Animal preparation and imaging

To prove the efficiency of the microbubbles in methylene blue and black ink as dual modal contrast agents in vivo imaging of rat urinary bladder was carried out. Animal experiments were performed in accordance with the approved guidelines and regulations, and were approved by the Institutional Animal Care and Use committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263). Healthy adult female Sprague Dawley rats of weight 300 ± 50 g (aged 12–14 weeks) were obtained from InVivos Pte. Ltd., Singapore. To prepare the rats for imaging, firstly they were anesthetized with a mixture of Ketamine (85 mg/kg) and Xylazine (15 mg/kg). An intraperitoneal injection of 0.2 mL of the mixture is administered per 100 g of the rat body weight. Then, the abdominal area is cleared of hair using commercial hair removal cream. Subsequently, a 23 G urinary bladder catheter was inserted through the urethra and was secured using tissue glue to avoid leakage. Finally, to maintain the animal under anaesthesia during experiments 0.75% of isoflurane gas (Medical Plus Pte Ltd, Singapore); was given through a nose cone covering the nose and the mouth of the rat. Also, to monitor the heart rate and peripheral oxygen saturation of the rat throughout the experiments a pulse oximeter (Medtronic, PM10N with veterinary sensor, Minneapolis, Minnesota, USA) was attached to the hind leg of the rat. After experiments, the rats were euthanized with a pentobarbital overdose.

For animal PA imaging a handheld probe holder was used. To couple the laser light and the ultrasound transducer a bifurcated optical fiber was used (Ceramoptec GmbH, Germany). It has 1600 fibers in total with a numerical aperture of 0.22 and a diameter of 200 µm was used. The fiber and the transducer are fixed into a custom designed probe holder with three slots (centre slot for transducer and the two sides for optical fiber) forming the handheld probe for combined US and PA imaging. The animal is placed facing upwards and a 0.5 cm thick chicken tissue slice is placed on the shaved region to mimic the distance of the urinary bladder from the skin for humans. The probe is placed on the animal and both US and combined US + PA images are obtained before injection in the transverse and sagittal planes for control images [shown in Fig. 5(b)]. 0.8 mL volume of microbubbles in MB and BI solutions are injected through the catheter. US, and combined US + PA images of the bladder were obtained in the transverse and sagittal planes. Saline was used as control. All the images were saved as beam-formed datatype. Total imaging depth was set as 3 cm. 50 imaging frames were saved for each experiment.