NanorulerQA: quantitative quality analysis of dual-color DNA nanorulers via single-molecule photobleaching step counting and spatio-temporal colocalization.

Dual-color DNA nanorulers are critical tools for characterizing the resolution of dual-color super-resolution imaging systems. However, existing quality analysis methods struggle to directly quantify the number of dye molecules (i.e., the degree of labeling) within these nanorulers, which precludes precise guidance for optimizing fabrication processes. Here, we present a novel quality analysis framework for dual-color DNA nanorulers, termed NanorulerQA. This framework employs a spatio-temporal colocalization algorithm to reduce the color crosstalk rate in dual-color nanoruler identification while improving throughput, and it utilizes single-molecule photobleaching step counting to calculate the absolute number of dye molecules. Experimental results demonstrate that our framework achieves a color crosstalk rate of less than 2%, comparable to state-of-the-art performance. Furthermore, applying this framework to compare fabrication protocols for dual-color DNA nanorulers (Cy3 & Atto647N) reveals that one protocol yields a significantly higher dual-color labeling degree (69.11% vs. 21.34%) and a greater proportion of intensity-matched nanorulers (34.15% vs. 11.30%). This work establishes a direct correlation between fabrication conditions and quality, providing precise feedback for optimizing preparation protocols and enabling practical quality spot-checking.