Chiral Single Molecule Localization Microscopy (chiralSMLM)

Chiral fingerprints are unique to a molecule and convey information related to binding sites, conformation, and its immediate chemical environment. Deciphering these information is expected to aid a better understanding of the biological processes at the very molecular scale. Here, we present a new technique called chiralSM LM , to selectively detect chiral enantiomers (left-handed and right-handed) of single molecules and generate a super-resolved map of their distribution in cell. Calibration, characterization, and optimization of the system is carried out using a polarized light source (left and right circularly) and by recording known structures (Actin filaments) in a cell. Subsequently, the distribution of Dendra2-Actin enantiomers on the actin filaments is mapped. Finally, chiralSM LM is employed to study the characteristic distribution of viral proteins (Hemaglutinin and NS3) in transfection studies of two diseases (Influenza type-A and Dengue type2) after 48 hrs of transfection. Single-molecule and cluster analysis reveals that left-handed HA and left-handed NS3 molecules have a larger footprint, suggesting the role of chiral molecules in promoting cluster formation during viral infection. This is amazing, considering the fact that Left-handed enantiomers are known to play a crucial role in a range of biological functions. The new classification of single molecules purely based on their chiral nature is expected to advance single-molecule imaging.