paraformaldehyde. All experimental procedures regarding the care and use of SD rat pups were reviewed and approved by the Animal Ethics Committee of Shanghai ninth People’s Hospital and the School of Biomedical Engineering, Shanghai Jiao Tong University. All efforts were made to minimize possible pain and discomfort of the animals during the experimental procedures.

Paraffin-embedded skin biopsies were cut into 5 µm sections and placed on glass slides. Slides were placed in a 60°C oven overnight, deparaffinized with xylene, then rehydrated with serial ethanol dilutions. The tissue section was placed in a repair box filled with EDTA antigen retrieval buffer (PH 8.0) in a microwave oven for antigen retrieval and were blocked using 10% rabbit serum. Sections were incubated with primary antibodies overnight at 4°C. Sections were double-stained for immunofluorescence of TNF-α (rabbit polyclonal anti-TNF-α, 1:200, Affinity Biosciences, AF7014) and IL-17 (mouse monoclonal anti-IL-17, 1:200, Santa Cruz, sc-374218), MMP3 (mouse monoclonal anti-MMP3,1:200, Proteintech, 66338-1-lg) and MMP9 (rabbit polyclonal anti-MMP9, 1:200, Proteintech, 10375-2-AP), and IL-6 (rat monoclonal anti-IL6R, 1:100, Abcam, ab83053) and TGF-β (rabbit polyclonal anti- TGF-β, 1:200, Proteintech, 21898-1-AP). Then the sections were washed with PBS for 5 min and incubated with diluted secondary antibody for 1.5 h at room temperature. Goat anti-Rat IgG secondary antibody conjugated with FITC (1:200, Servicebio), Goat anti-Mouse IgG secondary antibody conjugated with Alexa Fluor ^® 488 (1:400, Servicebio) and Goat anti-rabbit IgG secondary antibody conjugated with Cy3 (1:300, Servicebio) were used for microscopy. Sections were simultaneously counterstained with DAPI before fluorescent microscopy.

Images were acquired by a confocal microscopy system (Olympus FV3000). The power of three excitation lasers at 405 nm, 473 nm, and 543 nm was set at the level of 0.1 mW, which could excite fluorophores effectively with moderate photobleaching. The image size was set as 512 × 512 pixels and time interval for each pixel was 2 µs.

We investigated the responses of immune molecules in skin tissue to femtosecond laser micromachining at different powers. To eliminate the thermal accumulation between laser pulses, the pulse repetition rate was set at only 1 MHz. During the micromachining, the transient thermal effect of each femtosecond pulse can decay before the subsequent following pulse. In this regard, the average total thermal effect of the laser was little. At first, the skin tissue scanned by the focused femtosecond laser at 0.8 W line by line was dissected. The immunofluorescence microscopy of IL-17, TNF-α, IL-6, and TGF-β in the histosections of the tissue were performed respectively. Immediately it could be found TNF-α was significantly assembled in the epidermis (Figure 1B) compared with the control (Figure 1C). The immune molecules might come from the keratinocytes in epidermis that suffered the laser stimulation. No signals from IL-17, IL-6, or TGF-β that are usually involved in T cells could be found, indicating the acute response of the skin activated by the focused laser was only unspecific innate immune responses at epidermis (Figure 1D).

Specifically, we examined the matrix metalloproteinase 3 (MMP3) and 9 (MMP9) that are involved in tissue remodeling, wound healing, and breakdown of extracellular matrix proteins including collagen and elastin. As shown in Figures 1B,D, the MMP3 and MMP9 were both significantly upregulated in both epidermis and dermis, indicating the remodeling initiation of the laser-treated skin. The thickness of the skin with those molecular distribution was quantified in Figure 1E. Notably, MMP3 could be found highly in epidermis. In those processes, no physical or physiological damage could be found in the histosections. Still, the focused femtosecond laser could activate the collagen or elastin repair. This result suggests the upregulation of MMP family does not require physical injury by optical micromachining to collagen or elastin. The low level of TGF-β under this condition was consistent with this suspicion.

We then measured the molecular responses to the laser stimulation at different powers. As shown in Figures 2A,B, the TNF-α in epidermis increased moderately at 0.4 W but no signals could be found at 0.2 W. Compared with the control (Figure 1C), no significant differences could be found in IL-6, IL-17, or TGF-β (Figure 2D). The TNF-α level treated at 0.4 W was still significantly higher (Figure 2E). Consistently, the upregulation of MMP3 and MMP9 was also significantly higher at 0.4 W but no signals at 0.2 W (Figure 2E). The thickness of the skin stained with MMP9 and MMP3 signals was also consistent with the result of immunofluorescence (Figure 2F). Therefore, the immune process initiation requires a laser power threshold. At lower laser powers, cells deep in dermis could not be activated for MMP3 and MMP9 expression.

We investigated the long-term molecular responses of skin, in which the mice recovered 1 day after the laser micromachining at 0.8 W. The skin was then dissected for immunofluorescence microscopy, as shown in Figure 3A. The TNF-α level decreased significantly compared with the acute responses, indicating an anti-inflammatory and repair process of skin tissue. But it was still significantly higher than it of control (Figures 3B,C). No signals from IL-17, IL-6, or TGF-β were found either (Figures 3A,C). Therefore, the tissue damage was strictly confined in the laser scanning volume and only innate immune responses were activated. The expression level of MMP3 and MMP9 were still significantly high (Figure 3C). The thickness of the skin with positive MMP3 and MMP9 was consistently higher (Figure 3D). During the repair, the irradiated epidermis initiated cornification to detach from the skin, together with MMP3. Hence, taken the acute MMP expression together, MMP3 might be involved in the remodeling of both dermis, epidermis, and specifically, keratoderma. In general, the focused femtosecond laser could effectively activate the remodeling of collagen/elastin without complex immune responses of skin.

As a comparison, the mice were irradiated by the femtosecond laser at 1 W directly without any focusing for 15 s. The laser spot was around 2 mm in diameter. In this case, the laser could propagate into skin in a deeper distance while the photon density was lower (10⁶ fold lower). As shown in Figure 4A, no obvious physical damage could be found in those skin tissues. The immunofluorescence of TGF-β, IL-17, and IL-6 were all significantly higher (Figure 4B). The TGF-β generally distributed in the epidermis, suggesting the physical damage to skin. IL-6 was upregulated through the whole skin tissue and the muscle, fat under it. But the signals from TNF-α, MMP3, and MMP9 all decreased significantly (Figure 4C), indicating the tissue remodeling might not be initiated. The collagen and elastin repair was not activated either.

To compare with the clinic treatment, a widely-used picosecond laser in clinic (PicoSure, Cynosure) at 755 nm was used to treat skin of mouse. The laser spot was 2.5 mm in diameter and pulse width 750 ps. The micromachining was performed at 0.8 W (4.07 J/cm²) for 6 s. As presented in Figures 5A,B, the signals from TNF-α, TGF-β, IL-17, and IL-6 were all significantly higher. Unfortunately, the MMP3 and MMP9 did not show any increase (Figure 5C). Hence the skin suffered mostly damage with little remodeling or repairing.