A Spectral/Lifetime Microscope for in vivo Studies

描述(申请人提供)：通过荧光显微镜观察活细胞或组织提供了对动态行为和细胞生理学的洞察，这是通过观察固定样本无法获得的。这样的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质报告的使用相结合，为可视化任何蛋白质在活组织中的分布和动态提供了可能性。此外，特定荧光指示剂的使用使细胞的生理状态能够在体内可视化。然而，光谱重叠、低信号和光散射等问题严重限制了荧光显微镜在实践中的应用。这些问题将通过开发最大限度地利用荧光信号中存在的所有信息的多光子成像系统来解决。一种新型的光子计数探测器将被开发出来，它可以同时测量单个荧光光子的波长和寿命。将开发算法，从噪声有限的信号中分析和提取单个荧光成分的物理意义特征。该系统将应用于使用荧光报告的细胞生物学研究，以及识别癌症细胞和组织培养模型的潜在诊断光谱/寿命特征。

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.