2015/04/14 by Helen Miller, Miller, Helen, Zhaokun Zhou +5
Biochemistry, Genetics and Molecular Biology · Engineering · #Advanced Fluorescence Microscopy Techniques #Biological Physics (physics.bio-ph) #Biomolecules (q-bio.BM) #FOS: Biological sciences #FOS: Physical sciences #Near-Field Optical Microscopy #Plasmonic and Surface Plasmon Research
paper · pdf · doi:10.48550/arxiv.1504.03487
openalex publication_date 2015/04/14 · openalex created_date 2022/10/01 · openalex updated_date 2026/07/28
As proof-of-principle for generating superresolution structural information\nfrom DNA we applied a method of localization microscopy utilizing photoblinking\ncomparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13,\nusing a bespoke multicolor single-molecule fluorescence microscope. We used a\nfull-length ~49 kbp \λ DNA construct possessing oligo inserts at either\nterminus allowing conjugation of digoxigenin and biotin at opposite ends for\ntethering to a glass coverslip surface and paramagnetic microsphere\nrespectively. We observed stochastic DNA-bound dye photoactivity consistent\nwith dye photoblinking as opposed to binding/unbinding events, evidenced\nthrough both discrete simulations and continuum kinetics analysis. We analyzed\ndye photoblinking images of immobilized DNA molecules using superresolution\nreconstruction software from two existing packages, rainSTORM and QuickPALM,\nand compared the results against our own novel home-written software called\nADEMS code. ADEMS code generated lateral localization precision values of 30-40\nnm and 60-70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling,\nsimilar to rainSTORM, running more slowly than rainSTORM and QuickPALM\nalgorithms but having a complementary capability over both in generating\nautomated centroid distribution and cluster analyses. Our imaging system allows\nus to observe dynamic topological changes to single molecules of DNA in\nreal-time, such as rapid molecular snapping events. This will facilitate\nvisualization of fluorescently-labeled DNA molecules conjugated to a magnetic\nbead in future experiments involving newly developed magneto-optical tweezers\ncombined with superresolution microscopy.\n