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.
Muscle is a remarkable biological material. It contracts and shortens to produce the amazing and sometimes alarming animal motions we see in nature, such as cheetahs sprinting, birds flying, and snakes biting. We know a lot about the molecules inside muscles and how they work to produce muscle contraction, but many mysteries remain. For example, muscle tissue has a memory. It appears to "remember" events immediately before a contraction starts, such as being stretched by another muscle, and contraction speed and force can depend on those prior events. We currently can’t explain this memory effect, but recent characterization of a giant molecule inside muscle cells, called Titin, suggests that this long, spring-like molecule may be involved. So how can we find out what Titin is doing when muscles contract? Ideally we'd like to see it in action, but Titin, along with Actin and Myosin, the other main molecules that make up muscle, are too small to be seen in living tissue, even with the most powerful microscopes. We can get a glimpse of them in dead tissue using electron microscopes, but we need to catch them in the act of making muscle contract. Hence, the primary goal of this project is to develop and improve microscopy methods for visualizing Titin and probing other molecular mysteries of muscle. Developing new microscopy methods requires a team of scientists with diverse expertise. Our team includes a physicist, a chemist, a biomedical engineer, a biologist, and last, but not least, a science educator to help us all communicate with each other. It can be hard for scientists from different areas to understand each other, and our struggles and successes are all documented in over 700 blog posts on our web site, www.qstorm.org. The web site is a great place to get a glimpse into how science is really done, and it includes a glossary, a timeline and other tools to help visitors understand our research. The qstorm.org web site also includes photos and posts from all of the wonderful, curious and energetic students who have participated in this project and received hands-on learning about how scientific research is conducted. We call our research team QSTORM because STORM is a new and powerful microscopy method, and we are working to make it even better by using tiny particles, called Quantum Dots, rather than the more traditional fluorescent dyes to label structures within cells. The overall QSTORM project contains five separate projects, one each for the physicist, chemist, biomedical engineer, biologist, and science educator. This report covers the Biology section of the QSTORM collaboration, and specifically the development of QSTORM imaging for visualizing muscle molecules. Our goal is to spy on muscle contraction in action, so we started work with anesthetized zebrafish larvae (just hatched out of the egg). We perfected microinjection techniques for injecting dye into just one muscle cell at a time. These larvae are very small, so one muscle cell looks large (See Image 1). Then we tried the same injection with quantum dots, and found that they did not stay contained within one muscle cell (See Image 2). Then we needed to know whether the quantum dots could penetrate all the way down to the actual muscle proteins. This was a critical moment because if the quantum dots did not penetrate, we would have had to abandon the goal of QSTORM for muscle. We used electron microscopy to look at very thin slices of muscle tissue at very high magnification. We were pleased to find that the quantum dots did indeed penetrate well (Image 3), which was a key finding for the feasibility of QSTORM imaging for muscle. Then we found that muscle fibers from the leg of rabbits are better than the zebrafish for developing these methods because they still contract outside of the animal, and it is easier to work with isolated cells. We were able to use conventional dyes to label both Titin (green) and Myosin (red) in rabbit cells (Image 4). This was exciting because we could then image the muscle in both relaxed and contracted state (Image 5). Finally, we took the conventional dye techniques and used quantum dots to target specific proteins (Image 6). The broadest impact of this work comes from the improvement of technologies for visualizing molecules inside muscle with microscopes. These muscle-imaging techniques will be valuable for other researchers, and contribute to the overall infrastructure for advancing frontiers of knowledge. This project also contributes to the future of scientific research by providing hands-on research experiences for three PhD students and five undergraduate college students. Finally, all aspects of the research process are accessible for students and the general public through QSTORM.org, including 110 blog posts specifically about this muscle imaging section of the project.
