Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs.

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.