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.
C. elegans strains were grown on nematode growth medium (NGM) plates at 20°C with E. coli (OP50) as food source according to established protocols (reference https://doi.org/10.1093/genetics/77.1.71). Adult hermaphrodites were dissected in M9 buffer containing 25 mM levamisole hydrochloride (MP Biomedicals, Solon, USA). Isolated embryos were mounted on a 4% (w/v) agarose pad between a slide and a coverslip. For visualization of whole animals, we immobilized the worms in between an agar pad and a coverslip using M9 buffer containing 25 mM levamisole hydrochloride. Adult hermaphrodites were dissected in M9 buffer containing 25 mM levamisole hydrochloride (MP Biomedicals, Solon, USA). Isolated embryos were mounted on a 4% (w/v) agarose pad between a slide and a coverslip. For visualization of whole animals, we immobilized the worms in between an agar pad and a coverslip using M9 buffer containing 25 mM levamisole hydrochloride.
Fluorescence lifetime imaging microscopy was performed using an upright STELLARIS 8, Leica Microsystems, Germany with a pulsed supercontinuum laser (NKT Photonics, Germany) for single photon excitation at 40 MHz frequency, water immersion objective (40x/1.1, HC PL APO CS2, Leica Microsystems, Germany). Acquisition software was Leica Application Suite X (LAS X) (version 4.5.0.25531) including the FLIM/FCS license (version 4.5.0).
GFP and natural (gut) autofluorescence in the green spectrum were excited at 480 nm (approximately 16 µW in none-objective control) and emission detected in the range of 500 to 560 nm. mCherry and mKate2, both red fluorophores, were excited either alone or simultaneously at 590 nm (approximately 84 µW in none-objective control) and emission was detected in the range of 610 to 800 nm. The pinhole was set to one Airy unit at 600 nm reference. Both spectral ranges were imaged simultaneously. Emitted light was detected with hybrid HyD X detectors (Leica Microsystems, Germany).
The field of view and zoom factor of 4 were chosen to image whole embryos as z stacks of FLIM images of 256 x 256 pixels. The image stacks were recorded with 600 Hz speed of bi-directional scans and optical zoom of 4. This yielded a pixel dwell time of 2.4 µs, a frame time of 0.66 s and voxel sizes of 0.27 µm in x and y and 0.5 µm in the z dimension. To collect sufficient photons for FLIM imaging, three frames were summed per image yielding a total scan time of approximately 40 s or shorter per image stack. Acquisition was performed continuously from two-cell stage prior to the first division until condensed chromatin in the second division appeared (differing from 18 to 30 min between embryos).
For whole worms, we recorded several time points but analyzed only a single. Two channels z stacks of FLIM images were acquired with the same setup as embryos. Differences in central settings included optical zoom of 1, image sizes of 512 x 128 pixels resulting in voxel sizes of 0.54 µm x 0.54 µm x 0.5 µm in x, y, z. Pixel dwell times were 1.2 µs and frame times of 0.58 s with two added frames leading to 99.2 s (AZ212) and 116.5 s (JJ1473) per stack per time point.
The 6D FLIM image datasets were then saved in LIF format in a containerized data structure. We then exported 6D embryo datasets and 5D single time point adult datasets as individual 4D files in PTU format (dimensions being x, y, channels and photon counts in the FLIM dimension which we refer to as microtime) for each z plane and time point as sole export mode for downstream data processing and sharing. Data was then converted to 6D Zarr datasets and 5D OME-TIFF for further visualization, analysis and sharing using the napari-flim-phasor-plotter plugin for napari and custom scripts (see https://www.biorxiv.org/content/10.1101/2025.03.05.641717v1.full.pdf for details).