KSEF RDE: Imaging of surface nanostructures in a living cell

Grants and Contracts Details


The goal of this proposal is to develop a technique for imaging the dynamic behavior of protein complexes and other nanometer-scale structures at the surface of a living cell under physiological conditions. Imaging techniques with nanometer-scale resolution are generally unavailable for live cell studies. Electron microscopy requires fixing or freezing the sample. Significant sample-probe interaction restricts the implementation of atomic force microscopy in live cell imaging. Fluorescently labeled molecules can be localized with sub-wavelength resolution but cannot be easily related to the unlabeled surrounding structures. Given the current state of technology, a number of functionally important cellular processes are illustrated only as model cartoons supported by static electron microscopy images and/or various indirect techniques, but not by direct visualization. This project will develop the first non-contact nanoscale-resolution high-speed technique for imaging the cell surface in the physiological environment. It is based on the ideas that emerged from my longterm experience in developing of scanning ion conductance microscopy, a novel nanotechnology tool. The proposed study will bring this technology to a new level: nanoscale imaging with time resolution applicable to most physiological events. The ability to visualize spontaneous and evoked changes of nanostructures at the cell surface may lead to unanticipated discoveries in the mechanisms of cell motility, adhesion, mechanosensitivity, endocytosis, and exocytosis. It may also provide insight into the function of distinct plasma membrane microdomains that are involved in pathogenesis of human diseases including vascular, kidney, psychiatric and neurological disorders. The proposed study will result in development of the first (and the most advanced) prototype of scanning ion conductance microscopy for biological applications in the US. The existence of such technique at the University of Kentucky will be an addition of national prominence to the local research infrastructure. Technical Narrative Introduction and eXpected1i,ttigm~ afthewQI'k: . Several methods are currently available to visualize the cell surface with nanometer-scale resolution. Scanning Electron Microscopy (SEM) is able to resolve surface structures as small as single nanometers (Pawley, 1997), but it cannot be generally used for live cells. Recently, a special wet sample chamber was introduced to visualize fully hydrated samples by conventional SEM (Thiberge et aI., 2004). This technology does provide a potential for live cell imaging, but with relatively low resolution (-100 nm) and without possibility for changing the environment of the cell that is essential for physiological experiments. Freeze-fracture and freeze-etching are the "gold standard EM techniques for studying individual membrane proteins and protein complexes (Hawes and Martin, 1995; Meyer and Richter, 2001). However, the only way to study live cell behavior with these techniques is to design an experiment, in which the cells would be frozen before and during a physiological response. Sub-wavelength optical techniques (Liu et aI., 2007; Smolyaninov et aI., 2007) has a significant potential for live cell imaging but is yet very far from practical biological applications. So far, sub-wavelength resolution «200 nm) optical techniques, such as near-field total internal reflection microscopy (TIRF) (Axelrod, 1981), were applicable only to the imaging of fluorescently labeled structures/molecules, and did not provide any information about unlabeled structures. Atomic Force Microscopy (AFM) is able to resolve the surface of individual proteins with a lateral resolution of -0.5 nm and a vertical resolution of -0.1 nm (Engel and Muller, 2000). Similar to some other scanning probe techniques, AFM can visualize specimens in solutions, which allows studying proteins in their native environment and opens the possibility for imaging the surface of a living cell (Schneider et aI., 1997). However, the major problem for live cell imaging by AFM is the force applied to the sample by AFM cantilever. This problem is only partially alleviated by special imaging modes (Hansma et aI., 1994). Scanning Near-field Optical Microscopy (SNOM) is a scanninQ probe technique that explores the surface
Effective start/end date10/1/079/30/08


  • KY Science and Technology Co Inc: $20,000.00


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