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Updated: Mar 30, 2026
High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
Published on: July 25, 2014
Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in
Rolf Harkes1, Veer I P Keizer2, Marcel J M Schaaf2
1Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
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.
