We describe a ratiometric intravital two-photon microscopy technique for studying glomerular permeability and differences in proximal tubule cell reabsorption. This quantitative approach is based on using the Generalized Polarity (GP) concept, by which the intensity difference between two fluorescent molecules is normalized to the total intensity given by the two dyes. Following an initial intravenous injection of a mixture of 3, 40 and 70 kD fluorescently labeled dextrans to live Munich-Wistar Fromter (WMF) rats, we were able to monitor changes of the GP values between any two dyes within local regions of the kidney including the glomerulus, Bowman's capsule, proximal tubule lumens and proximal tubule cells, and individual capillary vessels. We were able to quantify accumulations of different dextrans in the Bowman's space and in tubular lumens as well as reabsorption by proximal tubular cells at different time points in the same rat. We found for 6-8 weeks old MWF rats that developed spontaneous albuminuria the 40 kD and the 70 kD dextrans, with hydrodynamic radii larger than albumin, were differentially filtered, but both were able to pass the glomerular filtration barrier and enter into the urinary space of the Bowman's capsule within a few seconds after their intravenous infusion. By using GP image analysis we found negatively charged dextrans, both 40 and 70 kD, were better reabsorbed by the proximal tubule cells than the neutrally charged 40 kD dextran. These results demonstrate the potential power of GP imaging technique for quantitative studies of glomerular filtration and tubular reabsorption.