Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy.
Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.
Blumberg
Seth
S
Department of Physics, Biophysics Research Division, Randall Laboratory, University of Michigan, Ann Arbor, 48109-1120, USA.
Gajraj
Arivalagan
A
Pennington
Matthew W
MW
Meiners
Jens-Christian
JC
D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D009038:Motion; D009690:Nucleic Acid Conformation; D049489:Stroboscopy / Q000295:instrumentation / Q000379:methods*
D004247:DNA