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

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.

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

“…Further experiments with state of the art super-resolution LM and staining with the most sensitive fluorescent dyes e.g. YOYO-1 could provide specific information already at the LM level during anaphase-A (Pyle and Chen 2017 ; Rocha et al 2017 ). These strands are withdrawn from the chromosomes, which could be explained by pulling forces from the MT-movement.…”

Three-dimensional super-resolution fluorescence imaging of DNA

“…1 a). SxO and YOYO-1 can be modelled as dissociating with mono and bi-phasic kinetics, as expected for mono and bis-intercalators 9 , 10 , respectively. YOYO-1 displayed slower dissociation compared to SxO due to an additional, slow, kinetic step (Fig.…”

Ultrafast transient absorption spectra of photoexcited YOYO-1 molecules call for additional investigations of their fluorescence quenching mechanism

“…It was carried out with a 473 nm solid-state excitation laser (Dragon Lasers, China) either under total internal reflection fluorescence (TIRF) or epifluorescence (EPI) mode; Nikon Ti-U inverted microscope with a Nikon 100× oil-immersed TIRF objective (CFI Apo 100×, NA 1.49, WD 0.12 mm); a fluorescent filter cube with ZET488/10 laser bandpass filter, ZT488rdc dichroic mirror, and ET500lp long pass filter (Chroma, USA); and an EMCCD camera (Andor iXon Ultra 897, USA). 21…”

“…Single-molecule fluorescent data were analysed using the open-source M TL B codes reported before. 21,44–46…”

“…16–20 YOYO-1, an oxazole yellow (YO) dimer and a member of the YOYO-TOTO family of cyanine dyes belonging to the polymethine group, is a widely used DNA fluorescent staining dye. 21–23 YOYO-1 has a high photon molar absorptivity, with peak extinction coefficient at a visible wavelength approaching 10 5 cm −1 M -1 . 22 The fluorescence quantum yield of YOYO-1 in water is usually very small (<0.1%) and thus is nonfluorescent.…”