Dm6000m

A preliminary ablation threshold study is conducted in order to determine the optimum wavelength for the femtosecond laser-based microstructuring of sapphire. Therefore, the planar substrates are locally irradiated with different wavelengths. While pulse duration and repetition rate are kept constant with values of 230 fs and 100 kHz, each emission wavelength features a different focal spot diameter. For wavelengths of 1030 nm, 515 nm, and 343 nm, the respective focal spot sizes are determined experimentally as 36 µm, 21 µm, and 20 µm. By adapting the laser’s average output power, the resulting peak fluence within the focal spot is varied. The diameter of the induced ablation area is then evaluated via bright-field microscopy (DM6000M, Leica, Wetzlar, Germany). Theoretically, the squared diameter d² of the ablation area correlates to the pulse peak fluence F according to
$${\mathrm{d}}^2=2{\mathsf{\omega }}_0^2\ln \left(\frac{\mathrm{F}}{{\mathrm{F}}_{\mathrm{th}}}\right),$$
whereas ${\mathsf{\omega }}_0$ represents the focal spot radius while ${\mathrm{F}}_{\mathrm{th}}$ is the ablation threshold fluence. Thus, it is possible to quantify the ablation threshold by plotting the squared diameter of the ablated areas as a function of the applied laser fluence in a semi-logarithmic manner. The ablation threshold is then determined by the respective linear least square fit function’s abscissa intersection [36 (link),37 (link)].

The β-galactosidase staining kit was used, which purchased from Genmed Scientifics Inc., USA(GMS10010.1). According to the experimental requirements, the Leydig cells were cultured primarily using Petri dishes. After the culture medium was aspirated, the cells were washed twice with 0.1 M PBS, and fixed using 1 mL β-galactosidase fixative for 15 minutes. Then, the fixative was aspirated, and the cells were washed with 0.1 M PBS three times for 3 minutes each. β-galactosidase dye working solution was added, 1 mL per well, along with 10 μL A solution, 10 μL B solution, 930 μL C solution, and 50 μL X-Gal solution. Cells were incubated at 37°C overnight. The six-well plates were covered with plastic wrap to prevent evaporation. Then, the working fluid was removed. Under the optical microscope (Leica DM 6000 M, Germany), the cytoplasm of β-galactosidase positive cells was blue. The number of blue cells out of 200 cells was observed and recorded. The experiments were repeated five times for robust statistical analysis.

Immunohistochemical staining was performed on 5-μm sections of paraformaldehyde- fixed, paraffin-embedded tumor samples, adjacent nontumoral liver tissue, normal liver tissue and mouse lung tissue. The details of the immunohistochemistry were described previously [28 (link)]. The slides were incubated with mouse anti-SIRT1 (Abcam), rabbit anti-TFAM (Abcam), rabbit anti-COXIV (Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-TOMM20 (Abcam) primary antibodies. The mouse lung tissue sections (5-μm thick) were stained with hematoxylin and eosin (H&E) so that tumor nodules could be observed. The slides were evaluated under a light microscope (Leica, DM6000M).

Mice were sacrificed on day 16, and the tumors and major organs were excised and fixed in 4% formalin for histology analysis. The tissues were subject to hematoxylin and eosin (H&E) staining using standard protocols and observed under a digital microscope (Leica DM6000M).

Fixed tissues were embedded in paraffin and were sectioned at a thickness of 3μm, and immunohistochemical staining was performed with antibodies to E-cadherin (abcam, ab76055, 1:100), HOXC10 (abcam, ab153904, 1:100), N-cadherin (abcam, ab76057, 1:100), Vimentin (abcam, ab8978, 1:100), DVL2 (abcam, ab137528, 1:100) and Wnt10B (abcam, ab70816, 1:100) at 4°C overnight. The sections were mounted in a mounting medium containing glycerol (Beyotime, P0126) and visualized with a light microscope (Leica, DM6000M).

Mice of control and treated groups were sacrificed after photodynamic therapy. Tumors and major organs (heart, liver, spleen, lung, and kidney) were collected and kept in 4% formalin followed by embedded in paraffin. Slices cut from the paraffin sections were stained by hematoxylin and eosin (H&E) before scanned with a fluorescence microscope (Leica DM6000M). Blood of mice was collected intravenously post-injection with PBS and IPA/LDH at day 1 and day 7. A standard biochemical procedure was carried out for the separation and analyzation of blood.