A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges.

A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges.
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DOI:
10.1111/jmi.13128
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发表时间:
2022-09
影响因子:
2
通讯作者:
Kaminski, Clemens F.
Kaminski, Clemens F.
中科院分区:
工程技术4区
文献类型:
--
作者:
Laine, Romain F.;Poudel, Chetan;Kaminski, Clemens F.

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荧光寿命成像(FLIM)允许在活细胞中原位定量亚细胞过程。已经开发了许多方法来从时域FLIM数据中提取寿命,但它们通常在速度,光子效率,精度或它们可以测量的寿命动态范围方面受到限制。在这里,我们专注于该领域中表现最好的方法之一,质心方法(CMM),它在速度和光子效率方面优于其他方法。然而,在本文中,我们确定了CMM的光子效率损失的短寿命时,存在背景噪声。随后,我们提出了一个新的发展和推广的CMM,提供了一个大的寿命动态范围内的荧光寿命的快速和准确的提取。我们提供软件工具来模拟、验证和分析FLIM数据集,并将我们的方法与标准CMM和常用的最小二乘最小化(LSM)方法的性能进行比较。我们的方法具有更好的光子效率比标准的CMM和LSM,并在存在背景噪声的情况下是强大的。该算法适用于任何时域FLIM数据集。
Fluorescence lifetime imaging (FLIM) allows the quantification of sub‐cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time‐domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre‐of‐mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least‐square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time‐domain FLIM data set.
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