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

作者:Boulanger Jerome*; Gueudry Charles; Muench Daniel; Cinquin Bertrand; Paul Gilloteaux Perrine; Bardin Sabine; Guerin Christophe; Senger Fabrice; Blanchoin Laurent; Salamero Jean
来源:Proceedings of the National Academy of Sciences, 2014, 111(48): 17164-17169.
DOI:10.1073/pnas.1414106111

摘要

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.

  • 出版日期2014-12-2
  • 单位中国地震局