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-01133826
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Submitted on : Friday, March 20, 2015 - 2:31:13 PM
Last modification on : Thursday, February 20, 2020 - 7:23:25 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 (48), pp.17164-9. ⟨hal-01133826⟩

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