Bioluminescent Imaging of Bacterial Colonization

All the animal-related procedures were performed strictly according to the guideline of Tianjin Committee of Use and Care of Laboratory Animals, and experimental protocols were approved by the Animal Ethics Committee of Nankai University. All animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg kg⁻¹) before imaging. Animals received substrates and bacteria by gastric inoculation before imaging, furimazine (1/20 dilution of Nano-Glo® Luciferase substrate, 200 µL gavage volume, Promega) is the substrate for NLuc system and d-luciferin (150 mg kg⁻¹, PerkinElmer) is for FLuc and CBRLuc systems.^{35,36 (link)} Bioluminescence images were acquired at room temperature on a Berthold NightOWL LB 983 Imaging System (Berthold Technologies, Bad Wildbad, Germany). The images were acquired with open filter and exposure time ranges from 1 to 3 min according to the signal intensity, the luminescence counts acquired under different exposure time were all normalized as the average value (total counts/exposure time) to ensure the reasonable comparison, and the pseudo colour images represented the light intensity (from red for most intense to blue for least intense). All bioluminescent mice had a region of interest (ROI), corresponding to the location of bioluminescent strain, which is manually determined.