Compensation-Free, Highly Multiplexed Flow Cytometer

项目摘要 流式细胞术在研究和开发以及临床实验室中是一项重要的技术 疾病的诊断和监测。它在区分免疫的种群之间特别有用 细胞基于其表达的细胞表面抗原。标准的流式细胞仪使用荧光标记，通常 与单抗结合，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 这种非常高的吞吐量是可能的(每秒可以分析30,000个单元)，使其成为强大的 技术。进一步多重(在单个细胞中测量各种不同分子物种)的能力 增加了它的实用性，并增加了可以使用这种技术解决的科学问题的复杂性。 然而，目前多路复用的水平存在局限性。通常，流式细胞仪分析仅依赖于 荧光标记的光谱信息，因此受到荧光团发射光谱重叠的限制。 目前，即使采用中等水平的多路复用来同时询问多个 单元格中的参数要求在检测和分析中具有很高的复杂性，并且需要仔细设计 以及实验的执行。相关的“补偿问题”(补偿信号的溢出 一个荧光团进入多个通道--由于大多数荧光团的广谱)也会导致显著的 仪器复杂，工作流程繁琐，结果不准确。这些因素严重限制了 可以使用当前技术解决的一系列科学问题，阻止了该技术的新手 从尝试更复杂但具有科学相关性的实验，以及集体被广泛认为是 这是目前流式细胞术的主要瓶颈。 为了克服这一限制，我们开发了一种创新的方法，使用荧光寿命作为 单独的、额外的辨别测量参数。我们使用荧光寿命的方案 多路复用是简单、可扩展的，并且得到了我们的原型仪器的初步数据的支持。这个 提出的项目将通过修改我们的实验来确定基于寿命的多路复用的可行性 具有关键硬件和算法改进的平台，用一个 越来越复杂的一套全面的验证和验证测试，最终以 将我们的技术与现有技术在标准四色细胞化验中进行比较。 成功的结果将为我们计划开发的商业票据(两者)奠定基础 分析器和分类器)，为最终用户提供两大好处：(A)简单、交钥匙、免费 具有中低级多路复用的仪器的操作；和(B)具有两个至两个至 是目前最大多路传输能力的三倍。

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 and that very high throughput is possible (30,000 cells per second can be analyzed), 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. Typically, flow cytometry analysis 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 for the simultaneous interrogation of multiple parameters within a cell requires high levels of complexity in instrumentation and analysis, and careful design and execution of experiments. The related "compensation problem" (compensating for spillover of signal from a fluorophore into multiple channels—due to the broad spectrum of most fluorophores) also causes significant instrument complexity, cumbersome workflow, and inaccurate results. These factors put severe limits on the range of scientific questions that can be addressed using current technologies, deter novices in the technique from attempting more complex yet scientifically relevant experiments, and collectively are widely regarded as the major current bottleneck in flow cytometry. To overcome this limitation, we have developed 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. The proposed project will establish the feasibility of lifetime-based multiplexing by modifying our experimental platform with key hardware and algorithm improvements, challenging the resulting prototype with a comprehensive set of verification and validation tests of increasing complexity, and culminating with a comparison of our technology to existing technology in a standard four-color cell-based assay. A successful outcome will lay the foundation for our planned development of commercial instruments (both analyzers and sorters) that offer two major benefits to end users: (a) simple, turnkey, compensation-free operation for instruments with low-to-medium levels of multiplexing; and (b) high-end instruments with two to three times the current maximum multiplexing capability.