Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging

Abstract : Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.
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https://hal.archives-ouvertes.fr/hal-01712220
Contributor : Perrine Paul-Gilloteaux <>
Submitted on : Monday, February 19, 2018 - 11:44:07 AM
Last modification on : Thursday, February 20, 2020 - 7:23:38 PM

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Jérôme Boulanger, Charles Gueudry, Daniel Münch, Bertrand Cinquin, Perrine Paul-Gilloteaux, et al.. Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2014, 111, pp.17164 - 17169. ⟨10.1073/pnas.1414106111⟩. ⟨hal-01712220⟩

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