Result: FLIM Playground: An interactive, end-to-end graphical user interface for analyzing single cells with fluorescence lifetime imaging microscopy.

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.

Competing Interests MCS is an adviser to Elephas Biosciences. All other authors declare they have no competing interests.