Dual-view microscopy of single-molecule dipole orientations

Measuring dipole orientations of fluorescent probes offers unique local structural insights of labelled biomolecules and has seen expanding applications in structural biology studies. Here we propose an alternative imaging geometry, dual-view microscopy, for single-molecule dipole orientation measurements. We develop a protocol capable of simultaneously measuring absorption and emission dipole orientations of single emitters. Further, through simulation, we demonstrate that absorption dipole orientation can be accurately measured with high and uniform precision in three dimensions, significantly outperforming epifluorescence microscopy. Meanwhile the emission dipole is independently narrowed down to four possible orientations and can be uniquely determined with the co-estimated absorption dipole. Dual-view microscopy represents a new paradigm in single-molecule orientation sensing and could have unique applications in imaging under cryogenic temperatures. WHY IT MATTERS?The transition dipole orientations of fluorescent molecules bound to target biomolecules give access to the targets local structural information, therefore can serve as unique probes in structural biology. This work proposes an alternative imaging geometry, referred to as dual-view microscopy for more efficient three-dimensional single-molecule dipole orientation measurements compared to existing schemes. We demonstrate using simulation that our scheme outperforms conventional epifluorescence microscopy in estimation precision and can simultaneously measure absorption and emission dipole orientations independently. Dual-view microscopy represents a new paradigm that will further advance single-molecule orientation imaging as an emerging structural biology tool.