Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

被引：2

作者：

Harkes, Rolf ^{[1
]}

Keizer, Veer I. P. ^{[2
]}

Schaaf, Marcel J. M. ^{[2
]}

Schmidt, Thomas ^{[1
]}

机构：

[1] Leiden Univ, Phys Life Proc, Huygens Kamerlingh Onnes Lab, Leiden, Netherlands

[2] Leiden Univ, IBL, Leiden, Netherlands

来源：

PLOS ONE | 2015年 / 10卷 / 11期

关键词：

TRANSCRIPTION FACTOR-BINDING; LIVING CELLS; PARTICLE TRACKING; PLASMA-MEMBRANE; HIGH-DENSITY; LIVE CELLS; MICROSCOPY; MICRODOMAINS; PROTEIN; DNA;

D O I：

10.1371/journal.pone.0141080

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：

Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49 +/- 0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.

引用

页数：13