Densely Multiplexed Fluorescence Imaging by Fourier Transform Fluorometry

描述(申请人提供)：这项K99/Roo提案的目标是开发一种用于高度复合荧光显微镜的新平台，该平台可以在活细胞中可视化数十到数百个生物标记物。目前，利用荧光发射波长的差异来识别多个荧光标记。在这里，我们建议通过同时检测具有独特的激发、发射和寿命特性的不同荧光标记来增加多路复用密度。使这成为可能的设备是一种新型的傅里叶变换荧光仪，它能够并行测量这些荧光信号。这项技术是由彭雷雷博士首创的，他目前是威尔曼光医学中心的研究员。目前，傅立叶荧光仪只能在有限的荧光波长和寿命范围内进行单点测量。对于这项提议，彭博士将进一步开发这项技术，以显著扩大可测量波长的范围和寿命。数据采集速度也将提高，使这种方法与显微成像兼容。在这些改进之后，荧光计将被整合到共焦显微镜成像系统中，并将测试和验证密集多路复用。这项研究将以体外多重显微镜的演示而达到顶峰。吉列尔莫·J·蒂尔尼(Guillermo J.Tearney)经营着一家开发疾病诊断显微光学方法的实验室，他将指导彭博士，并监督光谱仪和显微镜系统的开发。共同导师Tayyaba Hasan是光化学和光生物学领域的著名科学家，擅长光动力疗法和荧光成像。Hasan博士将协助开发和选择具有不同激发、发射和寿命特性的荧光标记，并将这些标记功能化，用于活细胞实验。这一奖项提供的机会不仅将使彭博士能够开发这一用于生物医学成像的新型光学技术，还将为她提供宝贵的指导、培训和资源，以开启她作为独立学术研究人员的职业生涯。

DESCRIPTION (provided by applicant): The goal of this K99/ROO proposal is to develop a new platform for highly multiplexed fluorescence microscopy, which could allow tens to hundreds of biomarkers to be visualized in living cells. At present, multiple florescent markers are identified using differences in fluorescence emission wavelength. Here we propose to increase multiplexing density by simultaneously detecting different fluorescent markers with unique excitation, emission, and lifetime properties. The device that makes this possible is a novel Fourier transform fluorometer that is capable of measuring these fluorescence signatures in parallel. This technology has been pioneered by Dr. Leilei Peng, who is currently a Research Fellow at the Wellman Center for Photomedicine. Currently, the Fourier fluorometer is only capable of making single point measurements over a limited range of fluorescence wavelengths and lifetimes. For this proposal, Dr. Peng will develop this technology further to dramatically expand the range of measurable wavelengths and lifetimes. Data acquisition speeds will also be increased to make this method compatible with microscopic imaging. Following these modifications, the fluorometer will be incorporated into a confocal microscopy imaging system and dense multiplexing will be tested and validated. The research will culminate with a demonstration of multiplexed microscopy in vitro. Guillermo J. Tearney, who runs a laboratory that develops microscopic optical methods for disease diagnosis, will mentor Dr. Peng and supervise development of the spectrometer and microscopy system. Co-mentor Tayyaba Hasan is a renowned scientist in photochemistry and photobiology with specialties in photodynamic therapy and fluorescence imaging. Dr. Hasan will assist with development and selection of fluorescent labels with distinct excitation, emission, and lifetime properties as well as functionalization of these labels for experiments in living cells. The opportunities provided by this award will not only allow Dr. Peng to develop this novel optical technique for biomedical imaging, but will also provide her with valuable mentorship, training, and resources to launch her career as an independent academic researcher.