Channel-linked multi-emitter fitting enables fast and accurate multi-channel single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) provides nanometer-scale spatial resolution for visualizing cellular structures. Multi-channel SMLM further extends its capabilities by incorporating single-molecule information from multiple channels, enabling more information-rich imaging modalities. However, both SMLM and its multi-channel variants rely on sparse activation, which limits imaging speed and labeling density. Here, we introduce climeLoc, a channel-linked multi-emitter fitter designed to correlate and localize emitters at high density across multiple channels. By integrating pre-calibrated channel transformations into a Bayesian-based multi-emitter fitting framework, climeLoc significantly enhances 3D localization accuracy for high-density multi-channel SMLM data. We show that climeLoc resolves the 3D hollow cylindrical structures of microtubules within 400 seconds, reducing imaging time by 70∼90% compared to conventional methods, and to resolve finer details of the densely packed chromatin structures in the nucleus. With a simple graphic user interface, climeLoc will empower a broad community to perform multi-channel SMLM on dense structures at optimal imaging speeds.