title: Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus.
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.
authors.affiliation: Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
authors.forename: Rolf
authors.initials: R
authors.lastname: Harkes
authors.affiliation: Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.
authors.forename: Veer I P
authors.initials: VI
authors.lastname: Keizer
authors.affiliation: Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.
authors.forename: Marcel J M
authors.initials: MJ
authors.lastname: Schaaf
authors.affiliation: Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmidt
mesh_terms: D000465:Algorithms; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004058:Diffusion; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009038:Motion; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013997:Time Factors; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria