Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going ‘beyond the diffraction barrier’ comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.
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We thank P. Sandoz and G. van der Goot for construction of Lifeact-Dreiklang (VDG-EPFL), the LSBG-EPFL for providing RAW 264.7 cells and M. Ricchetti from Institute Pasteur for human fibroblast cells. We are grateful to M. Sison for cell culture advice and assistance. We thank O. Peric and G. Fantner (LBNI-EPFL) for providing the technical sample and performing the AFM measurement. We acknowledge A. Radenovic and A. Nahas for support and discussion and G. M. Hagen for proofreading of the manuscript. This project has been partly funded from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. . The research was supported by the Swiss National Science Foundation (SNSF) under grant 200020_159945/1. T.L. acknowledges the support from the Horizon 2020 Framework Programme of the European Union via grant 686271/SEFRI 16.0047.
The authors declare no competing interests.
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The theory of partially coherent image formation, the retrieval of the complex three-dimensional cross-spectral density, the experimental and simulated results of the point-spread function in amplitude and phase, quantitative phase calibration using technical samples, the PRISM multi-plane platform for three-dimensional phase and SOFI imaging and supplementary figures and methods.
This video shows a living human fibroblast at an imaging speed of 200 Hz as it migrates on a glass substrate.
This video shows long-term three-dimensional imaging of a dividing HeLa cell undergoing mitosis from the metaphase to the telophase.
This video shows the imaging of the vimentin network in HeLa cells and the cell dynamics by longer-term phase imaging
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Descloux, A., Grußmayer, K.S., Bostan, E. et al. Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy. Nature Photon 12, 165–172 (2018). https://doi.org/10.1038/s41566-018-0109-4
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