Abstract
Fluorescence lifetime imaging microscopy (FLIM) is a cellular-resolution molecular imaging technique. Yet, the journey from raw photon decays to biological insight remains fragmented by multi-step data extraction and siloed analyses. This work presents FLIM Playground, the first interactive graphical platform that unifies single-cell FLIM workflows, embeds user checks at each stage, and offers diverse user options. Built in Python and available open-source, FLIM Playground runs on major operating systems as a ready-to-run application and is web deployable. Its Data Extraction section collects and checks field-of-view metadata, calibrates via instrument response function shift or fluorescence lifetime standard, and extracts single-cell fluorescence lifetime features, along with morphology and texture features across channels. Multiple datasets can be merged through an interface that assigns categorical labels. The Data Analysis section provides real-time visual analytic modules for outputs from Data Extraction or user-provided datasets. Lifetime extraction by fitting and phasor were validated by comparison with a commercial software and published results, respectively, and both sections were demonstrated on a FLIM dataset of cancer cell lines to obtain biological insights. By adopting best practices and offering interactivity, FLIM Playground accelerates hypothesis-driven discovery and promotes reproducibility, and its modular design can incorporate new imaging modalities, extraction methods, and analysis modules.