Abstract
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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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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Acknowledgements
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.
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Contributions
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
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A.R. declares his involvement in FLIM LABS Srl, Rome, Italy.
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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.
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Supplementary information
Supplementary Information
Supplementary Figs. 1–23, information on various technical aspects, Tables 1 and 2 and bibliography.
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).
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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). https://doi.org/10.1038/s41592-021-01108-4
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DOI: https://doi.org/10.1038/s41592-021-01108-4
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