Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells.
Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells.
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DOI：
10.1111/j.0022-2720.2004.01343.x
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发表时间：
2004-07
影响因子：
2
通讯作者：
Shipston MJ
中科院分区：
文献类型：
--
作者：
Duncan RR;Bergmann A;Cousin MA;Apps DK;Shipston MJ
我们提出了一种新的，多维的，时间相关的单光子计数（TCSPC）技术进行荧光寿命成像的激光扫描显微镜在微秒范围内的像素停留时间。这种方法结合了高检测效率的无与伦比的时间准确性被应用于测量增强型青色荧光蛋白（ECFP）的荧光寿命，并与EYFP（增强型黄色荧光蛋白）串联。该技术使多指数衰减分析在扫描显微镜具有高的固有时间分辨率，精度和计数效率，特别是在低激发水平所需的维持细胞活力，避免光漂白。使用编码由固定距离的氨基酸间隔区分开的两种荧光蛋白的构建体，我们能够测量由发色团间距离确定的荧光共振能量转移（FRET）效率。这些数据表明，ECFP表现出复杂的指数荧光衰减FRET和非FRET条件下，如前所述。两种方法计算施主和受主之间的距离从寿命交付值在10%的误差范围内。为了证实该方法也可用于定量分子间FRET，我们用苯乙烯基染料FM 1 -43标记培养的神经元，定量荧光寿命，然后使用FM 4 -64（FM 1 -43发射的有效能量受体）淬灭其荧光。这些实验首次直接证实了FRET发生在这两个发色团之间，表征了这些探针的寿命，确定了质膜中的发色团间距离，并提供了活神经元寿命分布的高分辨率二维图像。
We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1-43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4-64, an efficient energy acceptor for FM1-43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high-resolution two-dimensional images of lifetime distributions in living neurones.
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影响因子：
56.9
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