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(American Journal of Pathology. 2001;158:49-55.)
© 2001 American Society for Investigative Pathology

Technical Advances

Three-Dimensional Imaging of Embryonic Mouse Kidney by Two-Photon Microscopy

Carrie L. Phillips^*, Lois J. Arend, Adele J. Filson^*, Doug J. Kojetin^*, Jeffrey L. Clendenon^*, Shiaofen Fang and Kenneth W. Dunn^*

From the Department of Medicine,^*
Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana; the Department of Pathology and Laboratory Medicine,
University of Rochester Medical Center, Rochester, New York; and the Department of Computer and Information Science,
Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

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