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Background: The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in nonmammalian and mammalian whole-mount samples. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production. Results: In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples. Conclusions: Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging. [ABSTRACT FROM AUTHOR]