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Molecular & Cellular Analysis Technologies
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Methods using fluorescent probes to identify cancer signatures and biological activities of cancer cells hold great promise. Probes based on fluorescence resonance energy transfer (FRET) and techniques such as fluorescence correlation spectroscopy (FCS) and other similar technologies offer the ability to identify specific RNA or protein molecules that can identify a cancer and provide information on oncogenic pathways used by the tumor cells. Other probes can give insight into drug response by measuring apoptosis induction by chemotherapies and radiation. However, fluorescent analysis has several limitations. Most ex vivo analyses use a flow cytometer or complex, confocal microscope to perform analyses, and this requires that tissue be removed from the body and often disrupted into cells, then fixed and analyzed in a static manner. The problems with in vivo fluorescent analysis are even greater since background fluorescence and tissue scattering, even in the near-infrared range, limit signal acquisition to the skin. Two-photon excitation has been a critical advance in optics, facilitating FRET, FCS and CARS techniques in vitro. However, these applications are limited by the complex technology (confocal microscopy) necessary to employ these techniques. We have demonstrated the use of two-photon fluorescence analysis through optical fibers for analysis of cancer cells in vitro and human tumors in vivo in SCID mice. This prior work constitutes the equivalent of an R21 proposal, as we achieved our major objectives: to develop sensing system optics and electronics and to document the ability of this system to obtain and analyze fluorescence signals in vitro and in vivo. The primary goal of this R33 application is to develop a more sensitive prototype device based on a novel dual-clad photonic crystal fiber (DCPCF) that we hypothesize will provide the sensitivity and redundancy necessary for the clinical evaluation of fluorescence signals in vivo using several fluorescence techniques. We plan to carry out our studies in three Specific Aims: 1: Develop DCPCF for use in a two-photon optical fiber fluorescence probe (D-TPOFF). 2: Utilize the D-TPOFF to quantify cancer signatures in vitro and monitor drug effects in tumor cells using targeted nanoparticles ex vivo and in vivo. 3: Utilize D-TPOFF to adapt other fluorescent techniques to examine events in tumors in vivo. At the end of these studies, this technology will be at a point where it is ready for commercialization.