Fluorescence lifetime imaging microscopy (FLIM) is sensitive to molecular environments and enables high-resolution mapping of cellular heterogeneity. Yet, the journey from raw photon decays to biological insight remains fragmented by multi-step data extraction and siloed analyses, creating burdens for experts and non-experts alike. This work presents FLIM Playground, the first interactive graphical platform that unifies single-cell FLIM workflows. Modularly designed to encompass data extraction (if desired) and data analysis, FLIM Playground can check field-of-view metadata, calibrate and extract fluorescence lifetime features per region of interest along with morphology and texture features across channels, merge multiple datasets, and provide real-time visual analytic modules. Lifetime extraction was validated against commercial software and published results, and both data extraction and analysis were demonstrated on a FLIM dataset of cancer cell lines. By adopting best practices and offering interactivity, FLIM Playground promotes reproducibility, allows for expansion to new imaging modalities, and accelerates hypothesis-driven discovery.
Zhao et al. (Mon,) studied this question.