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

Abstract

AbbreviationsDIG -digoxigenin, FWHM -full width at half maximum, λ DNA -Lambda phage DNA PSF -point spread function, SNR -signal-to-noise ratio. Highlights: A bifunctional DNA construct tethers single molecules to paramagnetic beads  Intercalating (YOYO-1) and minor groove (SYTO-13) dyes are bound to DNA and photoblink  Stochastic dye photoblinking is used to obtain superresolution information via localization microscopy 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 fluorescentlylabeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.

Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements

“…[52]. To perform localization microscopy, we employed a scheme similar to PAINT [15,53] previously described in [54-56], in which dyes in solution continuously bind to the DNA, becoming bright spots which can be localized sequentially (see Supplement 1 ). Samples were imaged in 15.5 μm × 15.5 μm fields of view for periods of approximately 13.5 minutes (15,000 camera frames), yielding between 100,000-300,000 localizations and 10,000-30,000 single-molecule orientation measurements.…”

Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements

“…[52]. To perform localization microscopy, we employed a scheme similar to PAINT [15,53] previously described in [54][55][56], in which dyes in solution continuously bind to the DNA, becoming bright spots which can be localized sequentially (see Supplement 1). Samples were imaged in 15.5 μm × 15.5 μm fields of view for periods of approximately 13.5 minutes (15,000 camera frames), yielding between 100,000-300,000 localizations and 10,000-30,000 single-molecule orientation measurements.…”

Frequent exchange of the DNA polymerase during bacterial chromosome replication

“…Tracking was then done automatically using a previously developed custom program in MATLAB (Mathworks), ADEMS code (Miller et al, 2015) (freely available at https://sourceforge.net/projects/york-biophysics/). Most experiments analyzed had a pixel size of 100 nm, for which we used a search window with a radius of 5 pixels and an initial guess for the PSF of 3 pixels when fitting candidate spots.…”