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siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data


We developed single-image fluorescence lifetime imaging microscopy (siFLIM), a method for acquiring quantitative lifetime images from a single exposure. siFLIM takes advantage of a new generation of dedicated cameras that simultaneously record two 180°-phase-shifted images, and it allows for video-rate lifetime imaging with minimal phototoxicity and bleaching. siFLIM is also inherently immune to artifacts stemming from rapid cellular movements and signal transients.

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Figure 1: Single-image FLIM time-lapse experiment.
Figure 2: siFLIM detection of histamine-induced alterations in Gq activation and Ca2+ concentration in cultured HeLa cells.
Figure 3: Pushing siFLIM to its limits.


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We thank J. Klarenbeek for plasmids encoding EPAC sensors and W. Zwart and J. Neefjes for providing plasmids encoding GFP-Rab7 and RFP-RILP. This research is funded from ERC grant 249997, awarded to T. Sixma; KWF grant NKI2010-4626, awarded to K.J.; and IOP Project IPD083412A awarded by Agentschap NL to K.J. and I.T.Y. The LI2CAM FLIM-camera was purchased with help of the Maurits and Anna de Kock foundation.

Author information




M.R., K.M.K. and Q.Z. performed the experiments and M.R., K.M.K., B.v.d.B., I.T.Y. and K.J. analyzed the data. J.H., S.d.J., B.v.d.B. and K.J. optimized hardware and software for siFLIM. T.W.J.G., B.v.d.B., K.M.K. and K.J. developed the mathematical framework for siFLIM. K.M.K. and K.J. carried out the sensitivity analysis. I.T.Y. led the team developing the MEMFLIM chip; J.G. and M.M. developed and tested the Gq sensor; K.J., M.R., K.M.K., B.v.d.B. and I.T.Y. wrote the manuscript and K.J. conceived and supervised the study.

Corresponding author

Correspondence to Kees Jalink.

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Competing interests

M.R., K.M.K., Q.Z., B.v.d.B., J.G., M.M., T.W.J.G., I.T.Y. and K.J. declare no competing financial interests. J.H. and S.d.J. are employees of Lambert Instruments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Notes 1 and 2, and Supplementary Discussion (PDF 5440 kb)

Rapid Ca2+ oscillations as detected by siFLIM

Lifetime data are calculated from the MEMFLIM image pair recorded at Φ = 60°. HeLa cells loaded with fluorescent calcium indicator were stimulated with histamine (t = 20 s), with ionomycin (t = 210 s) followed by addition of extra Ca2+ (3 mM at t = 220 s). The intensity line-plots are taken from the ROIS with corresponding colors. The kymograph shows lifetime data taken from the rectangle indicated in white. Sampling rate is 6 Hz. (AVI 23186 kb)

siFLIM is immune to movement artefacts

Lifetime data recorded by 12-phase frequency-domain FLIM (left) and by siFLIM (right) from HeLa cells expressing GFPRab7 and mRFP-RILP. Note that fast-moving vesicles display artefacts in the 12-phase data but not in the siFLIM results. (AVI 1036 kb)

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Raspe, M., Kedziora, K., van den Broek, B. et al. siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data. Nat Methods 13, 501–504 (2016).

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