Single quantum dot tracking for quantitative molecular imaging

The application of semiconductor nanocrystals known as quantum dots (QDs) to biology in the past few decades has advanced the field of single-molecule biology by allowing for long time-scale tracking of individual biomolecules. However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste