This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-101: Structural analysis of macromolecular complexes. The focus of this proposal is the development and optimization of a novel approach for high-resolution direct imaging of biological processes as they occur in real-time. The """"""""live"""""""" movies will be acquired using a Dynamic Transmission Electron Microscope that couples the spatial resolution characteristics of an electron microscope with the temporal resolution of ultrafast Lasers. This first-of-its-kind instrument for imaging biological samples will be capable of sub-nanometer and nanosecond spatiotemporal resolution and will increase the combined attainable resolution five to eight orders of magnitude over current techniques. We further postulate that the resolution limiting effects observed during conventional cryo-EM imaging of biological samples arise from long-term specimen and electron beam interactions, and will be alleviated by using the Dynamic TEM with ultrafast imaging pulses. Mitigating these effects will result in improved three dimensional structure determination of macromolecular complexes. The impact of this research has the potential to elucidate new levels of understanding regarding microtubule and mitochondrial dynamics and the mechanisms involved with both normal and abnormal cellular processes.
Our work will provide the first high-resolution direct imaging of biological processes as they occur in real-time. These """"""""live"""""""" movies will be acquired using a Dynamic Transmission Electron Microscope that combines characteristics of an atomic resolution electron microscope with ultrafast Lasers to study the structure of macromolecular complexes. This first-of-its-kind instrument will increase the spatial and temporal resolution for observing dynamic biological processes by five to eight orders of magnitude over current techniques.
| Park, Chiwoo; Woehl, Taylor J; Evans, James E et al. (2015) Minimum Cost Multi-Way Data Association for Optimizing Multitarget Tracking of Interacting Objects. IEEE Trans Pattern Anal Mach Intell 37:611-24 |
| Woehl, Taylor J; Park, Chiwoo; Evans, James E et al. (2014) Direct observation of aggregative nanoparticle growth: kinetic modeling of the size distribution and growth rate. Nano Lett 14:373-8 |
| Pedrini, Bill; Tsai, Ching-Ju; Capitani, Guido et al. (2014) 7 Å resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source. Philos Trans R Soc Lond B Biol Sci 369:20130500 |
| Carlson, David B; Gelb, Jeff; Palshin, Vadim et al. (2013) Laboratory-based cryogenic soft x-ray tomography with correlative cryo-light and electron microscopy. Microsc Microanal 19:22-9 |
| Mehraeen, Shareghe; McKeown, Joseph T; Deshmukh, Pushkarraj V et al. (2013) A (S)TEM gas cell holder with localized laser heating for in situ experiments. Microsc Microanal 19:470-8 |
| Evans, James E; Browning, Nigel D (2013) Enabling direct nanoscale observations of biological reactions with dynamic TEM. Microscopy (Oxf) 62:147-56 |
| Acar, Seyda; Carlson, David B; Budamagunta, Madhu S et al. (2013) The bipolar assembly domain of the mitotic motor kinesin-5. Nat Commun 4:1343 |
| Woehl, Taylor J; Jungjohann, Katherine L; Evans, James E et al. (2013) Experimental procedures to mitigate electron beam induced artifacts during in situ fluid imaging of nanomaterials. Ultramicroscopy 127:53-63 |
| Welch, David A; Faller, Roland; Evans, James E et al. (2013) Simulating realistic imaging conditions for in situ liquid microscopy. Ultramicroscopy 135:36-42 |
| Woehl, Taylor J; Evans, James E; Arslan, Ilke et al. (2012) Direct in situ determination of the mechanisms controlling nanoparticle nucleation and growth. ACS Nano 6:8599-610 |
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