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Fit-free analysis of fluorescence lifetime imaging data using the phasor approach

Nature Protocols volume 13, pages 1979–2004 (2018)Cite this article

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Abstract

Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content. Phasor plots for fluorescence lifetime analysis were originally developed as a graphical representation of excited-state fluorescence lifetimes for in vitro systems. The method's simple mathematics and specific rules avoid errors and confusion common in the study of complex and heterogeneous fluorescence. In the case of FLIM, the phasor approach has become a powerful method for simple and fit-free analyses of the information contained in the many thousands of pixels constituting an image. At present, the phasor plot is used not only for FLIM, but also for hyperspectral imaging, wherein phasors provide an unprecedented understanding of heterogeneous fluorescence. Undoubtedly, phasor plots will be increasingly important in the future analysis and understanding of FLIM and hyperspectral confocal imaging. This protocol presents the principle of the method and guides users through one of the popular interfaces for FLIM phasor analysis, namely, the SimFCS software. Implementation of the analysis takes only minutes to complete for a dataset containing hundreds of files.

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Fig. 1: The reciprocity principle using the phasor approach.
Fig. 2: Expected position for fluorescence lifetimes in the phasor plot.
Fig. 3: Overview of protocol.
Fig. 4: Hardware connection of the FLIMbox.
Fig. 5: TCSPC versus FLIMBox data acquisition.
Fig. 6: Median filtering in the phasor approach to FLIM.
Fig. 7: Calibration of the phasor plot using a fluorescent standard or scattering sample.
Fig. 8: Phasor analysis of metabolism on the basis of free and enzyme-bound NADH FLIM data.
Fig. 9: Different FRET trajectories for increasing FRET efficiencies.
Fig. 10: Examples of the analysis of biosensor–FRET activity in cells by the phasor approach.
Fig. 11: Movement of the phasor plot based on addition of unmodulated light to calculate absolute concentration.
Fig. 12: Ion concentration and pH determination by the phasor plot approach.

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Acknowledgements

This work was supported in part by NIH grants NIH P41-GM103540 and NIH P50-GM076516. L.M. was supported as a full-time professor at the Universidad de la República-Uruguay.

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Author notes

  1. These authors contributed equally: Suman Ranjit, Leonel Malacrida

Authors and Affiliations

  1. Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California at Irvine, Irvine, CA, USA

    Suman Ranjit, Leonel Malacrida & Enrico Gratton

  2. Departamento de Fisiopatología, Hospital de Clínicas, Universidad de la República, Montevideo, Uruguay

    Leonel Malacrida

  3. Department of Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI, USA

    David M. Jameson

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Contributions

S.R. and L.M. wrote the manuscript and prepared the figures. E.G. and D.M.J. wrote the introduction and corrected all preliminary documents. All data in the article were collected by S.R. and L.M.

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Correspondence to Enrico Gratton.

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Key references using this protocol

1. Stringari, C. et al. Proc. Natl. Acad. Sci. USA 108, 13582–13587 (2011) https://doi.org/10.1073/pnas.1108161108.

2. Hinde, E., Digman, M. A., Welch, C., Hahn, K. M. & Gratton, E. Microsc. Res. Tech. 75, 271–281 (2012) https://doi.org/10.1002/jemt.21054.

3. Digman, M. A., Caiolfa, V. R., Zamai, M. & Gratton, E. Biophys. J. 94, L14–L16 (2008) https://doi.org/10.1529/biophysj.107.120154.

4. Ma, N., Digman, M. A., Malacrida, L. & Gratton, E. Biomed. Opt. Express 7, 441–2452 (2016) https://doi.org/10.1364/BOE.7.002441.

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Supplementary Figs. 1–18, Supplementary Methods and Supplementary Notes 1–6

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Ranjit, S., Malacrida, L., Jameson, D.M. et al. Fit-free analysis of fluorescence lifetime imaging data using the phasor approach. Nat Protoc 13, 1979–2004 (2018). https://doi.org/10.1038/s41596-018-0026-5

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