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，加上信息是逐细胞获得的事实 并且可以实现非常高的吞吐量（每秒可以分析30，000个细胞），这使得它成为一个强大的 法多路复用（在单个细胞中测量多种不同分子种类）的能力进一步 增加了它的实用性和使用这种技术可以解决的科学问题的复杂性。 然而，多路复用的水平目前具有限制。通常，流式细胞术分析仅依赖于 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用于多个信号的同时询问， 细胞内的参数要求在仪器和分析方面具有高度的复杂性， 和执行实验。相关的“补偿问题”（补偿来自 一个荧光团进入多个通道-由于大多数荧光团的广谱性）也引起显著的 仪器复杂性、繁琐的工作流程和不准确的结果。这些因素严重限制了 一系列的科学问题，可以解决使用目前的技术，阻止新手在该技术 从尝试更复杂但科学相关的实验，并共同被广泛认为是 流式细胞术中的主要当前瓶颈。 为了克服这一限制，我们开发了一种创新的方法，使用荧光寿命作为一个 单独的、额外的鉴别测量参数。我们的计划，使用荧光寿命为 多路复用是简单的、可扩展的，并且由来自我们的原型仪器的初步数据支持。的 拟议的项目将建立基于生命周期的复用的可行性，通过修改我们的实验 平台的关键硬件和算法的改进，挑战由此产生的原型， 一套全面的验证和确认测试，其复杂性不断增加，最终 在标准的基于四色细胞的测定中，将我们的技术与现有技术进行比较。 成功的结果将为我们计划开发的商业工具（包括 分析仪和分拣机），为最终用户提供两个主要好处：（a）简单，交钥匙，免补偿 用于具有低到中等水平的多路复用的仪器的操作;以及（B）具有两个到三个水平的多路复用的高端仪器。 是当前最大复用能力的三倍。

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.