  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 {\lambda} 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.
