A Comprehensive Design of Quantum Dots for Imaging

Quantum dots (QDs) have proved to be invaluable fluorophores for single molecule imaging at high spatial and temporal resolution because they are brighter and more photostable than organic dyes or fluorescent proteins. However, use of QDs in biomolecular applications is limited by their large size, frequent blinking of their fluorescence signal, and challenges in their conjugation to the molecules of interest. QDs are often too large for confined or crowded systems due to their bulky passivating layer. QDs suffer a further increase in diameter during bioconjugation. QDs cannot withstand traditional bioconjugation methods that utilize small molecules, and other approaches require the use of large proteins. Therefore, a unifying strategy for small probe size, bioconjugation, and elimination of QD blinking has not yet been achieved.In this dissertation, I chronicle the design of a new system for the application of QDs to the study of biological systems. I utilize core/shell CdSe/CdS structures that blink less frequently and are brighter than conventional CdSe/ZnS structures. I characterize the difference between these two types of QDs at the single-molecule level and highlight the advantages for single-molecule imaging at accuracies on the nanometer scale that are present with CdSe/CdS QDs. I upgrade an existing small molecule-fusion protein system for bioconjugation (SNAP-tag) that improves the labeling of protein targets tenfold over the conventional system. I apply SNAP-functionalized CdSe/CdS QDs to the study of human kinesin-1, a motor protein responsible for directional transport of cargos along cytoskeletal tracks in the cell. I use these QDs to explore the role of interhead tension in coordinated motion using a mutant protein in a geometry previously inaccessible with commercial QDs. I show that these QDs can target kinesins for labeling in live cells. Our QD design strategy will likely extend their use to previously inaccessible systems such as small individual proteins or membrane-embedded complexes