2  Overview

FLIM Playground transforms FLIM, intensity-only, and quantitative phase imaging (QPI) inputs into single-cell numerical features, then combines numerical tables and assigns categorical labels for downstream analysis.

2.1 Stages

Configure a profile for the channels, input types, file suffixes, and extractors in your experiment. Then open Data Extraction in a local installation. The hosted app provides Data Analysis only.

Numerical prepares the source, calibrates channels, and extracts features; Categorical combines and labels tables.

1. Numerical includes:
   • Field of View (FOV) metadata preparation scans a source folder, checks files and acquisition settings, and automatically records the metadata.
   • Interactive calibration (if needed): some channels may require IRF shifts or QPI background correction that need human inputs
   • Per-channel extraction: after calibration, each channel is processed independently to extract its features. Some example extractors are fitted lifetimes, phasors, morphology, intensity texture, and QPI dry-mass statistics and spatial texture.
2. Categorical combines numerical datasets and assigns labels such as treatment and time point from the FOV names.

2.2 Limitations

• To simplify the workflow and due to the framework chosen, FLIM Playground does not support pixel-level fitting and fit-free analysis, and there are many other open-source tools that can do so (for a comprehensive list, see here). Instead, it does cell-level fitting and fit-free (phasor) feature extraction by summing up all the decays belonging to the same cell ROI to get one cell-level decay as a preprocessing step¹. It also accepts pre-fit pixel-level lifetime features from SPCImage and aggregates pixel-level lifetime features to cell-level lifetime features.

1.

Samimi, K. et al. Segmentation-guided photon pooling enables robust single cell analysis and fast fluorescence lifetime imaging microscopy. bioRxiv https://doi.org/10.1101/2025.09.30.679660 (2025) doi:10.1101/2025.09.30.679660.