This award is for the development of a Thermal Noise Imaging Microscope for nanoscale imaging of the plasma membrane. The Thermal Noise Imaging Microscope will be capable of imaging soft dynamic nanostructures without damaging living cell membranes. Precise position tracking of an individual diffusing membrane molecule or a molecular complex labeled with a colloidal nanoparticle will be used to image membrane structures and produce an energy landscape of the membrane surface. This novel microscope will be useful for addressing questions of specific interest in the biological community, such as the possibility that lipid-protein clusters in the plasma membrane called lipid rafts can control cell response. Currently, no method allows such nanostructures to be visualized directly in a living cell. The core of the thermal noise imaging microscope will be an optical trap equipped with a 3D particle position detection system with >1 MHz bandwidth and 1 nm precision. The optical trap serves to localize the probe molecule-particle complex to a minute spot within the cell membrane and also to provide illumination for the position detector. Fluorescence and differential interference contrast microscopy will be integrated into the setup to observe the sample and probe and to quantify cellular response. Larger membrane areas will be explored by moving the sample with a precise piezo-scanner. Since thermal noise imaging differs from conventional scanning probe microscopy (it uses stochastic fluctuations instead of rigid scanning patterns) new ways of recording and displaying images are required. Specifically, feedback mechanisms for probe positioning have to be based on a complex statistical analysis of data acquired within the local environment. A digital feedback system will be developed to implement probe positioning. This system will be a part of an interface that enables the non-specialized user to perform experiments with a basic training. Thermal noise imaging will produce large amounts of data that contain a wealth of information. A userfriendly interface will be developed for off-line analysis of thermal noise imaging data with integrated image processing of differential interference and fluorescence images. The software will quantify accessible membrane areas, estimate the size, shape and dynamics of typical structural elements, and approximate physical properties of the membrane structures.
Determining the organization of nanostructures in the plasma membrane is crucial to understanding how drugs, hormones, and other molecules interact with the cell. With this new and novel microscope, thermal position fluctuations of probe molecules are used as natural scanners to explore their local environment. This research will contribute significantly to the understanding of the mode of action of membrane molecules, which in turn will stimulate many biomedical applications. In addition, the project is ideal for integrating research and education on the level of undergraduates, graduates, and postdoctoral students.
