Confocal and Two-Photon Microscopy

The majority of renal physiological processes, including glomerular filtrate formation, tubular reabsorption and secretion, and regulation of cortical and medullary blood flow, involve the complex interaction of a number of different cell types. This is exemplified by the juxtaglomerular apparatus (JGA), a highly complex structure that consists of the tubular epithelium; the macula densa (MD); vascular-endothelial, smooth-muscle, and renin granular epithelioid cells; and extra- and intraglomerular mesangial cells. These dissimilar cells interact with each other and form a functional syncitium to control glomerular hemodynamics (tubuloglomerular feedback) and renin release (1 ). Thus far, it has been difficult to visualize certain inaccessible cell types, such as MD cells or renal medullary interstitial cells in native kidney tissue, or to study cellular interactions given the constraints of existing technologies. Recently, multi-photon excitation fluorescence microscopy has been applied to kidney research (2 –4 ), and it offers a tremendous increase in optical resolution vs other imaging techniques, even over conventional confocal microscopy. Because of the nature and advantages of two-photon excitation, which are discussed in detail here, multi-photon microscopy is becoming increasingly used in biomedical research particularly for visualization of thick samples of living tissue. To date, two-photon microscopy has been applied in numerous in vivo experimental models, several applications have even used intact conscious whole animals—e.g., in neurobiological research, the mouse brain can be visualized to a depth of 1–2 mm into the cortex (5 ).