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Freeing the tip from the cantilever alleviates both of these problems. But now we need to implement new ways of force generation and position sensing. The current implementation of a 3D force microscope (3DFM) using a free particle uses an optical beam in a laser tweezers configuration (Optical Force Microscope, OFM [Ghislain1993]) both to apply forces and to track the particle position. Whereas the laser tweezers technique has made possible experiments in single molecule dynamics [Svoboda1994], the optical beam can generate only relatively small forces, normally up to several tens of picoNewtons [Mehta1998]. This is insufficient to break covalent bonds, or to measure the full mechanical properties of biological fibers such as microtubules. In addition, the method of applying the force is nonspecific (optical index contrast), and the trap can accumulate extraneous material from the cytosol in live cell studies.

We have designed and prototyped a novel alternative design for a 3D Force Microscope. In the Biomedical Research section, we describe three initial target research projects that will use the instrument: mucociliary clearance, biomotors, and microtubule mechanics (live cells). Here we describe the system design and prototype.

We have built a 3-D free-particle force microscope that combines and extends known technologies. Bausch has used magnetic beads to apply forces in one dimension to cell membranes and to measure the viscosity inside living cells [Bausch1998]. The Optical Force Microscope (OFM) monitors its particle position with optical light scattering, achieving a resolution on the order of 1nm in all three dimensions with a bandwidth exceeding 10kHz [Allersma1998; Gittes1998b; Gittes1998a]. The optical tweezers technique has been demonstrated as a 2D force microscope in the imaging of a surface [Ghislain1993]. Our design extends magnetic force control to a 3-D working volume and uses optical light scattering for tracking. Our system also includes an optical microscope system sharing the same optical path with the laser system, enabling the display of fluorescence and brightfield imaging superimposed with the tracked beam information. Such an approach promises substantial advantages. The force is applied selectively to the mag-netic bead, and so does not directly affect cell organelles or proteins. The magnetic field does not heat the medium while exerting forces, whereas heating limits the force that can be applied by an OFM.

Prototype Laser Tracking System: We have constructed a prototype laser tracking system (see the figure to the right). This system consists of a laser, optics, and quadrant photodiode detector as the optical path for tracking. It includes an XYZ translation stage, with the XY motion provided by a Queensgate NPS-XY-100A translation stage, and Z motion currently provided by an open-loop piezo stack that moves a sample stage relative to the main optical path. It also includes a magnified image of the surface captured on a CCD camera (illuminated through the same optical path used by the laser system). This system is intended as a proof-of-concept test fixture with which to verify design choices. With these in hand, we will design the initial implementation of a fully functional instrument. We have conducted measurements of system performance, simulations of the closed-loop performance of the prototype, and experiments of bead tracking using the system.

We have developed the 3DFM magnetic system to be a complete integrated system with high bandwidth forces in 3 dimensions, temperature control and high forces in the nanoNewton range. The system has been designed around single layer coils about the size of a dime, with twelve of them arranged in two layers of six coils each. These coils generate flux that is driven through pole tips fabricated from Permalloy foils, with micron scale tips defined by a chemical etching process. The flux from the coils, as driven through the pole tips to the sample region in the light microscope, is returned through a high permeability drive ring fabricated from metglass. In combination with our custom designed electronics, this provides high bandwidth force generation in excess of 3 kHz. For force magnitudes, we have tested our system to provide 800 pN with 1 micron diameter beads, and over 13 nN with 4.5 micron diameter beads. The system has incorporated a water cooled lid to cool the coils during operation, with the benefit of controlling the sample temperature. The pole plates are reconfigurable as they attach on cover slips to the 3DFM stage with reversible glue. The user can switch pole plate configurations, and hence magnetic field geometries, in minutes. These geometries include high force in a single direction, force within a single plane, large area force application for multi-cell assays, and a full 3D hexapole geometry.

3DFM system: The system includes a magnetics stage (a), shown with open lid, which contains 12 coils arranged in two levels. These coils (c), about the size of a dime, provide about 70 A-turns of flux into pole tips (at right) which are Permalloy foils with etched tips to focus flux into the specimen region.

In performing measurements using the magnetics, it is essential to prevent rises in temperature due to the application of significant currents, and to stabilize the temperature over a range that includes 37°C for mammalian cell studies. We have successfully developed a water cooled stage, designed from thermal simulations that incorporates heat loss through flowing water, and heat generation from coils. These calculations also included the deformation of the sample slide so that we could determine the expected bead motion due to the temperature change. This stage has been tested and has been confirmed to eliminate the sample movement during the application of full load currents, and to be able to stabilize the specimen chamber at 37°C. Simulation of a water cooled stage (below) showed that with only water flowing through the lid, a temperature variation reduction from (a) 6°C to (b) 2°C was achieved. By adding a copper plate as a heat spreader, the temperature variation was reduced to 0.1°C.

The study of single molecule forces would greatly benefit from high bandwidth force application to probe protein conformation dynamics and molecular loading. We have tested our high bandwidth magnetics and electronics and have measured a bandwidth of over 3 kHz.
Finally, we have built a laser tweezers system that incorporates a separate high speed tracking system and laser with our magnetic stage. This will allow us to directly compare the difference in biophysical measurements between the application of constant force (magnetics) and the application of forces through an effective spring (laser tweezers, AFM). This difference is clearly identified in our chromatin pulling experiments, where the magnetics experiments reveal step release of nucleosomes with multiple Nucleosome releases, while the laser tweezers more often shows single Nucleosome release events. Each of these classes of experiments would relate to a biological context where the application of force was through a very soft spring (long strand of DNA, magnetics) or a relatively stiff spring (short protein linkage, laser tweezers).

We have developed useful interfaces to go along with our Resource Tools which can be found in the Ideal User Interfaces section.