Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphologica l perturbations of nephrogenesis, such as those seen in inherited renal dis eases or induced in transgenic animals, require careful and often tedious d ocumentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dime nsional (3-D) imaging techniques to visualize and evaluate these complex ev ents, As compared with laser confocal microscopy, two-photon microscopy off ers superior optical sectioning deep into biological tissues, permitting an alysis of large, heterogeneous, 3-D structures such as developing mouse kid ney. Embryonic and newborn mouse kidneys were fluorescently labeled with le ctins, phalloidin, or antibody. Three-dimensional image volumes were then c ollected. The resulting volume data sets were processed using a novel 3-D v isualization technique. Reconstructed image volumes demonstrate the dichoto mous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple bra nches. Detailed morphology of in situ cysts was elucidated in a transgene-i nduced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a p owerful tool for illuminating a variety of complex developmental processes in multiple dimensions.