Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. – ScienceOpen
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      Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes.

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          Abstract

          As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49 kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30-40 nm and 60-70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.

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          Author and article information

          Journal
          Methods
          Methods (San Diego, Calif.)
          Elsevier BV
          1095-9130
          1046-2023
          Oct 15 2015
          : 88
          Affiliations
          [1 ] Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, United Kingdom.
          [2 ] Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, United Kingdom. Electronic address: mark.leake@york.ac.uk.
          Article
          S1046-2023(15)00016-X
          10.1016/j.ymeth.2015.01.010
          25637032
          bac01632-1b5a-48d6-8d07-832cb55ad8fd
          History

          BaLM/BALM,Magnetic tweezers,Photoblinking,SYTO,Single-molecule,YOYO

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