Multiphoton excitation fluorescence microscopy is a powerful noninvasive imaging technique for the deep optical sectioning of living tissues. Its application in several intact tissues is a significant advance in our understanding of organ function, including renal pathophysiological mechanisms. The glomerulus, the filtering unit in the kidney, is one good example of a relatively inaccessible and complex structure, with cell types that are otherwise difficult to study at high resolution in their native environment. In this article, we address the application, advantages, and limitations of this imaging technology for the study of the glomerular filtration barrier and the controversy it recently generated regarding the glomerular filtration of macromolecules. More advanced and accurate multiphoton determinations of the glomerular sieving coefficient that are presented here dismiss previous claims on the filtration of nephrotic levels of albumin. The sieving coefficient of 70-kD dextran was found to be around 0.001. Using a model of focal segmental glomerulosclerosis, increased filtration barrier permeability is restricted only to areas of podocyte damage, consistent with the generally accepted role of podocytes and the glomerular origin of albuminuria. Time-lapse imaging provides new details and important in vivo confirmation of the dynamics of podocyte movement, shedding, replacement, and the role of the parietal epithelial cells and Bowman's capsule in the pathology of glomerulosclerosis.