Molecular Foundry Seminar
"Time-Resolved Super-Resolution Imaging of
Biomolecular Processes in Two and Three Dimensions "
Peter M. Goodwin, CINT
Tuesday, January 31th @ 1:00 pm, Chemla Room, 67-3111
View the Foundry Seminar Schedule
Invited by Paul Ashby and the Imaging and Manipulation Facility
Abstract:
The emission of single fluorophores and fluorescent nanoparticles can be localized to well below the diffraction limit (~λ/2) encountered in conventional optical microscopy. I will discuss two research efforts at the Los Alamos Gateway to the Center for Integrated Nanotechnologies (CINT) on single-molecule imaging of biomolecular processes in two and three spatial dimensions.
We are using single-molecule imaging to characterize the two-dimensional motion of cellulases on cellulose. Cellulases, enzymes secreted by microbial organisms, catalyze the hydrolysis of recalcitrant cellulose to soluble sugars. The cellulose-catalyzed hydrolysis of cellulose is a complex heterogeneous reaction involving the synergistic action of several cellulase enzyme types. To date, due in large part to limitations of the bulk analysis methods used for its study, the underlying mechanisms of cellulase activity and synergy remain poorly understood. Our goal is to use single-molecule imaging to directly elucidate molecular-level details of cellulase activity that cannot be readily inferred from ensemble averages reported by conventional, bulk analysis.
We have developed a method for tracking individual quantum dot (QD) labeled proteins inside of live cells. This approach, based on confocal microscopy, uses four overlapping detection volumes coupled with active feedback to follow molecular motion in three dimensions over distances of ~10 microns. To demonstrate the power of this approach for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled receptors were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.
