Slides of the PoL-Bio-Image Analysis Symposium 2023: napari-flim-phasor-plotter: a Plugin to Generate Interactive Phasor Plots from Raw FLIM Data

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique used in various research
fields to study dynamic processes within biological samples. Among FLIM methods, Time-Correlated
Single Photon Counting (TCSPC) is widely employed due to its high sensitivity and accuracy in
measuring fluorescence lifetimes. TCSPC captures photon arrival times, allowing precise lifetime
determination. Researchers utilize FLIM to investigate molecular quantities like metabolism, FRET, pH,
and ion concentrations, offering new contrast in images. However, analyzing complex fluorescence
decays conventionally requires fitting models, which can be challenging. The Phasor Plot approach
revolutionizes FLIM data analysis by transforming complex fluorescence decays into a graphical
representation. The Phasor Plot is a polar plot, where each pixel's fluorescence decay is represented
by two coordinates. The plot provides a straightforward visualization of lifetimes and enables
unmixing different lifetime components without the need for fitting, making it a valuable tool for biological studies. A few different software were developed capable of analyzing FLIM data over the
last decades, however reading the raw FLIM data remains a non-trivial task for non-programmers.
We developed an open-source napari plugin called napari-flim-phasor-plotter, available
here: https://github.com/zoccoler/napari-flim-phasor-plotter. It is capable of opening and allowing
visualization of raw FLIM TCSPC data and generating the Phasor Plot inside the napari viewer. Since
the raw data can be very large, it can convert them to .zarr file format to allow lazy loading and
computing using the dask library. Beyond that, it integrates with another popular napari plugin, the
napari-clusters-plotter, thus, it opens the door for clustering algorithms to step into the universal
semi-circle. Segmentation results can be directly obtained from the phasor plot either manually, or
by means of a clustering algorithm.