Multi-photon microscopy and advances in optics, computer sciences, and the available labeling fluorophores now allow investigators to study the dynamic events within the functioning kidney with subcellular resolution. This emerging technology, with improved spatial and temporal resolution and sensitivity, enables investigators to follow complex heterogenous processes in organs such as the kidney. Repeated determinations within the same animal are possible minimizing their use and inter-animal variability. Furthermore, the ability to obtain volumetric data (3D) makes quantitative 4D (time) analysis possible. Finally, use of up to three fluorophores concurrently allows three different or interactive processes to be observed simultaneously. Therefore, this approach compliments existing molecular, biochemical, and pharmacologic techniques by advancing data analysis and interpretation to subcellular levels for molecules without the requirement for fixation.