Imaging Reaction Dynamics in Living Cells
Cells are dynamic environments that use carefully regulated mechanisms to maintain function and health. One example of this is the vesicle-mediated transport of lipids (shown to the right). Each bright spot shows a single vesicle as it transports lipids through the cell. Each step of this process; internalization, transport in the vesicle, and enzymatic degradation of the lipids, is controlled by chemical reactions within the cell. Understanding these dynamic processes requires a method that will provide both spatial and temporal information-the ability to watch each step as it occurs. To obtain this information the Payne Lab uses fluorescence microscopy to directly probe intracellular dynamics. The Payne Lab is interested in two related questions; what are the rates and mechanisms of these intracellular processes and how can we better image each event.
Imaging chemical reactions within a cell.
Cells control certain chemical reactions through the localization of substrates and enzymes within distinct vesicles that are actively transported through the cell. We are especially interested in the reactions that result from the interaction of substrate-containing vesicles with enzyme-containing vesicles. We are using two-color single particle tracking to address this question in standard cell lines and in a cellular model of the blood-brain barrier. In this case, motor proteins, rather than diffusion, bring together the substrate and enzymes. In the absence of this form of active transport, the interaction of substrate and enzymes is limited by diffusion in the crowded environment of the cell. We are using single particle tracking microscopy to characterize nanoparticle motion as a function of size, surface coating, and actin cytoskeleton to map the effective viscosity of the cell and understand the deviation from Stokes-Einstein behavior.
New methods for live cell imaging.
Intracellular delivery of nanoparticles. Nanoparticles have important biomedical applications ranging from the treatment of human disease with gene therapy to understanding basic cellular functions with fluorescent probes. For these applications to be fully realized it is necessary to deliver nanoparticles across the plasma membrane and into the cytosol of living cells. The Payne Lab is developing novel methods for the cytosolic delivery and targeting of nanoparticles in conjunction with the use of advanced microscopy techniques to understand the mechanisms of nanoparticle delivery.
Fluorescence microscopy in challenging environments. While recent developments in fluorescence microscopy make it possible to image many of the dynamic events that are essential to cellular function, new methods are necessary to observe the dynamics of single molecules inside living cells. Imaging within live cells is difficult as the emission from fluorescent probes competes with the autofluorescence of the cell. The Payne Lab is developing new optical techniques for quantitative cellular imaging. Optical methods of interest include nanometer-level imaging, spectroscopic single-particle tracking, and multiphoton total internal reflection microscopy.