This proposal would fund an advanced optical trapping microscope facility. The recent success of one- and two-beam optical traps in cell biology has led to breakthroughs in our understanding of how biological motors work, and provided scientists with unprecedented means of micromanipulation. The time is ripe to extend the utility of optical trapping in interdisciplinary directions, particularly towards the interface of molecular biology and materials science, to address outstanding problems in such areas as biophysics and nanotechnology. The Multiple Beam Optical Trapping (MBOT) microscope is a versatile, third-generation device, designed ab initio for micromanipulation and micromechanical measurement. It will be capable of generating several simultaneous trapping beams under computer control, each equipped with independent controls, and will incorporate position sensors to detect displacements of objects down to the subnanometer level. The beams can be alternately configured to grasp, manipulate. develop tension, determine position, image, or, in feedback mode, to measure force. Aspects of the microscope hardware will be linked to microprocessors that will control the operation, collect data, and perform image processing.. A set of four auxiliary microscopes will constitute an integral part of the overall facility, serving double-duty as teaching tools and as test beds for prototyping and developing equipment destined for the central microscope, as well as to explore new approaches to imaging and optical trapping. A key feature of multiple independent traps is that they permit two or more objects to be freely manipulated, oriented, and juxtaposed in all three dimensions, without necessarily passing laser light directly through the objects themselves, which might not tolerate the exposure. In addition, experimental geometries can be achieved that are quite impossible with conventional, single- or double-beam optical tweezers. A way to generate multiple traps and avoid optical interference effects is to time share the traps. In this scheme, all traps are produced by a single laser beam that is scanned rapidly among different positions, pausing at those locations where traps are desired. A prototype for this concept was developed in the Netherlands, where galvanometer mirrors were used to scan the beam. The MBOT design calls for the use of acousto-optic beam deflectors to achieve faster scan rates with inherently less jitter. Among the potential uses of the MBOT instrument are: (1) Single-motor motility assays, to measure the tiny stepwise displacements and forces produced by individual mechanoenzymes, such as kinesin and myosin, as well as those produced by processive nucleic acid enzymes, such as RNA polymerase; (2) Picotensiometry, to measure the molecular elasticity of biopolymers such as microtubules and actin filaments, as well as the micromechanical properties of biomimetic and other novel materials; (3) Optical force microscopy (OFM), a promising variation of scanned-probe microscopy that uses an optically-trapped stylus to obtain images under physiological conditions with a resolution exceeding that possible with the conventional light microscope; (4) Characterization of coupling forces involved in protein receptor-ligand binding, cell-cell and cell-substrate recognition, and RNA/DNA base-pairing; and (5) Creation of miniature hybrid devices consisting of both biological components (cells, microtubules, enzymes) and synthetic components (glass, silicon, etc.) on rnicrofabricated chips. As proposed, the instrument would be jointly funded by the .NSF and a private foundation, and housed in the Princeton Materials Institute. It is anticipated that the MBOT facility will serve as a catalyst to stimulate a range of interdisciplinary studies on rnicrostructures and nanodynamics.
