Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxy-ethylcellulose solution

Three-dimensional (3-D) video fluorescence microscopy is demonstrated for t he investigation of biopolymer electrophoretic migration using double-stran ded (ds)DNA in semidilute hydroxyethylcellulose (HEC) as a test system. It is shown that 3-D imaging enables visualization of segmental motion with gr eater detail than is available in conventional video microscopy. A high fra me rate (50-110 frames per second (fps)) intensified progressive scan camer a is used to acquire fifteen axial sections focused at different depths thr ough the DNA molecule. A 3-D DNA image is generated from these sections usi ng blind deconvolution image reconstruction and motion is represented as a succession of volume images. A 3-D extension of the Doi/Oana ellipsoidal mo del is used to fit the DNA envelope, allowing simple quantitative descripti ons of the changing shape of the DNA as it interacts with the sieving polym er solution. With 3-D views of migrating DNA molecules we observe U-shaped conformations oriented at an angle to the microscope plane. We are also abl e to resolve ambiguities and artifacts resulting from loss of information f rom DNA segments that are not in focus.