A Spectral/Lifetime Microscope for in vivo Studies

描述（由申请人提供）：通过荧光显微镜观察活细胞或组织，可以深入了解固定标本观察无法获得的动态行为和细胞生理学。这些信息可以向医生揭示组织的病理状态，或者向研究科学家揭示细胞骨架动力学的信息。全基因组测序与荧光蛋白报告基因的使用相结合，开辟了可视化活组织中任何蛋白质的分布和动态的可能性。此外，特异性荧光指示剂的使用允许在体内可视化细胞的生理状态。然而，重叠的光谱，低信号和光散射的问题造成了严重的限制，在实践中可以实现与荧光显微镜。这些问题将通过开发多光子成像系统来解决，该系统最大限度地利用荧光信号中存在的所有信息。一种新型的光子计数探测器将被开发，同时测量单个荧光光子的波长和寿命。将开发算法来分析和提取噪声限制信号中单个荧光成分的物理意义特征。该系统将被应用于细胞生物学研究，使用荧光报告，并确定潜在的诊断光谱/寿命签名的细胞和组织培养模型的癌症。

DESCRIPTION (provided by applicant): Observations of living cells or tissues through fluorescence microscopy provide insights into dynamic behavior and cellular physiology that cannot be obtained from observations of fixed specimens. Such information may reveal the pathological state of a tissue to the physician or information on cytoskeletal dynamics to the research scientist. Whole genome sequencing combined with the use of fluorescent protein reporters has opened up the possibility of visualizing the distribution and dynamics of any protein in living tissue. Furthermore, the use of specific fluorescent indicators has allowed the physiological state of a cell to be visualized in vivo. However, problems of overlapping spectra, low signal and light scatter pose serious limitations on what can be achieved in practice with fluorescence microscopy. These problems will be addressed by the development of a multi-photon imaging system that makes maximum use of all the information present in the fluorescence signal. A novel photon-counting detector will be developed that simultaneously measures the wavelength and lifetime of individual fluorescent photons. Algorithms will be developed to analyze and extract physically meaningful characteristics of individual fluorescent components from noise-limited signals. The system will be applied to cell biological studies using fluorescent reporters and to the identification of potentially diagnostic spectral/lifetime signatures of cells and tissue culture models of cancer.