Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging.

Joseph R Pyle,Jixin Chen

doi:10.3762/bjnano.8.229

Abstract

Super-resolution imaging of single DNA molecules via point accumulation for imaging in nanoscale topography (PAINT) has great potential to visualize fine DNA structures with nanometer resolution. In a typical PAINT video acquisition, dye molecules (YOYO-1) in solution sparsely bind to the target surfaces (DNA) whose locations can be mathematically determined by fitting their fluorescent point spread function. Many YOYO-1 molecules intercalate into DNA and remain there during imaging, and most of them have to be temporarily or permanently fluorescently bleached, often stochastically, to allow for the visualization of a few fluorescent events per DNA per frame of the video. Thus, controlling the fluorescence on–off rate is important in PAINT. In this paper, we study the photobleaching of YOYO-1 and its correlation with the quality of the PAINT images. At a low excitation laser power density, the photobleaching of YOYO-1 is too slow and a minimum required power density was identified, which can be theoretically predicted with the proposed method in this report.

Highlights

Fluorescence imaging of DNA with intercalating dyes is important for DNA sensing [1,2], nucleic acid imaging inside cells and viruses [3,4,5], DNA protein studies [6,7], and optical mapping [8,9,10]
We study the effect of laser power on both conventional fluorescent imaging and point accumulation for imaging in nanoscale topography (PAINT) imaging of single DNA molecules using the intercalating dye YOYO-1
Because single-molecule separation is still clearly seen at equilibrium (Figure 8b), the whole video can be used to generate the PAINT image (Figure 8c) and the video can run even longer until the DNA is saturated with YOYO-1

Summary

Introduction

Fluorescence imaging of DNA with intercalating dyes is important for DNA sensing [1,2], nucleic acid imaging inside cells and viruses [3,4,5], DNA protein studies [6,7], and optical mapping [8,9,10]. We study the effect of laser power on both conventional fluorescent imaging and PAINT imaging of single DNA molecules using the intercalating dye YOYO-1. YOYO-1 (5 nM) was flowed through the channel at 0.05 mL/min while recording 5100 frame videos of binding to DNA under TIRF illumination from the 473 nm laser with an exposure time of 50 ms and an EM gain of 200.

Results

Conclusion