A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

Abstract

Photobleaching limits extended imaging of fluorescent biological samples. Here, we developed DNA origami-based "Fluorocubes" that are similar in size to the green fluorescent protein (GFP), have single-point attachment to proteins, have a 50-fold higher photobleaching lifetime and emit 40-fold more photons than single organic dyes. We demonstrate that DNA Fluorocubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometerprecision.

Multiple-labeled antibodies behave like single emitters in photoswitching buffer

“…11a). As recently observed for multiple-labeled DNA origami 12 , the total photon output and on-time as a measure of photostability increased with increasing DOL in trolox buffer. On the other hand, in photoswitching buffer containing 100 mM MEA and oxygen scavenger multiple-Al647-labeled antibodies displayed on/off switching independent of the DOL of the antibody (Figs.…”

“…To minimize photobleaching especially in single-molecule tracking experiments semiconductor quantum dots (QDs) and fluorescent nanoparticles have been developed [9][10][11] . Furthermore, multiple dyelabeled probes such DNA-based FluoroCubes 12 , bottlebrush polymers with DNA-tipped bristles 13 and biotin-avidin signal amplifications 14 have been introduced in combination with immunolabeling and chemical tags to reduce photobleaching in highly sensitive fluorescence imaging experiments.…”

“…On the other hand, intermolecular quenching interactions between fluorophores, e.g. via formation of nonfluorescent dye aggregates should be avoided 12,13,[15][16][17] . Generally, standard immunlabeling with a primary and secondary antibody is used for imaging of endogenous proteins.…”

A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging

“…Example raw datasets of DNA FluoroCubes with six dyes, Single Dye Cubes, one dye double-stranded DNA constructs, single, biotinylated dyes, and Compact Cube used to determine the photophysical properties are hosted on Zenodo: https://doi.org/10.5281/zenodo.3561024 36 . All other data files are available from the authors upon request.…”

“…The latest version for MacOS and Windows can be downloaded here: https://micro-manager.org/wiki/Download%20Micro-Manager_Latest%20Release (version 2.0 gamma). The custom written Python code used to determine the total number of photons, the average number of photons per frame, and the half-life (photostability) for all fluorescent samples used in this study is hosted on Zenodo: https://doi.org/10.5281/zenodo.3629746 37 . All other code is available from the authors upon request.…”