Molecular resolution imaging based on two-color single-molecular localization microscopy (SMLM)

Single-molecule localization microscopy (SMLM) breaks the diffraction limit, significantly enhancing optical resolution and providing a powerful tool for imaging complex biological structures in life sciences. However, imaging biological structures below 10 nm and studying protein-protein interactions remains a challenge for optical super-resolution microscopy. Here, we introduce a microscopy based on two-color single-molecule localization microscopy, we call it molecular Imaging by two-color imaging (MITI), which improves resolution to the molecular level, reaching several nanometers. We demonstrate MITI is suitable for samples labeled with organic dyes and photoactivatable/photoswichable fluorescent proteins. MITI can resolve two dyes separated by ssDNA with 2-10 nm length as well as ffDronpa and PAmCherry1 fused to the N- and C-terminal of FtsZ (ffDronpa-FtsZ-PAmCherry1). MITI is versatile and easy to realize, and thus, it will expand the applications of molecular scale super-resolution microscopy in life sciences.