High Resolution Single Molecule Optical Localization of Multiple Fluorophores on DNA Origami Constructs Fluorophores on DNA Origami Constructs
Date of Award
College of Science
Type of Degree
Michael L Norton
Brian Scott Day
An ongoing challenge in the development of nanoelectronics and nanophotonics is the nondestructive, high-resolution localization in space of single molecules and multi-molecular assemblies. The apparent barrier to the use of optical microscopy at the sub-100 nm scale is the well known Abbe Limit, the diffraction limit to resolution. This laboratory has adapted a technique called Single-molecule high resolution imaging with photobleaching (SHRIMP). We have developed methods for utilizing SHRIMP for the determination of the separation of two fluorophores in single DNA origami constructs.
DNA Origami is extremely useful because it can address nanocomponents down to 2nm separation. It should be possible to prepare standards for the measurement of distances on the order of 10-200nm, between light emitting particles in these DNA self-assembled nanoconstructs, by incorporating two fluorophores of the same color or different colors.
Particularly in this work, 1) two channel fluorescence imaging has been implemented using an Optosplit equipped Nikon microscope; 2) four types of DNA origami test objects have been constructed and characterized; 3) a highly effective system to remove adventitious fluorescent contamination from substrates and from buffer solutions has been constructed, because a pre-requisite for single molecule microscopy is the production and maintenance of coverslips and solutions with essentially no fluorescent contamination; 4) a software based protocol for Gaussian fitting single molecule images to find the center of fluorophores and to determine the distance between the fluorophores has been developed. The application of this software to an Origami construct with two rhodamine labels is presented.
DNA -- Research.
DNA -- Structure.
Rajulapati, Anuradha, "High Resolution Single Molecule Optical Localization of Multiple Fluorophores on DNA Origami Constructs Fluorophores on DNA Origami Constructs" (2009). Theses, Dissertations and Capstones. 805.
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