Departamento de Física, FCEyN, UBA and IFIBA, CONICET, Pabellón 1, Ciudad Universitaria, 1428 Buenos Aires, Argentina.
Methods Appl Fluoresc. 2017 Jun 26;5(2):024016. doi: 10.1088/2050-6120/aa72ab.
Förster resonant energy transfer measured by fluorescence lifetime imaging microscopy (FRET-FLIM) is the method of choice for monitoring the spatio-temporal dynamics of protein interactions in living cells. To obtain an accurate estimate of the molecular fraction of interacting proteins requires a large number of photons, which usually precludes the observation of a fast process, particularly with time correlated single photon counting (TCSPC) based FLIM. In this work, we propose a novel method named pawFLIM (phasor analysis via wavelets) that allows the denoising of FLIM datasets by adaptively and selectively adjusting the desired compromise between spatial and molecular resolution. The method operates by applying a weighted translational-invariant Haar-wavelet transform denoising algorithm to phasor images. This results in significantly less bias and mean square error than other existing methods. We also present a new lifetime estimator (named normal lifetime) with a smaller mean squared error and overall bias as compared to frequency domain phase and modulation lifetimes. Overall, we present an approach that will enable the observation of the dynamics of biological processes at the molecular level with better temporal and spatial resolution.
荧光寿命成像显微镜（FRET-FLIM）测量的Förster 共振能量转移是监测活细胞中蛋白质相互作用时空动力学的首选方法。为了准确估计相互作用蛋白质的分子分数，需要大量的光子，这通常排除了对快速过程的观察，特别是基于时间相关单光子计数（TCSPC）的 FLIM。在这项工作中，我们提出了一种名为 pawFLIM（通过小波进行相分析）的新方法，该方法通过自适应和选择性地调整空间和分子分辨率之间所需的折衷，允许对 FLIM 数据集进行去噪。该方法通过对相图应用加权平移不变 Haar 小波变换去噪算法来实现。与其他现有方法相比，这会导致显著更低的偏差和均方误差。我们还提出了一种新的寿命估计器（称为正常寿命），与频域相位和调制寿命相比，它的均方误差和整体偏差更小。总的来说，我们提出了一种方法，该方法将能够以更好的时间和空间分辨率观察分子水平上的生物过程动态。
