Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 ± 0.4 to 59.5 ± 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased ∼10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared with peritubular vessels (4.7 ± 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times (∼45 s) to glomerular filtration, with a delay of ∼5–10 and 25–30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology.