3D Force Microscopy for Microrheology & Active Transport

Principal Investigator: Richard Superfine
Funding Agency: National Institute of Biomedical Imaging and Bioengineering 
Agency Number: 1-R01-EB00761-01

Abstract
The importance of the rheological properties of biological media, including the cytoplasm, the extracellular matrix and biological gels such as mucus is being appreciated in understanding intracellular transport, pathogen clearance, drug delivery, to name a few. Correspondingly, forces in biological contexts, as driven by molecular motors and filament polymerization, is understood as essential for the understanding of cell division and motility, intracellular trafficking of vesicles, and the beating of cilia that are responsible for bacterial locomotion and mucus hydrodynamics. We propose to develop magnetic bead manipulation into a new microscopic technology for studying forces and rheology in biological systems. There are three trends that make now the opportune time to make rapid progress. First, there has been a growing development of magnetic separation technology that has led to the availability of a wide range of magnetic particles, in size and functionality, and the incorporation of micro-fabricated magnetic pole systems onto silicon wafers. Second, while bead rheology is a decades old technology, there have been recent developments in the application of submicron-sized beads for measuring the complex viscoelastic moduli of biological gels, including the analysis of Brownian motion and two-particle correlation functions. The third development has been in the area of user interfaces. The application of advanced 3D visualization in real time, combined with haptic (touch-sensitive) control and display of force probes has been demonstrated in atomic force microscopy. We propose to bring these three developments into a system that a) applies forces to magnetic beads suspended inside biological media using micro-fabricated pole geometries that will allow b) the simultaneous use high numerical aperture optics for nanometer scale particle tracking and 3D confocal microscopy, and c) advanced user interfaces for the immediate understanding of complex data sets and control of the instrument. The last feature of the system, the user interface and instrument control, will be supported almost entirely from an existing NCRR grant. We believe in developing systems to solve specific driving problems, while understanding the wider applicability of the technology. Our magnetic bead system, which we call a 3 Dimensional Force Microscope (3DFM), will be developed to address a) the rheological properties of mucus, the periciliar layer and the glycocalyx of lung epithial cultures and b) kinetochore motility in cell extracts. These driving problems are of broad importance, and drive the technology in the range of capability (small bead size, high force) and system applicability (purified gel, cell extract and cell culture). One of our primary goals is the development of an integrated system that will be usable on a daily basis for biology studies, and therefore a great deal of attention and resources will be devoted to the design of sample cells that facilitate ease of sample preparation and the conduct of the experiments on the microscope.