Residual blinking-driven channel alignment for multicolor single-molecule localization microscopy

Given the nanometer-scale resolution of single-molecule localization microscopy (SMLM), sequential multicolor SMLM is inherently challenged by inter-channel misalignment arising from unpredictable sample drift during channel switching. Traditional fiducial-based strategies are limited by added complexity in sample preparation. Here, we report a residual blinking-driven method for channel alignment in multicolor SMLM, termed ReBling alignment. By exploiting residual blinking events under dual-wavelength excitation, our method extracts shared localizations as intrinsic registration anchors and performs accurate cross-correlation-based alignment. We validated ReBling alignment across biological systems featuring distinct spatial patterns, including continuous patterned structures (mitochondria-microtubule co-imaging, dual-labeled microtubules), and discrete point-cluster assemblies (dual-color nuclear pore complex components), demonstrating its broad applicability and reliability. These results establish ReBling alignment as a practical, versatile, fiducial-free solution for accurate channel registration in sequential multicolor SMLM, enabling more precise and reliable characterization of complex cellular architecture and molecular interactions.