uFLIM --FLIM-FRET显微镜数据的无监督分析
uFLIM -- Unsupervised analysis of FLIM-FRET microscopy data
摘要：
尽管荧光寿命成像显微镜（FLIM）数据集在细胞生物学中广泛使用，但其分析仍具有挑战性，因为每个空间位置都可能包含多个荧光成分的叠加。在这里，我们提出了一个数据分析方法，采用所有的信息，在可用的光子预算，以及快速。该方法称为uFLIM，可以在没有先验知识的情况下确定多个荧光成分的空间分布和时间动态。它大大超越了当前的方法，这些方法要么假设动态的函数依赖性，例如指数衰减，要么要求动态是已知的或校准的。其高效的非负矩阵分解算法允许实时数据处理。我们通过计算机验证uFLIM能够从仅两个激发和检测通道以及每个像素100个检测光子的光子预算中解开五个荧光探针的空间分布和光谱特性。通过适应的方法，表现出福斯特共振能量转移（FRET）的数据，我们检索的空间和转移速率分布的结合物种，没有约束的供体和受体的动态。
英文摘要：
Despite their widespread use in cell biology, fluorescence lifetime imaging microscopy (FLIM) data-sets are challenging to analyse, because each spatial position can contain a superposition of multiple fluorescent components. Here, we present a data analysis method employing all information in the available photon budget, as well as being fast. The method, called uFLIM, determines spatial distributions and temporal dynamics of multiple fluorescent components with no prior knowledge. It goes significantly beyond current approaches which either assume the functional dependence of the dynamics, e.g. an exponential decay, or require dynamics to be known, or calibrated. Its efficient non-negative matrix factorization algorithm allows for real-time data processing. We validate in silico that uFLIM is capable to disentangle the spatial distribution and spectral properties of five fluorescing probes, from only two excitation and detection channels and a photon budget of 100 detected photons per pixel. By adapting the method to data exhibiting Forster resonant energy transfer (FRET), we retrieve the spatial and transfer rate distribution of the bound species, without constrains on donor and acceptor dynamics.
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