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

被引：69

作者：

Boulanger, Jerome ^{[1
]}

Gueudry, Charles ^{[2
,3
]}

Muench, Daniel ^{[2
,3
]}

Cinquin, Bertrand ^{[1
]}

Paul-Gilloteaux, Perrine ^{[1
,2
]}

Bardin, Sabine ^{[1
]}

Guerin, Christophe ^{[4
]}

Senger, Fabrice ^{[4
]}

Blanchoin, Laurent ^{[4
]}

Salamero, Jean ^{[1
,2
]}

机构：

[1] Inst Curie, CNRS UMR144, F-75005 Paris, France

[2] Inst Curie, Plateforme Imagerie Cellulaire & Tissulaire Infra, F-75005 Paris, France

[3] Roper Sci, F-91017 Evry, France

[4] Univ Grenoble 1, INRA,CEA, CNRS,Inst Rech Technol & Sci Vivant, Lab Physiol Cellulaire & Vegetale, F-38054 Grenoble, France

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2014年 / 111卷 / 48期

关键词：

TIRFM; high resolution; living cells; 3D image reconstruction; membrane recycling; LIMIT; ILLUMINATION; GRANULES; LANGERIN; RECEPTOR; TIRF;

D O I：

10.1073/pnas.1414106111

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

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.

引用

页码：17164 / 17169

页数：6

共 37 条

[1] Visualization of regulated exocytosis with a granule membrane probe using total internal reflection microscopy [J].