Mehta, S.B. et al. Proc. Natl. Acad. Sci. USA 113, E6352–E6361 (2016).

Many biological structures depend on the proper orientation of molecular building blocks. However, studying an object's position and orientation in living cells can be challenging. Mehta et al. developed an instantaneous fluorescence polarizing microscope (instantaneous FluoPolScope) to enable tracking of single-molecule positions and orientations in living cells. The instantaneous FluoPolScope uses total internal reflection excitation with a custom image-splitting device that enables sorting of emitted fluorescence along four polarization orientations. The researchers show that their microscope can be used to track the positions and orientations of sparsely labeled proteins in order to study dynamic structural rearrangements, and it can also be used to track the position and orientation of protein subunits as they interact with higher-order assemblies. The microscope can potentially shape understanding of how relatively large ordered assemblies are formed from individual molecules.