The development of single-molecule localization microscopy (SMLM) provides a powerful tool for exploring nanostructures in cells that were previously concealed by diffraction limits. Besides the spatial resolution, multicolor imaging in SMLM is another important aspect critical for investigating protein colocalizations and organelle interactions in biological research.

Recently, researchers from the Institute of Biophysics (IBP) of the Chinese Academy of Sciences (CAS) have developed a new multicolor SMLM method called excitation-resolved stochastic optical reconstruction microscopy (ExR-STORM). The work was published in Light Science & Applications.

The newly developed method utilized three excitation lasers (620 nm, 639 nm and 671 nm), which were synchronized with a resonant scanner. In this manner, the single molecule image under three excitation lasers could be obtained in one frame. The resonant mirror is the key point in the method since single molecules blink much faster than the exposure time of the camera, and the right photon number distribution without this fast-switching technique cannot be recorded.

To verify the capability of this method, microtubules, intermediate filaments, endoplasmic reticulum and the outer mitochondrial membrane were labeled with DyLight 633, Alexa Fluor 647, Dyomics 654 and CF660C, respectively. And tetra-color super-resolution images were reconstructed successfully with negligible cross-talk.

This method yields several advantages. First, dyes with similar emission spectra could be resolved, resulting in negligible chromatic aberrations. Second, it resolved four far-red dyes with only one objective, extending color channels with cross-talk of less than 3%. Third, the high scanning speed could eliminate the brightness fluctuation of single molecules, compatible with fast dynamics of STORM imaging.

In short, this work provides a powerful tool for high-throughput investigation of the 3D nanostructures in cells. This capability shows its great potential in the biological and medical research. The researchers believe that ExR-STORM allow further investigation of organelle organization and interactions in cell stress responses, migration, division and differentiation.