6D Single-Molecule Orientation-Localization Microscopy: Fundamental Limits for Visualizing the Dynamic Organization of Biomolecule

Fluorescent molecules are uniquely versatile quantum emitters whose emissive properties can be exquisitely sensitive to the surrounding environment. The position, wavevector, polarization, and wavelength of each photon collected from a single fluorophore can yield detailed information on the nanoscale organization and structure of biomolecular assemblies, but imaging systems must be carefully designed to encode this information efficiently into the images captured by a camera. Here, I will introduce the concept of 6D single-molecule orientation-localization microscopy (SMOLM), in which the dipole-spread function (the image of a dipole-like emitter) is specifically engineered to enable sensitive measurements of the 3D position and 3D orientation of any fluorescent emitter. I will show how classical and quantum estimation theories reveal the fundamental best-possible limits of accurately and precisely measuring molecular orientation and dynamic rotation. I will also discuss how to engineer imaging systems whose performance approaches these limits in practical experiments. Finally, I will cover recent work in my lab on how single fluorogenic molecules can be used to sense protein architectures within biological targets without the need for genetic or covalent modifications of the proteins themselves. We use fluorogenic dyes like Nile red and merocyanine 540 to image the helical organization of peptide assemblies and the network architecture of proteins within biomolecular condensates.