Multi-photon excitation fluorescence microscopy is a state-of-the-art confocal imaging technique ideal for deep optical sectioning of living tissues. It is capable of performing ultra-sensitive, quantitative imaging of organ functions in health and disease with high spatial and temporal resolution that other imaging modalities cannot achieve. Since the low cytotoxicity of multi-photon excitation allows continuous imaging of living tissues, real-time imaging of the tubuloglomerular feedback (TGF) and renin release mechanisms became possible. Novel, TGF-associated morphological findings include significant cell volume changes of the macula densa under isotonic or hypertonic conditions, the existence of bulk fluid flow in the JGA, a sphincter-like contraction of the terminal, intraglomerular afferent arteriole, and the TGF-associated contraction of not only the afferent arteriole, but the entire intraglomerular mesangium. Spreading of the TGF vasoconstrictor signal in the JGA and beyond involves an extracellular ATP-mediated purinergic calcium wave. This wave was directly visualized with confocal microscopy to propagate from the macula densa and extraglomerular mesangial area to the afferent arteriole, along the vasculature to adjacent glomeruli, and also to all cells of the glomerulus including the most distant podocytes. Propagation of the TGF calcium wave from afferent arteriole smooth muscle cells to the underlying endothelium was also observed in these studies. This phenomenon may provide negative feedback and helps to balance the TGF vasoconstriction by triggering endothelium-derived vasodilator mechanisms. These imaging studies further emphasized the roles of both gap junctional communication and extracellular ATP as integral components of TGF. In addition, these studies provided functional evidence that complementing the afferent arteriolar vasoconstriction, all cells of the glomerulus actively participate in TGF by contracting the glomerular tuft, thereby helping to reduce the rate of glomerular filtration. The unexpected finding that the calcium wave of TGF was mediated by extracellular ATP provided further support that ATP itself is directly involved in TGF and not only through its breakdown to adenosine. Renin release is the first, and at least initially, the rate-limiting step in the activation of the renin-angiotensin system which helps to maintain body salt and water balance. Additional details of the renin release mechanism were also observed using the multi-photon imaging approach. Acidotropic fluorophores including quinacrine and LysoTracker dyes (Invitrogen) are highly membrane permeant weakly basic compounds that rapidly accumulate in acidic cellular organelles. They have been successfully used to label renin granular content both in vitro and in vivo, and even as a counter stain on histological sections. Imaging the entire granular content as opposed to labeling specific molecules of interest (renin itself) is of great advantage when studying the mechanism and regulation of renin granule exocytosis. For example, there is a renewed interest in the enzymatically inactive prorenin, which is part of the granular contents and therefore its release may also be visualized even though it cannot be detected by existing assays measuring renin activity. Renin exocytosis has been visualized in real-time and on the individual renin granule level in response to a number of physiological stimuli including beta-adrenergic activation, low perfusion pressure and the macula densa mechanism. Dimming and disappearance of the entire granular content (quantal release) was observed within 2-300 ms. A significant number of renin granules was released into the interstitial side of the JGA, in addition to the vascular lumen. Not only the degranulation process, but enzymatic activity of the released renin (angiotensin I generation) was visualized in real-time using a FRET-based renin substrate. In vivo visualization of cellular variables like cytosolic calcium in the collecting duct, intracellular pH in the proximal tubule, cell-to-cell communication and signal propagation will be shown. Basic kidney functions that can be measured by in vivo quantitative multi-photon imaging include glomerular filtration and permeability, concentration, dilution, and activity of the intra-renal renin-angiotensin system. New visual data challenge a number of existing paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function with multi-photon microscopy has tremendous potential to eventually provide novel non-invasive diagnostic and therapeutic tools for future applications in clinical nephrology.