Scanning Laser Force Microscope with Nanometer Resolution for Dynamic Imaging of Single Biomolecules Under Physiological Conditions

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The ideal biological microscmpe should have enough resolution to image biological macromolecules on a nanometer scale, should image in physiological, watery conditions, and should have sufficient temporal resolution to follow crucial dynamic processes on the scale of macromolecules, macromolecular assemblies and cells. An important unsolved problem in biology that highlights the potential usefulness of such an instrument is the micromechanical function of the ubiquitous motor proteins. Motor proteins are the driving force behind processes such as muscle contraction, cell locomotion, ciliar and flagellar beating, mitosis, meiosis, intracellular transport, DNA replication and transcription and protein synthesis. The goal of the proposed feasibility study is to develop a microscope with molecular resolution, based on scanning a nanofabricated silicon tip over a protein sample using a single beam laser optical trap (optical tweezers). The trapping laser will double as displacement/force sensor, recording the interaction force between tip and sample. The displacement signal will be fed into a feed-back loop keeping the tip at a constant distance from the sample, thus imaging its contours. As proof-of-concept, cytoskeletal protein polymers will be imaged to demonstrate spatial resolution. The motor proteins kinesin and ncd will be imaged on microtubules, with the goal of resolving the movement of the individual heads of the motors and their tracking on the microtubule surface. Single headed truncated motors will be imaged in comparison, with the goal of understanding the cooperativity of the two heads of native kinesin and ncd, and to test if a single head can produce movement at all. The project will be an interdisciplinary collaboration between i) a nanofabrication laboratory, providing expertise to develop a process to manufacture exactly defined silicon tips with nm features and a controlled surface chemistry, ii) a physics/biophysics laboratory providing expertise in instrument building, microscopy and laser technology, and iii) a molecular biology laboratory providing the expertise in protein biochemistry and developing procedures to produce well characterized cytoskeletal and motor proteins and modified proteins using an E.coli expression system. If successful, the proposed instrument would constitute a major advance in microstructure determination, uniquely matched to the requirements of biological research. It would make it possible to dynamically image the fundamental functional units of biology, proteins, DNA, RNA, "at work". The instrument would have a broad range of applications, especially in the life sciences, and a multitude of fundamental questions appear solvable, by "looking and seeing how things work".