To assess the ability of multiphoton microscopy (MPM) to visualise, differentiate and track periprostatic nerves in an in vivo rat model, mimicking real‐time imaging in humans during RP and to investigate the tissue toxicity and reproducibility of in vivo MPM on prostatic glands in the rat after imaging and final histological correlation study.

In vivo prostatic rat imaging was carried out using a custom‐built bench‐top MPM system generating real‐time three‐dimensional histological images, after performing survival surgery consisting of mini‐laparotomies under xylazine/ketamine anaesthesia exteriorising the right prostatic lobe. The acquisition time and the depth of anaesthesia were adjusted for collecting multiple images in order to track the periprostatic nerves in real‐time. The rats were then monitored for 15 days before undergoing a new set of imaging under similar settings. After humanely killing the rats, their prostates were submitted for routine histology and correlation studies.

In vivo MPM images distinguished periprostatic nerves within the capsule and the prostatic glands from fresh unprocessed prostatic tissue without the use of exogenous contrast agents or biopsy sample. Real‐time nerve tracking outlining the prostate was feasible and acquisition was not disturbed by motion artefacts. No serious adverse event was reported during rat monitoring; no tissue damage due to laser was seen on the imaged lobe compared with the contralateral lobe (control) allowing comparison of their corresponding histology.

For the first time, we have shown that in vivo tracking of periprostatic nerves using MPM is feasible in a rat model. Development of a multiphoton endoscope for intraoperative use in humans is currently in progress and must be assessed.