Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxyethylcellulose solution.

Three-dimensional (3-D) video fluorescence microscopy is demonstrated for the investigation of biopolymer electrophoretic migration using double-stranded (ds)DNA in semidilute hydroxyethylcellulose (HEC) as a test system. It is shown that 3-D imaging enables visualization of segmental motion with greater detail than is available in conventional video microscopy. A high frame rate (50-110 frames per second (fps)) intensified progressive scan camera is used to acquire fifteen axial sections focused at different depths through the DNA molecule. A 3-D DNA image is generated from these sections using blind deconvolution image reconstruction and motion is represented as a succession of volume images. A 3-D extension of the Doi/Oana ellipsoidal model is used to fit the DNA envelope, allowing simple quantitative descriptions of the changing shape of the DNA as it interacts with the sieving polymer 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 able to resolve ambiguities and artifacts resulting from loss of information from DNA segments that are not in focus.