Wang, G. et al. Cell 167, 1839–1852.e21 (2016).
Single-molecule imaging allows scientists to probe the conformation, dynamics, and activities of biological molecules. However, the axial resolution of single-molecule setups is typically lower than the lateral resolution, making isotropic 3D measurements challenging. Wang et al. addressed this challenge by using interferometry to gain information about a fluorophore's position in the z-dimension, thereby developing multicolor single-molecule interferometric super-resolution microscopy. The team validated the method using dsDNA rulers and demonstrated the power of the approach by examining nuclear pore complex proteins in mammalian cells. To demonstrate the full versatility of the approach, the researchers studied the transcription cycle of the Escherichia coli RNA polymerase with good spatiotemporal resolution.