"Real-time 3D microsopy methods"

Single molecule spectroscopy elucidates the internal dynamics of immobilized molecules in vitro by collecting a continuous stream of emitted photons on a single photon counting detector, allowing continuous observation of the real-time molecular state, temporally limited only by the photon emission rate. In live cells, molecules rapidly diffuse out of the observation volume, precluding continuous observation. Moreover, in contrast to in vitro experiments, the molecule’s cellular environment becomes critical to its dynamics. To enable continuous observation of molecular species in their live-cell context we developed 3D Multi-resolution Microscopy (3D-MM). 3D-MM utilizes real-time 3D single-particle tracking to “lock-on” to a single diffusing nanoparticle using optical feedback and a 3D piezo-electric stage, permitting continuous observation of its dynamics. The cellular context is collected via a two-photon laser scanning microscope, which constructs a 3D map of the nanoparticle’s surroundings. The 3D-MM has enabled observation of the cellular uptake of Tat-modified nanoparticles, capturing, for the first time, the transient landing of a nanoparticle on the cell surface. To advance the application of 3D-MM to single molecules, we have developed a single-beam and single-detector tracking mechanism by applying a 3D time varying excitation beam with a 2D electro-optic deflector and an acoustically tunable (TAG) lens. This method improves the photon-efficiency and robustness of real-time 3D single-particle tracking, paving the way towards contextualized single molecule spectroscopy in live cells.

Assistant Professor Kevin Welsher is starting his second year in the Duke Department of Chemistry. Prior to coming to Duke, he obtained his PhD from Stanford University under the guidance of Prof. Hongjie Dai, studying the near infrared (NIR) luminescence of single-walled carbon nanotubes and their use as biological imaging agents in the second NIR transparency window. After his graduate studies, he went on to work as a postdoctoral researcher with Prof. Haw Yang at Princeton University where he focused on developing new 3D microscopy methods.