Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution
Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution
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DOI：
10.1046/j.1365-2818.2002.01031.x
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发表时间：
2002-06-01
期刊：
影响因子：
--
通讯作者：
Van Sark, WGJHM
中科院分区：
文献类型：
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作者：
Gerritsen, HC;Asselbergs, MAH;Van Sark, WGJHM
本文详细讨论了影响单光子计数、二通道到八通道时间门控检测系统的扫描显微镜荧光寿命成像性能的因素。我们特别讨论了灵敏度、寿命分辨率、采集速度和可测量的最短寿命。配备四到八个时间门的检测系统比两个时间门系统要灵敏得多。在具有四个或更多时间门的系统之间只发现微小的灵敏度差异。实验证实，寿命分辨率主要由光子统计量决定。探测器的时间响应决定了可分辨的最短寿命；快速mcp - pmt为25 ps，其他检测器为300-400 ps。然而，快速MCP-PMTs的最大计数率比快速PMTs低10-100倍。因此，基于MCP-PMT的系统的采集速度受到限制。使用接近其最大计数率的快速PMT，我们能够在不到一秒的时间内记录跳动的肌细胞的荧光寿命图像。
In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two- to eight-channel, time-gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time-gates are significantly more sensitive than the two time-gate system. Only minor sensitivity differences were found between systems with four or more time-gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP-PMTs and 300-400 ps for other detectors. The maximum count rate of fast MCP-PMTs, however, is 10-100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP-PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.