105 Simultaneous fluorescence imaging of bowel perfusion and ureter delineation using methylene blue (ICG mode) while the 700 nm channel was used for MB fluorescence imaging (MB mode). The information captured during recording was visualized within several different fluorescence formats and displayed onto a screen while performing the procedures. First, a color image of the surgical field was presented together with the NIRF image to allow surgical guidance. Additionally, two overlay modes, including a fluorescence intensity map and a NIRF image that is projected over the colored image, were projected onto the same screen. Anesthesia All surgical procedures were performed under general anesthesia. Standard medication used to ensure proper sedation and analgesia was as follows: intramuscular injection of Zolazepam/Tiletamine (6 mg/kg, Virbac, Barneveld, The Netherlands) and Thiopental (10 mg/ kg, Panpharma SA, Trittau, Germany), a combination of sufentanyl (0.01 mg/kg/h, Hameln Pharma GmbH, Hameln, Germany), Propofol (9 mg/kg/h, B. Braun Melsungen AG, Melsungen, Germany), and Midazolam (1 mg/kg/h, Aurobindo, Baarn, The Netherlands) intravenously. All alterations in vital parameters were monitored by the animal anesthesiologist. Surgical procedure and measurements After general anesthesia, a midline laparotomy was performed by an experienced surgeon. First, a loop of the small bowel with a length of approx. 15 cm, at 250 cm measured from the gastric pylorus, was selected as a region of interest. The camera system was switched to ICG mode followed by ICG injection. Bowel perfusion imaging was performed for at least 120 seconds. The same procedure was repeated under MB mode whereafter MB was administered. Consequently, the area where the left ureter would be expected was identified after 120 seconds and continuous left ureteral imaging in MB mode was performed until 5 minutes after dye administration. The latter was repeated every 10 minutes for a total of minimum 120 minutes (T120) after MB dye injection. Bowel perfusion imaging in both ICG and MB modes was performed in parallel for every 10 minutes in two pigs to investigate the washout pattern. In the other four pigs, only T0 measurements were taken. The identification of the right ureter occurred in the meantime, without any further recordings. An overview of the surgical procedure and measurements is shown in Figure 1. At the end of the protocol, animals were euthanized with a lethal dose of pentobarbital (200 mg/kg). Statistical analysis and quantification of the fluorescence imaging All NIRF videos were post-analyzed with Quest Spectrum software (ResearchTool v4.3 and TBR tool v1.0). The ureteral fluorescence imaging was assessed and quantified by calculating the fluorescence intensity (FI) and the target-to-background ratio (TBR; FI of target/FI background) 13, 17. Background values were calculated approximately 1 cm on either side of the ureter, with solely retroperitoneal tissue. The FI of target was calculated based on the fluorescence signal during peristaltic contractions of the ureter by drawing a circle of interest in the corresponding region. Numerical variables were presented as means and standard deviation (SD) or median and interquartile range (IQR) where appropriate. To evaluate the 5
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