The use of electrochemical probes for sensing the action potentials of neuron cells in vitro and in vivo has led to much of our basic understanding concerning the mechanisms of nerve cell signaling. These probes are difficult to employ in vivo, especially when the simultaneous detection of numerous neuron cells is required, as each neuron requires a connection to an electrochemical probe, and subjects must be anesthesized. Our long term goal is to develop a much less invasive technology to monitor action potentials, in vivo, from individual nerve cells, that has potential for clinical application. The specific hypothesis behind the proposed research is that semiconductor nanoparticle Fluorescence Resonance Energy Transfer (FRET) dyes which emit and are excited in the near IR spectrum, can enhance the signal to noise of previously developed optical action potential sensing techniques to allow the noninvasive in vivo mapping of voltages from individual nerve cells. First, in the proposed scheme, single-neuron spatial resolution is achievable by using dye materials that fluoresce via a two-photon excitation process. Second, the optical properties of quantum dots have numerous demonstrated advantages over the organic dyes that have been typically employed, including orders of magnitude improvent in the one and two-photon absorption cross- sections, enhanced photostability, high quantum efficiencies, and a broad above band-gap absorption. Third, these nanoparticle materials have short fluorescence lifetimes between 20-50 ns, indicating that this technique could be used to map out the voltage-signals from large areas with millisecond time resolution via raster-scanning instrumentation. The experimental focus of this proposal is on the design and in vitro characterization of highly luminescent nanoparticle FRET donors with covalently bound mobile lipophilic acceptor pairs that can be stimulated with and emit near IR light.
The specific aims are to: 1. Design and synthesize tethered FRET based donor and acceptor dyes. This will be accomplished through the (i) synthesis of various dyes consisting of a nanoparticle donor covalently linked to a lipophilic organic acceptor, (ii) optimization of the nanoparticle surface chemistry to encourage maximum adhesion to artificial phospholipid bilayers, and (iii) characterization of the dye FRET properties on these bilayers. 2. In Vitro optical signaling and cytotoxicity studies using mammalian nerve cells, (i) The dye surface functionalization will be evaluated to encourage nonspecific binding to cell membranes without endocytosis. Then, we will investigate the FRET dye (ii) optical characteristics on in vitro neural networks via simultaneous optical and electrical voltage sensing, and (iii) adhesion kinetics and cytotoxicities. ? ? ?
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