Houston Jessica P, Naivar Mark A, Jenkins Patrick, Freyer James P
Department of Chemical Engineering, MSC 3805, New Mexico State University, P.O. Box 30001, Las Cruces, NM 88003-8001, (575) 646-5563, fax: (575) 646-7706.
DarklingX, 1199 45 Street, Los Alamos, NM 87544.
Curr Protoc Cytom. 2012;59(125):1.25.1-1.25.21. doi: 10.1002/0471142956.cy0125s59.
In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.
在流式细胞术中，可激发物质的荧光衰减时间在很大程度上未得到充分利用，并且在任何成像细胞仪、分选或分析系统上都不太可能作为标准参数出现。大多数细胞仪缺乏荧光寿命硬件，主要是由于两个核心问题。首先，寿命技术的研发缺乏对现代激光系统、数据采集板和信号处理技术的适当利用。其次，在更大的细胞术领域，人们对细胞中荧光寿命应用以及基于微珠的检测方法一直缺乏热情。在本单元中，我们描述了针对这些问题的新方法，并展示了在流式中数字获取荧光弛豫速率的简便性。本单元分为方案和评论部分，以便就使用频域方法获取荧光寿命提供最全面的论述。本单元涵盖：（i）基于方案的标准荧光寿命获取，采用调频激光激发；（ii）数字频域细胞术分析；（iii）将荧光寿命测量与分选系统连接。本单元还讨论了如何使用数字方法进行混叠以利用更高频率范围。此外，还提供了关于外差检测和处理波形以提取多指数衰减的最终讨论。
