A fluorescence lifetime separation approach for FLIM live-cell imaging

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Fluorescence lifetime imaging microscopy (FLIM) provides contrast by measuring fluorophore excited-state duration.
• Distinguishing spectrally similar fluorophores is challenging in biological imaging.
• Phasor analysis offers a method for FLIM data interpretation.

Purpose of the Study:

• To demonstrate lifetime separation using FLIM phasor analysis for distinguishing fluorescent molecules in live-cell imaging.
• To showcase applications in tracking proteins and separating autofluorescence.
• To introduce an open-source software plugin for FLIM data analysis.

Main Methods:

• Utilized FLIM phasor analysis for lifetime separation of fluorophores.
• Applied the technique to Caenorhabditis elegans for live-cell imaging.
• Developed and used the open-source napari-flim-phasor-plotter plugin.

Main Results:

• Successfully separated highly spectrally overlapping fluorophores mCherry and mKate2 in developing embryos.
• Differentiated tagged proteins from natural autofluorescence in adult hermaphrodites.
• Enabled distinct tracking of tagged proteins in six-dimensional datasets.

Conclusions:

• FLIM phasor analysis provides effective lifetime separation for complex biological samples.
• The developed open-source software facilitates FLIM data management and analysis.
• This approach advances live-cell imaging capabilities in life science research.