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Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging

A potent tool for studying differentiation and development in vitro has been offered to researchers by the isolation of mouse embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos. Two unique characteristics distinguish them from all other organ-specific stem cells identified to date. First, they can be maintained and expanded as pure populations of undifferentiated cells for extended periods of time retaining normal karyotypes. Second, they are pluripotent, capable to generate every cell type in the body. In vitro differentiation of embryonic stem (ES) cells recapitulates early events in the development of the mammalian nervous system: ES cells can both generate and respond in vitro to signals that normally regulate embryonic development. Cellular three- dimensional structures can be analyzed with different imaging techniques.Taking advantage of the optical transparency of cells, light microscopy uniquely provides noninvasive imaging of the interior of cells. Here we describe how combining three different imaging techniques (time-lapse DIC imaging, AFM on both fixed and living neurons and fluorescence confocal microscopy) used separately, at different times, on different instruments - but on the same samples it was possible to provide a morphological characterization of ES-derived growth cones related to their movement (Ban et al., 2011) and hypothesize a possible functional role of the fragmented structures observed only in a fraction of growth cones.

embryonic stem cells ; confocal microscopy ; atomic force microscopy

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