Murine bladder imaging by 2-photon microscopy: an experimental study of morphology

Purpose: We developed 2-photon laser scanning microscopy analysis of the native murine bladder. Materials and Methods: Bladder tissue from wild-type mice was imaged by 2-photon laser scanning microscopy autofluorescence and second harmonic generation microscopy. Bladder wall layers and structures were analyzed using differences in color, size, shape and morphology. Results: Autofluorescence of the urothelium, nerve structures and muscles was visible in the green spectral channel due to autofluorochromes such as NAD(P)H and elastin. Second harmonic generation of collagen was seen in the blue spectral channel. Imaging from the mucosal side revealed umbrella cells at 0 and 30 mm, of which the high cellular NAD(P)H content allows autofluorescence detection. Below that a network-like connective tissue layer was visualized up to 50 mm that contained vessels with a diameter of 10 to 40 mm and nerves with a diameter of 1 to 6 mm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 mm. Below at 20 to 25 mm we visualized a thick muscle layer containing elastic fibers and macrophages. Findings were also represented in 3-dimensional reconstructions, providing information on structure localization, orientation and interconnection. Conclusions: Two-photon laser scanning microscopy imaging using autofluorescence of the murine bladder is a promising technique to provide new insight into structures and morphology. It opens avenues to identify structural changes in bladder pathology.