Different segments of the nephron appear to be differentially sensitive to mitochondrial cytopathy; clinically the proximal tubule (PT) appears particularly vulnerable, and dysfunction of the PT can lead to the renal Fanconi syndrome (1). We have developed a method, using multi-photon microscopy (2), to investigate underlying differences in mitochondrial function between the PT and other nephron segments in live rat kidney tissue, which might explain this clinical observation. Adult male Sprague-Dawley rats were killed humanely and their kidneys were immediately removed and placed into ice-cold Krebs solution gassed with oxygen. Live 200μm slices of kidney were produced using a Microm 650V tissue slicer. The slices were imaged with a Zeiss LSM 510 upright multi-photon microscope, coupled with a Coherent Chameleon tunable laser. An onstage perfusion system was used to infuse metabolic substrates, dyes and mitochondrial reagents; thus allowing changes in mitochondrial function in different cell types to be imaged in real time. We have found that renal tubular cells emit a large background auto-fluorescence signal when excited at a range of wavelengths from 720-850nm. With excitation at 720nm, the blue fluorescence emitted is predominantly mitochondrial NADH. Green fluorescence arises partly from oxidized flavoproteins. The identity of the signal can be confirmed by a change in response to either respiratory chain inhibitors or uncoupling agents. Different parts of the tubule can be distinguished by their auto-fluorescence signals. Calcein labels living cells, confirming viability, and is also useful to identify structures. Tetramethyl rhodamine methyl ester (TMRM) is used to visualize variations in mitochondrial location, potential difference and morphology in different cell types (3). TMRM signal is brighter in more distal parts of the tubule compared with the PT, suggesting that mitochondria in these regions may be more polarized. Multi-photon imaging of live slices of rat kidney is a useful technique to investigate mitochondrial function in the kidney. It allows direct comparison of different nephron segments in primary, non-immortalized tissue, with preserved architecture. Furthermore, it permits access to structures that are difficult to visualise in the whole intact organ. Early results suggest differences between the PT and other nephron segments, which may prove to be important in the nature and localization of renal cell injury.