CAREER: Target-locked single molecule spectroscopy and super-resolution microscopy

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With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, and co-funding from the Molecular Biophysics and Cellular Dynamics Clusters in the Division of Molecular and Cellular Biosciences, Dr. Kevin Welsher and his group at Duke University are seeking new ways to enhance understanding of chemical and biological processes by measuring the function of single molecules with high precision. Specifically, they are developing "target-locked" microscopy tools to follow the rapid motions of single proteins and other cellular matter within cells in real time. The information developed in this project will help us Understand the Rules of Life by helping us see what goes on inside biological cells both at high speed and with high resolution. The Welsher group is also developing a set of experiments for the "home spectroscopists" aimed at using smartphones to illustrate the principles of spectroscopy and optics. These experiments illustrate the color components of various samples using simple red-green-blue values. Online videos "Teachable Tidbits" are being developed with an aim of demystifying chemistry concepts that are frequently obscured by mathematics. Finally, the research is guiding graduate students into the field of microscopy, with an emphasis on learning microscopy through relatively simple numerical simulations that the students build from scratch. Internal molecular motions of single proteins have traditionally been investigated by single molecule Forster Resonant Energy Transfer (smFRET), which requires that the protein be tethered to a surface so that it remains in the focal volume of a microscope. The Welsher group is developing target-locked smFRET to probe untethered single cytosolic proteins in real-time, thereby enabling studies of the effect of cellular crowding and non-equilibrium environments on fast protein domain motions which have eluded traditional smFRET. The work should provide a more complete picture of the molecular underpinnings of cellular cargo transport by providing simultaneous measurement of "tug-of-war" motions of individual motor proteins and the local nanometer-scale cytoskeletal structure using target-locked super-resolution (TL-SR) microscopy. The high-speed, high-precision tracking and local sub-diffraction imaging of TL-SR may enable distinction between existing models of cargo trafficking. The broader impacts of this CAREER proposal focus on making microscopy and spectroscopy accessible to students at all levels.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.