Boulanger Jérôme, Gueudry Charles, Münch Daniel, Cinquin Bertrand, Paul-Gilloteaux Perrine, Bardin Sabine, Guérin Christophe, Senger Fabrice, Blanchoin Laurent, Salamero Jean
UMR144 CNRS/Institut Curie, 75005 Paris, France;
Plateforme Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie Institut Curie, 75005 Paris, France; Roper Scientific SAS, 91017 Evry, France; and.
Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17164-9. doi: 10.1073/pnas.1414106111. Epub 2014 Nov 17.
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.
全内反射荧光显微镜（TIRFM）是一种用于可视化各种细胞过程的首选方法，尤其适用于实时贴壁细胞质膜附近发生的特定事件。这种成像技术不依赖于特定的荧光探针，具有很高的切片能力。然而，它仅限于单个平面。我们在此提出一种基于通用设计的方法，该方法能够进行快速多波长方位平均和入射角扫描，以通过计算重建三维图像序列。我们在盖玻片上方800纳米的范围内实现了前所未有的50纳米轴向分辨率。我们应用这种成像方式来获取有关三维肌动蛋白结构的结构和动态信息。我们还以每秒七组图像堆栈的速度，从时间上解析三维空间中不同的Rab11a依赖性胞吐事件。
