FLIM Playground: An interactive, end-to-end graphical user interface for analyzing single-cell fluorescence lifetime data

Fluorescence lifetime imaging microscopy (FLIM) is sensitive to molecular environments and enables high-resolution mapping of cellular heterogeneity. Yet, the analytical journey from raw photon decays to biological insight remains fragmented by multi-step data extraction and siloed downstream analyses that introduce friction and propagate errors. We present FLIM Playground, the first interactive graphical platform that unifies single-cell FLIM workflows, embeds user checks at each stage, and offers diverse options for user preferences. Built in Python and available open-source, FLIM Playground runs on all major operating systems as a ready-to-run app and can also be deployed on the web. The Data Extraction section interactively collects and checks field-of-view metadata, calibrates either via instrument response function (IRF) shift or reference dye, and extracts single-cell fluorescence lifetime features, both fitted and phasor, along with morphology and texture features across all channels. Multiple datasets spanning time, samples, or treatments can be merged through an interface that assigns categorical labels for downstream analyses. The Data Analysis section accepts either outputs from the Data Extraction or user-provided datasets. It provides visual analytic methods that share common interactive controls to visualize and filter large datasets in real time through a graphical user interface (GUI). Validation of lifetime fitting with FLIM Playground was performed by comparison with SPCImage using two-photon autofluorescence FLIM of Panc1 cells. Additionally, two-photon autofluorescence FLIM of Panc1 and MCF7 cells treated with metabolic inhibitors were used to demonstrate FLIM Playground features. By adopting best practices and offering flexibilities through interactivity, FLIM Playground accelerates hypothesis-driven discovery and promotes reproducibility.