uFLIM -- Unsupervised analysis of FLIM-FRET microscopy data.

Despite their widespread use in cell biology, fluorescence lifetime imaging microscopy (FLIM) data-sets are challenging to analyse. This is because, in each spatial position, there is typically a superposition of multiple fluorescent components. Here, we present a data analysis method making the most out of the information in the available photon budget, while being fast and unbiased. 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. Its efficient non-negative matrix factorization algorithm allows for real-time data processing. We show that uFLIM is capable to disentangle the spatial distribution and spectral properties of 5 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 F\"orster resonant energy transfer (FRET), we retrieve the spatial and transfer rate distribution of the bound species, without constrains on donor and acceptor dynamics. The deployment of this powerful unsupervised method allows to extract the maximum information from FLIM-FRET data.