Compensation-Free, Highly Multiplexed Flow Cytometer

项目摘要 流式细胞术是研究和开发以及临床实验室中的主力技术，用于 疾病的诊断和监测。它特别适用于区分免疫系统的人群， 细胞基于其表达的细胞表面抗原。标准流式细胞仪通常使用荧光标记， 与单克隆抗体偶联，以提供有关特定分子的定性和定量信息 在牢房里这种分子特异性，再加上信息是在逐个细胞的基础上获得的， 具有非常高的吞吐量（高达每秒30，000个细胞），使其成为一种强大的技术。的能力 多重（测量单个细胞中的多种不同分子种类）进一步增加了其实用性， 科学问题的复杂性，可以使用这种技术来解决。 然而，多路复用的水平目前具有限制。流式细胞术分析通常仅依赖于 荧光标记的光谱信息，并因此受到荧光团发射的光谱重叠的限制。 目前，即使采用中等水平的多路复用也需要复杂的仪器和仔细的测试。 实验设计、执行和分析，以补偿信号的频谱溢出， 渠道这严重限制了利用现有技术可以解决的科学问题的范围 技术，阻止新手尝试更复杂，但科学相关的技术 实验，并被广泛认为是该领域的主要瓶颈。 为了克服这一局限性，我们建议以第一阶段的结果为基础，在第一阶段，我们证明了可行性 对于一种创新的方法，使用荧光寿命作为一个单独的，额外的鉴别 测量参数我们使用荧光寿命进行复用的方案简单、可扩展， 得到了我们的原型仪器的初步数据的支持。在此，我们建议将我们的第一阶段 增加多路复用能力仪器，用一组验证测试来挑战仪器， 使用相关的生物测定法进行验证，将结果与常规流式细胞仪进行基准比较。 其结果将是一个系统，使补偿免费流式细胞术实验的12种颜色，而 需要比类似装备的商业系统更少的激光器和探测器。这样的系统将 服务于很大一部分市场，包括临床细胞计数，预计将得到广泛采用。这 将为进一步开发适用于以下方面的超高（30+）参数仪器铺平道路 免疫表型，但需要比目前的系统少得多的补偿，并推动 实验复杂性的边界。鉴于流式细胞术的广泛使用和重要性，本项目 将在许多生物医学和临床应用中产生重大影响。几家大型仪器仪表公司 已经表示渴望参与旨在商业化的战略伙伴关系。

PROJECT SUMMARY Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of disease. It is particularly useful in distinguishing between populations of immune cells based on their expressed cell surface antigens. Standard flow cytometers use fluorescent tags, often conjugated to monoclonal antibodies, to give qualitative and quantitative information about specific molecules in the cell. This molecular specificity, coupled with the fact that information is obtained on a cell-by-cell basis with very high throughput (up to 30,000 cells per second), make this a powerful technique. The ability to multiplex (measure a variety of different molecular species in a single cell) further adds to its utility and to the complexity of the scientific questions that can be addressed using this technique. However, the level of multiplexing currently has limitations. Flow cytometry analysis typically relies solely on spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions. Currently, employing even moderate levels of multiplexing requires complex instrumentation and careful experimental design, execution and analysis to compensate for spectral spillover of signal into multiple channels. This severely limits the range of scientific questions that can be addressed using current technologies, deters novices in the technique from attempting more complex yet scientifically relevant experiments, and is widely regarded as the major bottleneck in the field. To overcome this limitation, we propose to build on our results from Phase I where we demonstrated feasibility for an innovative approach that uses fluorescence lifetime as a separate, additional discriminating measurement parameter. Our scheme for using fluorescent lifetime for multiplexing is simple, scalable, and supported by preliminary data from our prototype instrument. Here we propose to upgrade our Phase I instrument to increase multiplexing capability, challenge that instrument with a battery of verification tests, and validate using a relevant biological assay, benchmarking results against a conventional flow cytometer. The result will be a system enabling compensation-free flow cytometry experiments of 12 colors, while requiring fewer lasers and detectors than similarly equipped commercial systems. Such a system would serve a large segment of the market, including clinical cytometry, and is expected to see broad adoption. This would pave the way for further development of an ultra-high (30+) parameter instrument suitable for immunophenotyping, yet requiring significantly less compensation than current systems, and pushing the boundaries of experimental complexity. Given flow cytometry’s wide-spread use and importance, this project will have a high impact in many biomedical and clinical applications. Several large instrumentation companies have already indicated an eagerness to engage in strategic partnerships aimed at commercialization.