3. Biodistribution of needle-injections and needle-free jet-injections visualized by a 3D- Fluorescent Imaging Cryomicrotome System 77 3. preserved in any preservative fluid, or (3) the patient had objected to the use of discarded material for scientific purposes. The keloids were obtained from patients who underwent elective keloid excision and adjuvant radiotherapy (Department of Plastic Surgery Amsterdam UMC, Department of Dermatology Erasmus Medical Centre, The Netherlands). Normal skin was obtained from patients who underwent abdominoplasty (Department of Plastic Surgery, Jan van Goyen Medical Center, The Netherlands). After removing excessive subcutaneous fat, the keloids and normal skin tissues were stored at -80 ⁰C or at -20 ⁰C, the latter for a maximum of 6 months. Experiments Prior to the experiments, the normal skin and keloid samples were thawed to room temperature, fixed under mild tension, kept moist with wet gauzes, and marked with 1.5 x 1.5 cm zones. All experiments were conducted in triplicate, by a dermatologist experienced in ICA in keloids (AW). For the experiments with ‘conventional’ needle, a 25-gauge needle and 1 mL syringe were used to administer 100 µL fluorescent-labeled TCA 40 mg/mL per sample. For the experiments with the jet injector, a needle-free jet injector (Enerjet 2.0, Perfaction, Rehovot, Israel) was used to administer 100 µL (device range: 50-130 µL) fluorescentlabeled TCA 40 mg/mL with pressures of 4, 5 and 6 bar (device range: 2-6 bar) per sample. All jet-assisted injections were administered perpendicularly in the center of the sample. Image acquisition The biodistribution of fluorescent-labeled TCA suspension was visualized using the 3DFICS (Figure 1). Firstly, all samples were embedded in a 3% carboxymethylcellulose with black ink to reduce background signaling. All samples were sectioned vertically into slices of 48 µm thickness. After each section, images were taken from the remaining bulk with a camera with an in-plane resolution of 13.66 x 13.66 µm. Prior to the experiments, wavelengths and exposure times were optimized using test samples. Eventually, a 595 nm excitation and 620 nm emission wavelength with an exposure time of 500 ms were used
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