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Link to original content: https://pubmed.ncbi.nlm.nih.gov/29181286
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. 2017 Nov 2:8:2296-2306.
doi: 10.3762/bjnano.8.229. eCollection 2017.

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

Joseph R Pyle  1 , Jixin Chen  1
Affiliations

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

Joseph R Pyle et al. Beilstein J Nanotechnol. .

Erratum in

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.

Keywords: PAINT; diffusion; single-molecule photophysics; super-resolution imaging.

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Figures

Figure 1
Figure 1
Scheme of super-resolution imaging of DNA with PAINT. (a) A scheme depicting the localization of single molecules in each frame. (b) Scheme of the reconstruction of a super-resolution image on top of a regular fluorescence image from the super-localized molecules in each frame.
Figure 2
Figure 2
Scheme and image of the optical microscope.
Figure 3
Figure 3
(a) Scheme of glass surface modification and DNA immobilization. (b) Fluorescent image of stretched DNA molecules on the substrate that have been labeled with YOYO-1. The inset shows a droplet of water on the substrate before DNA immobilization, giving a water contact angle of 48 ± 4°.
Figure 4
Figure 4
(a) A single DNA molecule (top), the mask used (middle), and overlay (bottom) to select the DNA and filter the other events (the arrow). (b) The intensity (total photocounts) per micrometer of single DNA molecules as a function of laser power density. The images are obtained under TIRF mode but the power densities are measured under epifluorescence mode. YOYO-1 molecules are premixed with λ-DNA at a dye/base pair ratio of 1:10. The error bars represent the standard deviation between at least 25 DNA for two samples.
Figure 5
Figure 5
(a) The time trace of the photocounts per micrometer of a single DNA strand for power densities of 62 W cm−2 (red), 38 W cm−2 (orange), 23 W cm−2 (green), 10 W cm−2 (blue), and 1.9 W cm−2 (violet). (b) Image of a DNA molecule at different times of the video. All images have the same color scale. A laser power density of 38 W cm−2 was used.
Figure 6
Figure 6
(a) The fast component of the bleaching lifetime for YOYO–DNA as a function of power density. (b) The slow component of the bleaching lifetime for YOYO–DNA as a function of power density. The error bars represent the standard deviation between at least 20 DNA for two samples.
Figure 7
Figure 7
(a) Fluorescence images of regular and (b) super-resolved YOYO–DNA (inset shows a line profile across two nearby DNA molecules). Laser power density 23 W cm−2. (c) A single frame of the video of fluorescent images. The circles are events chosen and the arrows are events discarded in generating the image in (b). (d) PSF peak intensity as a function of laser power density (inset shows histograms of the background and PSF peak intensities, see Supporting Information File 1, Figure S4 for larger images). The error bars represent the standard deviation of the photocounts of YOYO-1 molecules for three samples.
Figure 8
Figure 8
Single-frame images of a single DNA molecule at (a) 1.9 W cm−2 and (b) 38 W cm−2 power densities (time shown is in unit of seconds) and (c) corresponding PAINT images. Red circles show events selected and arrows show the events discarded by the code and bleached later. All scale bars are 5 μm. (d, e) Normalized total photocounts of 1 μm of DNA in each frame at 1.9 W cm−2 and 38 W cm−2 power densities, respectively. The red double arrows indicate where equilibrium is maintained. (f) The average number of dye molecules selected in the frames during the PAINT analysis. The error bars represent the standard deviation between ≈20 DNA for two samples.
Figure 9
Figure 9
Scheme of molecular diffusion in the solution.
See this image and copyright information in PMC

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