Mutiphoton & Confocal

The ability to observe the dynamics of neuronal proliferation, migration and differentiation in real time and in situ revolutionizes our ability to study the function of specific genes underlying neurodevelopmental disorders and neurodegenerative syndromes. Over the past few years, NINDS-funded and NDRC investigators at UNC-CH have developed genetic and cell biological approaches to characterize some of the key cellular and molecular mechanisms underlying the initial construction and subsequent maintenance of forebrain circuitry. Core 5 has been instrumental to establishing the technology necessary to image these dynamic events in organotypic slice cultures and in the intact developing mouse brain. Long-term confocal time-lapse imaging has allowed the laboratories of Drs. Anton and Polleux to study in detail the cellular mechanisms associated with the development of neuronal morphology and migration. To acquire additional insights into the molecular mechanisms underlying neuronal development, these Investigators are now using functional statespecific fluorophores, such as genetically-encoded calcium sensors, bioprobes for small-GTPases activity or photoactivatable fluorophores to perform tracking of subcellular protein localization (Knopfel et al., 2006; Lukyanovet al., 2005). In our renewal, we propose to increase our capacity to offer long-term, time-lapse confocal imaging to other NINDS-funded investigators who now want to use this technology in support of their Qualifying Projects. To achieve this, we will acquire a new Zeiss LSM 510 on the Axio Observer Z1 MOT microscope and controller (see below) in order to perform long-term time-lapse imaging of living neurons at multiple locations. This system will take advantage of the Image J scripts that the Polleux laboratory has developed to track cells and quantify cell migration. Our goal is not only to dynamically image neural function and differentiation at high spatial and temporal resolution, but also to comprehensively image large cohorts of neurons and glia as they undergo development and differentiation with the aim of gaining system wide insights into the construction and maintenance of neural circuitry. The usage of the existing Zeiss 510 NLO has increased greatly due to its improvements for fixed sample imaging (see progress report), including upgrades of a 633nm laser, a third PMT, a X-Y motorized stage, and new software. We now plan to separate this system (see below) into two confocal microscopes, one dedicated to fixed single photon confocal imaging (Zeiss LSM 510) and one dedicated to multiphoton confocal imaging (Olympus FV1000MPE).