Imaging is one of the most important tools in biology. However, observing biological structures and processes in living cells at a resolution below the diffraction limit of light microscopy (~200 nm) remains extremely challenging. Recently, several super-resolution techniques have been introduced to improve the resolution of optical fluorescence, with reported static and dynamic resolutions reaching ~20 nm and ~60 nm, respectively. However, these techniques have yet to be translated to the live cell because of difficulties caused by limitations of fluorescent probes and optical aberrations and light scattering in tissues. Thus researchers must extrapolate information from images of fixed specimens to the living state. This project proposes a new super-resolution imaging technology: QSTORM, which combines user-controlled, switchable quantum dots (QDs) with specialized computer-based algorithms (STORM) and adaptive optics to enhance images. QSTORM will, for the first time, enable imaging in living cells with a resolution superior or comparable to other super-resolution techniques. QSTORM will be evaluated in two models systems: the structure and function of muscle myofilaments in zebrafish and the intracellular transport of vesicles in fruit fly neurons. Normal muscle function depends on the highly organized multi-scale architecture of muscle tissue. QSTORM will enable simultaneous imaging of functioning myofilaments, sarcomeres, and whole muscle cells within the same sample. Similarly, axonal transport of cargo by vesicles is critical to the survival and function of neuronal cells. QSTORM will permit observation of the movements of individual vesicles and the mapping of the underlying cytoskeletal structures that enable this transport. Additionally, the QSTORM team will collaborate with the Museum of Science in Boston to share the results of this research broadly through science education programs, museum demonstrations, and Web-based multimedia projects.
Intellectual merit. If fully successful, QSTORM will harness the superior imaging capabilities of quantum dots and adaptive optics for live cell imaging at a super-resolution of less than 50 nm. QSTORM will transform imaging of biological processes, particularly those involving the cytoskeleton and motor proteins. In the models to be studied, QSTORM will permit three-dimensional high resolution imaging of intact live muscle without the destructive processing required for transmission electron microscopy (TEM), thus potentially leading to new hypotheses of how muscle proteins such as actin, myosin, and associated proteins interact. Similarly, QSTORM will permit, for the first time, imaging the movements of neuronal vesicles over complete transport cycles along the entire length of the axon at single nanometer resolution, thus potentially transforming current understanding of the fundamental molecular mechanisms of transport and its regulation.
Broader impacts. QSTORM will contribute a powerful new microscopy tool to the scientific community. Not only will this research produce extraordinary images that offer visual insight into fundamental biological processes, but also the broader dissemination of results and educational activities will widely advance subcellular biological research and training. Researchers, students, educators, and public audiences will benefit from the potentially extraordinary visualizations produced by QSTORM. This research will be incorporated into graduate and undergraduate courses in a wide-range of disciplines. Collaboration with the Museum of Science will provide broadly accessible and nationally disseminated educational materials. The proposed QSTORM Web site will present these extraordinary images as part of a lively multimedia story of high-risk, interdisciplinary scientific and technical collaboration in pursuit of a grand challenge.
In 2006, a new microscopy technique was developed that allowed fluorescence microscopy to achieve resolutions below 50nm, an order or magnitude better than the resolution of a conventional fluorescence microscope. This technique is commonly referred to as either Photo-Activated Localization Microscopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM) depending on the details of the implementation. STORM has been applied to a variety of imaging problems in single tissue culture cells using conventional fluorophores, but has not been applied that successfully in thicker samples, such tissue sections, fruit flies and other model organisms. In thicker tissue samples, background light and optical aberrations will degrade the performance of STORM. The goal of the QSTORM project has been to improve the performance of STORM in thicker tissue by using quantum dots as fluorophores to provide brighter markers and by using adaptive optics to correct for optical aberrations. Quantum dots are semiconductor crystals a few nanometers in diameter that are brighter and more stable than the fluorescent dyes commonly used in biology. Adaptive Optics is a technique used for correcting optical aberrations commonly used in astronomy to correct ground-based telescopes for the aberrations caused by imaging through the earth’s atmosphere. The QSTORM project was a collaboration between a chemical engineering group developing quantum dots (Dr. Jessica Winter’s lab at Ohio State University), two biology research groups (Dr. Elizabeth Brainerd’s lab at Brown University and Dr. Ge Yang’s lab at Carnegie Mellon University), and the Kner lab at the University of Georgia. The Kner lab was responsible for developing the STORM imaging and the Adaptive Optics technology. We have developed multicolor Quantum Dot STORM and applied this approach to imaging microtubules and mitochondria in tissue culture cells. This is the first multicolor STORM imaging using Quantum Dots. We demonstrate that quantum dots produce more photons than conventional fluorophores and we develop techniques for optimizing the performance of quantum dots for STORM imaging. To image in thicker tissue samples, we have developed an Adaptive Optics approach that allows the optical aberrations to be corrected based on the weak, strongly fluctuating raw images that are the basis for STORM. We have developed an image quality metric that is optimized for raw STORM images, and we demonstrate this approach by correcting images of microtubules below the nucleus in tissue culture cells and by correcting images of neuropeptides in nerve cells in the intact fruit fly central nervous system. The QSTORM project has provided training for four graduate students and a postdoc at UGA in modern microscopy techniques at a time when biological microscopy is experiencing a wave of new developments. An undergraduate student has also been involved in adaptive optics research related to the QSTORM project. As part of this collaborative project, a team from the Boston Museum of Science has been documenting the research as it has been developing, allowing the public to learn about how research progresses in real time. The Kner lab has contributed to QSTORM website (www.qstorm.org) developed by the Museum of Science team. This website allows the public to learn about the scientific goals of the project and to learn about how the project is developing by reading blog posts from the researchers as they try different experiments.
