125 Quantitative analysis of intestinal perfusion with ICG and MB Figure 1. Schematic overview of the surgical procedure and measurements. T = 0 represents the timepoint at which the ischemic loop was created. After 10 minutes (T = 10) MB was injected. One hour after the creation of the ischemic bowel loop (T = 60), ICG was injected. NIRF imaging continued until at least 120 minutes. Fluorescence imaging system The commercially available Quest Spectrum Fluorescence Camera (QUEST SPECTRUM®, Quest Medical Imaging, Middenmeer, The Netherlands) was used for NIRF imaging. The camera was fixed with a custom-made mechanical arm, which was connected to the surgical table to ensure a stable vision and distance to the ROI. The camera tip was fixed at 15cm from the target organ in all operations. To avoid ambient light interference, all lights of the operating room were dimmed. As introduced, this camera system has bimodal properties and both channels were used in the current study; an 800 nm channel to capture ICG fluorescence (ICG mode) and a 700 nm channel for MB fluorescence imaging (MB mode). During the surgical procedure, four images were displayed on the screen (Supplementary S1). First, a standard color image of the surgical field was displayed alongside a grayscale NIRF image to aid surgical guidance. Moreover, two overlay modes were utilized, comprising a fluorescence intensity map and a NIRF image projected onto the colored image, all presented on the same screen. Statistical analysis and quantification All NIRF images and videos were post-analyzed with Quest Spectrum software (ResearchTool v4.7, Quest Medical Imaging, Middenmeer, The Netherlands). As all ROIs were intraoperatively marked with a surgical marker, this was visible on all recordings and the exact same ROIs could be used during the analysis. A tracker synchronized the ROI with movement, and afterwards the software created a time-intensity curve of the measured intensity of the specific ROIs. The measured fluorescence intensity is displayed in arbitrary units (a.u.). Baseline subtraction was 6
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