Orientation Single Molecule Localization Microscopy (oSMLM) for Decoding Orientation of Single Molecule

Abstract

Standard SMLM facilitates the reconstruction of super-resolution map (both location and localization precision) of the target single molecules. In fact, single molecule data does provide information related to the orientation of single molecules, which can be derived from the knowledge of PSF shape and its direction. This information is vital to probe the sub-domain of macromolecules that undergo orientation and conformational changes and provides essential clues on their catalytic activity. Accessing this information in real-time opens up a powerful new window to look into the link between the orientation of macromolecules and the output function. Here, we decode the orientation of single molecules from the knowledge of PSF shape and its direction. The method is primarily based on field-dipole interaction and the fact that the distribution of emitted photons strongly depends on the orientation of the dipole (fluorophore) with respect to the polarization of light. Accordingly, the photon emission from the specimen and the resultant PSF distribution model is developed. Computational studies show changes in the shape and orientation of the recorded PSF (in the image / detector plane). Specifically, a set of three distinct distributions (Gaussian, bivariate-Gaussian and skewed-Gaussian) are recognized from the study, apart from a superset of all possible (a total of 16) distributions. Experiments were conducted on Dendra2-Actin and Dendra2-HA transfected cells that validate the emission model. We report a localization precision of∼20nmand an orientation precision of±5°. In addition, the distinct orientation of single molecules is noted for Actin and HA in a cell (Influenza type-A model). Further analysis suggests a preferred directional distribution of Dendra2-Actin single molecules, while Dendra2-HA molecules seem to be randomly oriented in a cluster. The availability of orientation information in SMLM without the need for additional optics adds a new feature, which can be explored to reveal the state of a single molecule (orientation and conformational changes) in cellular sub-domains / partitions. The study implies that the orientation of single molecules that has more profound implications for the functioning of macromolecules. The orientation information revealed byoSM LMtechnique gives it a wide-spread appeal and expands the reach of localization microscopy.