TIME-RESOLVED FLOW-CYTOMETRY FOR THE MEASUREMENT OF LANTHANIDE CHELATE FLUORESCENCE .1. CONCEPT AND THEORETICAL EVALUATION
TIME-RESOLVED FLOW-CYTOMETRY FOR THE MEASUREMENT OF LANTHANIDE CHELATE FLUORESCENCE .1. CONCEPT AND THEORETICAL EVALUATION
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DOI：
10.1002/cyto.990160302
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发表时间：
1994-07-01
期刊：
影响因子：
--
通讯作者：
ANLIKER, M
中科院分区：
文献类型：
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作者：
CONDRAU, MA;SCHWENDENER, RA;ANLIKER, M
提出并评价了一种适用于衰减时间为10亩S至2ms量级的稀土元素络合发光的时间分辨测量的流式细胞仪的概念。该仪器包括一个用于激发荧光的连续波激光器和一个用于消除在发光检测期间在1 ns到1亩的范围内衰减的细胞自体荧光的光开关，S。缓慢衰减的荧光由光子计数系统定量，而光散射和即时荧光参数由常规检测系统获取。根据每细胞可检测到的最小荧光络合物数量，考察了该方法的检出限。它是近30,000个Eu的络合物，其衰减时间为1.6ms，量子效率为17%，与快速衰减的细胞自发荧光或瞬时染料发射强度无关。估计了激光关闭时细胞通过仪器而未被检测到的概率，并评估了对细胞吞吐量和仪器分选性能的影响。在500亩S至1ms的典型荧光检测间隔，每秒100-200个颗粒的细胞流速导致检测概率超过90%，分选纯度可与传统的荧光激活细胞分选相媲美。(C)1994年Wiley-Liss，Inc.
The concept of a flow cytometer suited for the time-resolved measurement of lanthanide chelate luminescence with a decay time on the order of 10 mu s to 2 ms is presented and evaluated. The instrument proposed encompasses a continuous-wave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 mu s during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon-counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system.The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 mu s to 1 ms, cell flow rates of 100-200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence-activated cell sorting. (C) 1994 Wiley-Liss, Inc.