## 2 Materials and Methods
### In Vivo--Feline Case Study  
A 14-year-old male domestic shorthair cat was referred to the veterinary center for removal of the mast cell tumor (MCTs) localized under the metatarsal pad of the left hind limb (Figure 1). The owner refused radical excision; therefore, alternative treatment with surgical excision of the mass supported by intraoperative ECT and removal of the sentinel lymph node was proposed. The owner signed the agreement consent for the proposed treatment. The study was approved based on consent issued by the Bioethical Committee of Wroclaw University of Environmental and Life Sciences (Wroclaw, Poland) no. 49/2020 and 50/2020 (valid for five years). Surgery was conducted under short-infusion anesthesia and epidural analgesia. The cat was given dexmedetomidine (Dexdomitor, Orion Pharma, Espoo, Finland) at a dose of 40 mcg/kg intramuscularly in premedication. General anesthesia was induced using propofol (Propofol-Lipuro 1%, B. Braun Melsungen AG, Melsungen, Germany) until the desired level of unconsciousness was achieved. The cat was intubated with a cuffed endotracheal tube (internal diameter 4.0 mm) and was breathing spontaneously with oxygen-enriched air. Additional propofol was administered as a bolus of 1-2 mg/kg when needed. The cat was clipped and washed to allow visualization of the suspected draining tracts, and the surgical field was routinely disinfected. The visualization of the BLM-ICG fluorescence was conducted with VS3 Iridium(tm) (Philadelphia, PA, USA). The excitation source is a laser tuned in the NIR range (\(\lambda_{\text{exc}}\) = 805 nm, and \(\lambda_{\text{em}}\) = 835 nm) with an incident excitation power of 11 mW/cm\({}^{2}\) at 30 cm from the tissue (Figure 1b,c). The level of the infrared signal (IR) captured by the camera from the surgical field was visible on the screen as a scale of IR intensity. The absolute intensity of the IR signal was visible at a given point (Base Point) on the screen ranging from 0 to 255 (Figure 2). The relative intensity of the IR signal at user-specified points was calculated by the system with reference to the base point (in %). The tumor mass was injected with bleomycin (Bleomedac, medac GmbH, Germany) at a dose of 1 mL/cm\({}^{3}\) of the tumor mass mixed with 0.1 mL of indocyanine green 5 mg/mL (Verdye, Diagnostic Green, GmbH, Kirchheim bei Munchen, Germany) and exposed to the ESOPE protocol. Figure 1: Visualization of the tumor: (**a**) Tumor mass before the ECT treatment, (**b**) tumor mass after infiltration with BLM-ICG, (**c**) tumor mass after infiltration with BLM-ICG with fluorescence green overlay. Figure 2: The screenshot from the NIRF imaging after ECT treatment shows the fluorescence of ICG in three modalities—IR, color fused, and green overlay, and the scale with strength of the IR signal. For pulse delivery, the ELECTROcell B15 (Leroy Biotech, Saint-Orens-de-Gameville, France) and an applicator with a contact L-shape electrode 10 mm gap was used. A standard ESOPE pulsing protocol was employed. The electroporation was made immediately after drug administration, directly on the tumor mass and at a distance ranging from 5-15 mm. After ECT, the tumor mass was excised routinely with a CO\({}_{2}\) laser (Aesculight, Bothell, WA, USA) using 0.25 mm spot size, working in super pulse mode and 10 W of power output. Due to finding the draining node, the lymphatics were followed until the point at which they were no longer visible in the skin. Then, the suspected lymph node exhibiting fluorescence was removed. The wounds were sutured with monofilament nonabsorbable suture material (Figure 2b). Tumor tissue and lymph node were directed for CLSM (Leica TCS SP8 MP) imaging, similarly as for the in vitro. For the ICG detection, the following wavelengths were used \(\lambda_{\text{exc}}\) = 638 nm and \(\lambda_{\text{em}}\) = 660-788 nm. Images were captured with a 25\(\times\) (numerical aperture, NA 0.95) water objective.