DNA-PAINT (points accumulation for imaging in nanoscale topography) is a single-molecule localization microscopy (SMLM) technique that takes advantage of the high specificity of DNA hybridization for super-resolution imaging. The docking strand, linked to an affinity agent such as an antibody, localizes to a target in the cell. The localizations arise from the reversible hybridization between a docking strand and the freely diffusing fluorescently labeled imaging strand. DNA-PAINT thus increases the number of observations possible compared to other super-resolution techniques such as STORM or PALM, which are constrained by the photon budget of the fluorophore. However, DNA-PAINT suffers from slow imaging speed inhibiting its potential for observing biological systems.
In a recent publication in Nature Communications, Civitci et al. developed a method that improves the imaging speed of DNA-PAINT by increasing the localization kinetics of the imaging strand labeled with the photostable CF®660R. The goal was to devise a strategy compatible with a large panel of oligonucleotide pairs validated for DNA-PAINT and can be readily adopted into the existing workflows.
The authors hypothesized that increasing the imaging strand’s off-rate without slowing down the binding would improve the localization kinetics and improve the imaging speed. The authors demonstrate that the addition of ethylene carbonate (EC), a water-soluble aprotic solvent shown to speed up hybridization between target DNA and oligonucleotides, in the imaging buffer increases the off-rate without significantly affecting the on-rate. The authors further optimized DNA hybridization kinetics and efficiency by implementing tandem sequence repeats (R) in the docking strands and a PEG spacer (S) between the docking oligonucleotide and antibody. Cumulatively, these modifications (ERS) to DNA-PAINT increased both super-resolution imaging speed and quality.
Learn more about our bright and photostable CF® dyes for super-resolution applications, including single-label secondary antibodies for STORM. Also, learn more about our rapid and versatile Mix-N-Stain™ Antibody Labeling Kits for labeling antibodies with CF® dyes and other labels.
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Full Citation
Civitci, F., Shangguan, J., Zheng, T., Tao, K., Rames, M., Kenison, J., Zhang, Y., Wu, L., Phelps, C., Esener, S., Nan, X. (2020). In Vivo Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow. Nature Communications. https://doi.org/10.1038/s41467-020-18181-6