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Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging


Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

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Fig. 1: Phasor S-FLIM workflow and robustness to noise of the phasor approach.
Fig. 2: Phasor S-FLIM spectral and lifetime blind unmixing of cellular samples.
Fig. 3: Phasor S-FLIM approach to FRET standards and biosensors.
Fig. 4: Phasor S-FLIM characterization of solvatochromic probes in living cells.
Fig. 5: Phasor S-FLIM single-cell physiological profiling of living tumor spheroids.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The code for performing blind unmixing is provided, together with a script for simulating S-FLIM data for testing and an example of experimental S-FLIM data. Further code is available from the corresponding author upon reasonable request.


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We thank F. Palomba, A. Vallmitjana Lees, C. Gohlke and A. Dvornikov for the useful discussion and D. Jameson for his input on the paper. This work was supported by grant no. NIH P41-GM103540.

Author information

Authors and Affiliations



L.S. and E.G. conceived the idea. A.R. developed and wrote FPGA code. L.S., A.R. and E.G. wrote code. L.S. designed electronics, built the microscope and performed simulations and experiments. G.T. prepared all biological samples. L.S. wrote the paper with input from all authors

Corresponding author

Correspondence to Enrico Gratton.

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Competing interests

A.R. declares his involvement in FLIM LABS Srl, Rome, Italy.

Additional information

Peer review information Nature Methods thanks Luis Alvarez, Mantas Zurauskas and Francesco Cutrale for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–23, information on various technical aspects, Tables 1 and 2 and bibliography.

Reporting Summary

Supplementary Software

File contains example of S-FLIM dataset (SFLIM_Dataset.mat), calibration file (20200319.mat) and script to run the unmixing (SFLIM_Unmixing_Solution.mat).

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 3

Statistical source data.

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Scipioni, L., Rossetta, A., Tedeschi, G. et al. Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging. Nat Methods 18, 542–550 (2021).

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