Fluorescence microscopy enables simultaneous observation of the dynamics of single molecules in a large region of interest. Most traditional techniques employ either the geometry or the color of single molecules to uniquely identify (or barcode) different species of interest. However, these techniques require complex sample preparation and multicolor hardware setup. In this work, we introduce a time-based amplification-free single-molecule barcoding technique using easy-to-design nucleic acid strands. A dye-labeled complementary reporter strand transiently binds to the programmed nucleic acid strands to emit temporal intensity signals. We program the DNA strands to emit uniquely identifiable temporal signals for molecular-scale fingerprinting. Since the reporters bind transiently to DNA devices, our method offers relative immunity to photobleaching. We use a single universal reporter strand for all DNA devices making our design extremely cost-effective. We show DNA strands can be programmed for generating a multitude of uniquely identifiable molecular barcodes. Our technique can be easily incorporated with the existing orthogonal methods that use wavelength or geometry to generate a large pool of distinguishable molecular barcodes thereby enhancing the overall multiplexing capabilities of single-molecule imaging.
Keywords: DNA barcodes; DNA kinetics; TIRF; nanoscale fingerprinting; single-molecule imaging; temporal patterns.