Dual-color single-molecule localization microscopy with a sub-nanometer channel misalignment and sub-1% color...

Multicolor single-molecule localization microscopy (SMLM) plays a critical role in studying biomolecular interactions at the subcellular organelle scale, owing to its nanoscale resolution. However, the study of interactions between proteins only a few nanometers in size (such as globulins and insulin) requires colocalization analysis with sub-nanometer precision. Although existing super-resolution imaging techniques have achieved breakthroughs in resolution (sub-nanometer) and color crosstalk rate (<1%), channel registration errors remain at the nanometer scale (3-10 nm). This registration error would cause nanometer-scale deviations between channels, and ultimately compromise the reliability of sub-nanometer colocalization analysis. Here, we propose a dual-color single-molecule localization imaging method based on periodic trigger synchronization. In this scheme, an optical chopper is used to make two CW lasers alternately illuminate the samples at millisecond intervals, while the signals are collected synchronously, thus reducing channel misalignment to a sub-nanometer level (<1 nm). Fluorescent microsphere model experiments show that compared with the sequential imaging strategy, this method increases the Spearman correlation coefficient describing the spatial correlation of colocalization by 18% to 24%, and increases the Manders coefficient evaluating the degree of molecular overlap by 4% to 7%. Meanwhile, it is verified that the crosstalk rate between channels is less than 1% by the dual-channel imaging experiment of the single-color labeled samples. In summary, this method is expected to achieve sub-nanometer channel registration accuracy while maintaining low crosstalk, offering a robust approach to advance the channel registration accuracy of multicolor SMLM from the nanometer level to the sub-nanometer level.