2025-0607_IN2SIGHT_IMAGES_UNIMIB_TPE_exvivo

Description

This is a set of images taken on In2Sight devices coupled to PVC (polyvinylchloiride), implanted in mice. -- Animal implants. Each mouse (BALB/cAnNHsd, Envigo) received one In2Sight device, which was implanted subcutaneously in the inner part of the hind leg. Mice were anesthetized intraperitoneally with 100mg/kg ketamine and 4mg/kg xylazine. The operating field was prepared by hair trichotomy followed by appropriate disinfection with 70° ethanol or betadine. Two incisions were then made using a sterile scalpel. After positioning the device, a tight suture (single stitch) of the incision was performed using a sterile 5/0 absorbable suture thread. For the first 2 days, pain control was foreseen by administering opioids (Buprenorphine hydrochloride 0.16mg/kg subcutaneously) and infections avoided by administering an antibiotic (Enrofloxacin 5 mg/kg for 5 days). All the implants were done under the animal permit n. 551/2023-PR of the Italian ministry for health. The device, once coupled with acrylic glue to a slab of PVC (Polyvynil chloride, 5 mm in diameter, 0.3 mm thickness), was sterilized by STERRAD before the implant that lasted up to 6 weeks. The mice were kept in a pathogen‐free conventional animal house facility. The animal house is run by professional employees fully equipped with state‐of‐the‐art instrumentation in order to maintain the standard of animal welfare at the maximum levels. All mice were housed in individual, ventilated cages with 12 h light/dark cycles with food and water ad libitum. The mice were euthanized at the end of the implant by cervical dislocation right before the imaging session. Experiments were performed using protocols approved by the Institutional Animal Care and Use Committee of the University of Milano‐Bicocca and by the Italian Ministry of Health. -- Chemicals. Hoechst 33342 (Sigma-Aldrich Chemical Company (Steinheim, Germany)) was used to stain nuclei in ex-vivo experiments. -- Multiphoton imaging. Third harmonic generation and 3-photon excitation fluorescence were selected by the Semrock filters FF02-447/60 and FF562-Di03-25, respectively. For SHG the exc. wavelength was 800 nm (emission 400/40 nm). λexc was 770 nm in case of Hoechst nuclei staining (485/30 nm). Auto-fluorescence in the green channel was collected at 535/50 nm. FILE NAME FORMAT for folder TPE_SHG_6weeks: name@800P140mWBP400BP535z200um50piani_Ch2 is taken as an example. name: if "lente#" (#=1,..4) means one of the 4 lenses on top of the microatlas; if "spacer" means the In2Sight spacers; if "fralenti" means the space between the lenses and therefore in the free tissue 800P140m: excitation wavelength = 800 nm, average power = 140 mW, WBP400BP535z200um50piani: means emission filters at 400 nm (Ch1) and 535 nm (Ch2); 200 micrometer full depth of stack, 50 layers. FORMAT in THG_3PE_data_1week: modality, exc. , through the lenses or control, FOV in um, image size in pixels, the depth, the number of averages (#avg).

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• University of Milano-Bicocca

  Lombardy, Milan

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