Abstract
Measuring forces within living cells remains a technical challenge. In this Tutorial, we cover the development of hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing. Flipper probes can therefore be used as reporters for membrane tension via the measurement of changes in their fluorescence lifetime. We describe the technical optimization of the probe for imaging and provide working examples for their characterizations in a variety of biological and in vitro systems. We further provide a guideline to measure biophysical parameters of cellular membranes by fluorescence lifetime imaging microscopy using Flipper probes, providing evidence that flippers can report long range forces in cells, tissues and organs.
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Acknowledgements
We thank S. Srinivas and C. Royer (University of Oxford) for isolating and staining the mouse embryos. F.S. is grateful for the support from the EMBO (ALTF 849-2020) and HFSP (LT000404/2021-L) long-term postdoctoral fellowships. F.S. thanks S. Fraser (University of Southern California) for the support and supervision and acknowledges the Translational Imaging Center (University of Southern California) for access to instrumentation and expertise. A.C. also acknowledges funding from MCIU, PID2019-111096GA-I00; MCIU/AEI/FEDER MINECOG19/P66, RYC2018-024686-I and the Basque Government T1270-19. V.D. thanks P.-F. Lenne (IBDM Marseille) for supervision and resources. V.D. acknowledges support by an HFSP long-term postdoctoral fellowship (HFSP LT0058/2022-L) and the France–BioImaging infrastructure supported by the French National Research Agency (ANR–10–INBS- 04-01, Investments for the future). V.D. thanks F. Schnorrer (IBDM Marseille) for access to the FLIM system. S.M. thank the University of Geneva, the National Centre of Competence in Research Chemical Biology (51NF40-185898), the National Centre of Competence in Research Molecular Systems Engineering (51NF40-182895) and the Swiss NSF (Excellence Grant 200020 204175; SNSF-ERC Advanced Grant TIMEUP, TMAG-2_209190) for financial support. A.R. acknowledges funding from the Swiss National Fund for Research Grants nos. 310030_200793 and no. CRSII5_189996 and the European Research Council Synergy Grant no. 951324 R2-TENSION.
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The project was designed by C.R., A.R. and V.M. C.R. and V.M. carried out most of the experiments and analyses. J.M.G.-A. performed the experiments on bacteria, P.C. performed some experiments on MDCK, C.T. performed experiments on alginate tubes, F.S. performed the biphoton experiments, I.D.M. performed experiments with alginate capsules, A.C. performed experiments on Arabidopsis, K.B. and V.D. performed the experiments on Xenopus and J.L.-A. and S.M. designed and synthesized the Flippers molecules. C.R., V.M. and A.R. wrote the paper, with corrections from all co-authors.
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Extended data
Extended Data Fig. 1 FLIM of Flipper-TR using two-photon microscopy.
(a) Absorption and excitation spectrum of the Flipper-TR. (b) Two-photon excitation spectra of Flipper-TR, acquired in 48h gastruloids. Five gastruloids were imaged in total at each wavelength. The acquired spectra were normalized to the maximum intensity value. Dots represented the average value, scattered lines the minimum/ maximum values of the 5 recorded spectra. (c) and (d): Lifetime of Flipper-TR in GUVs of different compositions DOPC (c) and 1:4 DOPC:Cholesterol (d) at different laser repetition rates and under two-photon excitation. Every dot represents fitted lifetime for 1 GUV, error bars are standard deviation and line is mean. (e) Fluorescence lifetime images of GUVs from (c, d) under typical excitation conditions (20 MHz, 488 nm, top panel) and under two-photon excitation (80 MHz, 900 nm, bottom panel). Longer lifetime component is shown (shorter lifetime component fixed). Image size is 36.4 × 36.4 µm2.
Extended Data Fig. 2 Effect of linearly polarized excitation light on Flipper-TR lifetime measurements in GPMVs and GUVs.
(a) Intensity and (b) Lifetime images of cell-derived vesicles. Photoselection of the fixed excitation dipole of the Flipper-TR probe (using a linearly polarized excitation laser) causes bright rings with higher intensity. Graphs show (c) Lifetime and (d) intensity analysis of bright vs dark regions. Every dot represents the lifetime fit across one vesicle. The line represents the mean and error bars are standard deviation. (e) Intensity and (f) lifetime and image of artificial vesicle composed of DOPC and Cholesterol (4:1). Image size is 36.4*36.4 µm2. The graphs represent (g) lifetime and (h) intensity analysis of bright vs dark regions. Every dot represents the lifetime fit across one vesicle. The line represents the mean and error bars are standard deviation.
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Roffay, C., García-Arcos, J.M., Chapuis, P. et al. Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes. Nat Protoc 19, 3457–3469 (2024). https://doi.org/10.1038/s41596-024-01027-6
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DOI: https://doi.org/10.1038/s41596-024-01027-6
