Comprehending the appearance of structure in the developing brain is central to an understanding of its function, yet imaging the key events with any single technique is difficult because the events take place over a wide range of spatial and temporal scales. No one imaging technique combines the needed spatial resolution, field of view temporal resolution, and depth of penetration to follow brain development. The goal of the work proposed is two-fold: 1. To meld the complementary imaging modalities of two-photon laser scanning (TPLS) and magnetic resonance imaging (MRI) microscopies to provide coherent four dimensional (3 space and 1 time) information on the emergence of and changes in biological order. The applicant proposes to capitalize on the strengths of each technique and maximize the quality of the resulting data by integrating the imaging modalities at both the experimental and analysis levels; and 2. To apply this melded technology to follow cell lineages and tissue movements that contribute to morphogenesis of avian forebrain. The experimental aim is to directly test hypotheses that lineage restrictions at neuromere boundaries generate regionalization in the forebrain. Implementing the technology within the context of this problem will assure that the technological developments are truly applicable to brain science by driving them with actual research needs. The research plan will entail technological developments in three specific areas: 1.) Hardware and protocols for time course imaging of the same samples with both TPLS and MRI microscopies; 2.) Cellular labels and labeling techniques that are bifunctional, generating image contrast in both modalities; and 3.) Computational tools for combining the image sequences from each modality into an integrated rendering. Although this combination of TPLS and MR imaging will be directed at brain development, the approach is general and the multimodal imaging instrumentation tools will be readily applicable to other problems in neuroscience and biomedical research.
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