Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus |
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| Authors: | Rolf Harkes Veer I P Keizer Marcel J M Schaaf Thomas Schmidt |
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| Institution: | 1. Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.; 2. Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.; University of Ulm, GERMANY, |
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| Abstract: | 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. |
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