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.
A. Overview The neuron is a highly polarized cell. A hallmark of its polarized structure is its axon, which in humans can extend more than one meter. Because of its polarized structure, the neuron depends critically on transport of materials, packaged in different forms of cargoes, through the axon for their survival and function. This transport process is referred to as axonal transport. A fundamental yet unanswered question regarding axonal transport is how it is controlled so that the right cargo is delivered to the right destination at the right time. Answering this question is crucial to understanding the biology and physiology of the neuron and related human neurodegenerative diseases such as Alzheimer’s disease. To this end, imaging techniques are frequently used for visualizing and analyzing axonal transport. However, conventional imaging techniques can only resolve features larger than ~200 nanometers. This imposes a basic constraint on the ability of researchers to study fine details of axonal transport. The main outcome of this research project is that we have developed new super-resolution imaging techniques and related image analysis software tools for visualizing axonal transport up to a resolution of ~20 nanometers (see image 1). Our techniques are based on STORM (stochastic optical reconstruction microscopy) and make use of quantum dots, which are light-emitting semiconductor nanocrystals. To test and validate our techniques and software tools, we have also used them to study a specific biological question, namely how tau, a key axonal protein, may play a role in controlling the transport of individual cargoes. Although the focus of this research project is on developing technologies for studying axonal transport, the techniques and software tools we developed can be used to study other cellular processes as well as related human diseases. B. Major research outcomes B.1 Major technological contributions - We have developed a cell penetrating peptide based technique for reliable and efficient delivery of quantum dots into mammalian cells (see image 2). It provides significant advantages over microinjection techniques, which are more invasive, less efficient, and less reliable, in studying axonal transport. - We have developed sample preparation and image acquisition protocols for super-resolution imaging of axonal transport. - We have developed computational techniques and software tools for reconstructing and analyzing STORM images of axonal transport (see image 3). B.2 Major biological findings - Using the techniques and tools we developed, we found that cytoskeletal protein tau serves as a key regulator that modulates kinesin-1 mediated axonal transport spatially. B.3 Research publications Research results related to this project have been published in 6 journal papers, 3 book chapters, and 10 peer-reviewed conference papers. We have also made our software tools freely available through the internet. C. Major education and outreach outcomes This project provided research training and support to 1 postdoctoral research associate, 3 PhD students, 2 MS students, and 2 undergraduate students. Some of them went on to pursue doctoral training in biomedical engineering at leading research universities, including MIT and UC Berkeley. Others went on to work in the pharmaceutical or biotechnology industry. Funding of this project has also supported outreach activities to promote science and engineering education, including providing information regarding our research on the web page www.qstorm.org. D. Critical assessment of research outcomes This project was funded by the NSF Ideas Lab mechanism, which supports high-risk high-reward research projects. Over the past four years, we have largely achieved the research aims we proposed initially. However, we also experienced some setbacks. For example, our aim of implementing STORM imaging in live animals was only partially achieved due to technical difficulties. Despite these setbacks, we have developed the techniques and software tools that are applicable to studies of axonal transport and other cellular processes. E. List of journal publications (Lists of book chapters and conference papers are omitted due to space limitation) J.Q. Xu, S. Rastogi, Y. Yu, and G. Yang (2015). Quantitative characterization of size-dependent efficiency of cell penetrating peptide mediated delivery of nanoparticles into mammalian cells, under review. M. Qiu., Y. Yu, H.-C. Lee, and G. Yang. Tau mediates spatial organization of axonal transport, under review. S. Gunawardena S., G. Yang G., and L. S. B. Goldstein L. S. B. (2013) Presenilin controls kinesin-1 and dynein function during AP vesicle transport in vivo, Human Molecular Genetics, vol. 22, pp. 3838-3843. G. Yang (2013) Bioimage informatics for understanding spatiotemporal dynamics of cellular processes, Wiley Interdisciplinary Reviews Systems Biology and Medicine, vol. 5, pp. 367-380. E. A. Booth-Gauthier, T. A. Alcoser T. A., G. Yang, and K. N. Dahl (2012) Force-induced changes in subnuclear movement and rheology, Biophysical Journal, vol. 103, pp. 2423-2431. S. Roy S., G. Yang G., Y. Tang, and D. Scott (2012) A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution, Nature Protocols, vol. 7, pp. 62-68, 2012.
