Multiphoton microscopy allows investigators the opportunity to study dynamic events within the functioning kidney and during acute kidney injury. This enables investigators to follow complex multifactorial processes in the kidney with improved spatial and temporal resolution, and sensitivity. Furthermore, the ability to obtain volumetric data (3-D) makes quantitative 4-D (time) analysis possible. Finally, use of up to three fluorophores concurrently in multiphoton microscopy allows for three different or interactive processes to be observed simultaneously. Therefore, this approach compliments existing molecular, biochemical and pharmacologic techniques by allowing for direct visualization at the cellular and subcellular levels for molecules without the requirement for fixation. Its use in acute kidney injury is in its infancy but offers much promise for unraveling the complex interdependent processes known to contribute to cell injury and organ failure.