National Changhua University of Education Institutional Repository : Item 987654321/11938
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题名: Fiber-optic multiphoton fluorescence spectroscopy for biosensing and in vivo flow cytometry
作者: Chang, Yu-Chung
贡献者: 電機工程學系
关键词: Fiber probe;Multiphoton process;Fluorescence spectroscopy;Biosensing;Cancer metastasis;Flow cytometry
日期: 2009
上传时间: 2012-07-03T01:49:30Z
出版者: University of Michigan, Ann Arbor, Michigan
摘要: There is considerable interest in developing real-time diagnostic tools to detect disease signatures and therapeutic responses in vivo; however, tissue scattering and absorption limit the capability of traditional optical techniques for quantitative biosensing in deep tissue. To address these limitations, we have developed a fiber-optic two-photon-excited fluorescence probe system to quantify deep tissue fluorescence in situ. A double-clad fiber (DCF) was employed to increase the sensitivity of the probe. The probe was used to quantify targeted delivery of biocompatible dendrimer nanoparticles into tumor cells. Exploiting the ability of two-photon excitation to excite multiple fluorophores, we were able to quantify few-nanomolar concentrations of different emission-wavelength antibody conjugates in mouse tumors. To enable time-resolved spectroscopic measurements, a time-correlated single-photon counting (TCSPC) module was incorporated into the system. Fluorescence lifetime changes due to quenching of the fluophores on the dendrimer conjugates were observed. In addition, fiber-optic two-photon fluorescence correlation spectroscopy (FCS) was demonstrated for the first time with this system. Fluorescent nanoparticles as small as 7 nm in radius were measured. We also demonstrated the technique’s ability to measure the flow velocity of fluorescent species. When applying the technique to measure flow cells, distinct FCS curve behaviors were observed in differently labeled cells; this may enable cell differentiation by in situ FCS measurements. The minimally invasive nature of the single-fiber probe geometry is suitable for in vivo long-term monitoring of circulating cells. We applied the fiber probe to implement flow cytometry in vivo and in vitro. With dual-channel detection, we conducted quantitative ratiometric measurements on the detection efficiency of dual-labeled fluorescent protein-expressing cells. In the in vitro studies, our system showed about one order of magnitude higher detection sensitivity for green fluorescent protein (GFP)-expressing cells in whole blood when compared to the sensitivity of a free-space detection scheme. In the in vivo studies, cancer cells were injected into different locations in mice, and the cell circulation dynamics were monitored. The high detection sensitivity of GFP-expressing cells in vivo may help the study of cancer metastasis in mouse models by fluorescence techniques.
關聯: Ph.D. Dissertation, University of Michigan, Ann Arbor, Michigan, USA
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