SSBD:database

Name

ssbd-repos-000438

URL

DOI

Title

Fluorescence lifetime separation of fluorophores in C. elegans embryos and hermaphrodites

Description

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime separation, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we separated the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we separated fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-FLIM-phasor-plotter to implement conversion, visualization, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

Submission date

2025-05-26

Release date

2025-09-30

Update date

-

License

Funding information

C.W. is funded by the German consortium NFDI4BIOIMAGE (Deutsche Forschungsgemeinschaft, grant number NFDI 46/1, project number 501864659). Research in the Müller-Reichert lab is supported by grants from the Deutsche Forschungsgemeinschaft (MU1423/8-3, grant number 258577783 and MU1423/10-3, grant number 282354882). The Müller-Reichert lab also acknowledges support from the CCBx program of the Center for Computational Biology of the Flatiron Institute (NY, USA).C.W. is funded by the German consortium NFDI4BIOIMAGE (Deutsche Forschungsgemeinschaft, grant number NFDI 46/1, project number 501864659). Research in the Müller-Reichert lab is supported by grants from the Deutsche Forschungsgemeinschaft (MU1423/8-3, grant number 258577783 and MU1423/10-3, grant number 282354882). The Müller-Reichert lab also acknowledges support from the CCBx program of the Center for Computational Biology of the Flatiron Institute (NY, USA).

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LIF, xml, PTU (7-Zip), Zarr (7-Zip), OME-TIFF

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294 files / 49.69 GiB

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2 files / 17.14 GiB