Fluorescence Lifetime Imaging in Biological Systems

Fluorescence lifetime imaging microscopy (FLIM) measures the decay time of excited fluorophores rather than their intensity, providing a robust indicator of local molecular environments that is independent of dye concentration and excitation intensity. In biological systems, FLIM has become a vital tool for probing cellular metabolism, monitoring protein–protein interactions via Förster resonance energy transfer, and assessing tissue health through endogenous biomarkers such as NADH and FAD. Both time-domain and frequency-domain approaches enable high spatial resolution and quantitative differentiation between free and protein-bound fluorophores. Advanced FLIM configurations now permit deep-tissue multiphoton imaging, high-throughput flow cytometry applications and simultaneous multicolour lifetime measurements, opening new avenues for real-time in vivo studies. Integration with spectroscopic techniques such as Raman and coherent anti-Stokes Raman scattering further enhances chemical specificity, while phasor-based analysis simplifies complex decay data into intuitive graphical formats. Together, these developments underscore the global significance of FLIM for biomedical research, drug discovery and potential clinical diagnostics.

Research from Nature Portfolio

Recent studies have demonstrated high-throughput FLIM flow cytometry capable of imaging over ten thousand cells per second by employing dual intensity-modulated beam arrays, allowing rapid discrimination of cancer cell subpopulations and monitoring of drug-induced nuclear changes. Foundational work has also used FLIM to distinguish between NADH and NADPH in intact tissues by systematically perturbing metabolic pathways, enabling quantitative maps of cofactor distributions in complex organs. Multicolour two-photon FLIM based on wavelength mixing has further enabled simultaneous lifetime imaging of NADH and FAD without motion artefacts, facilitating ratiometric redox analyses in reconstructed human skin and live model organisms and revealing metabolic gradients associated with differentiation and development.

Fluorescence Lifetime Imaging in Biological Systems publication trend

The graph below shows the total number of articles in fluorescence lifetime imaging in biological systems across all publications each year (not limited to Nature Index journals).

Technical terms

Fluorescence lifetime: The average time a fluorophore remains in the excited electronic state before photon emission, typically measured in picoseconds to nanoseconds. Phasor approach: A model-free graphical representation that maps fluorescence decay profiles to points in a Cartesian coordinate system, simplifying the analysis of mixtures and heterogeneous environments. Förster resonance energy transfer (FRET): Non-radiative energy transfer between two chromophores in close proximity (<10 nm), used to report on macromolecular interactions. Time-correlated single-photon counting (TCSPC): A high-precision time-domain method recording the arrival time of individual photons relative to an excitation pulse to reconstruct fluorescence decay kinetics.

References

1. High-throughput fluorescence lifetime imaging flow cytometry. Nature Communications (2024).
2. Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. Journal of Biomedical Optics (2020).
3. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nature Communications (2014).
4. Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing. Scientific Reports (2017).
5. Fast fluorescence lifetime imaging techniques: A review on challenge and development. Journal of Innovative Optical Health Sciences (2019).
6. Measurements of absolute concentrations of NADH in cells using the phasor FLIM method. Biomedical Optics Express (2016).