Combinatorial Microscopies for Super-Resolution Imaging of Molecular and Cellular Dynamics

理解并最终控制分子如何组装成功能结构在材料科学、化学、结构和细胞生物学等领域都是至关重要的。我们对了解自组装的基本机制有着长期的兴趣，从染料分子的自结合到激子结构，到蛋白质和肽的聚集。我们最近的工作集中在活细胞中分子组装的动力学，特别是膜受体的异质和同质结合如何影响细胞信号传导和细胞间相互作用。确定这些复杂组装过程的结构和构象要求及其动力学，最好是通过在分子尺度上实时和名义上的现实世界条件下检查这些现象来完成。我们专注于设计和优化组合显微镜，用于直接询问分子和生物分子的自组装。***我们的技术目标是实现：***用于跟踪亚细胞动力学，细胞器重塑和活细胞中的蛋白质组装的高光谱超分辨率3d光学显微镜；***用于探测细胞和组织动力学和结构的超分辨率薄片显微镜，具有亚微米空间分辨率和高时间分辨率；***这些平台和我们实验室的其他系统具有独特的能力，可以在长度和时间的几个数量级上评估取向、构象和结构在自组装中的作用。以前，他们已经允许我们确定与二维分子固体中激子产生相关的关键尺寸，并确定活细胞中膜受体的空间，构象和动态异质性。在本提案中，该平台独特的超分辨率能力将使我们能够(1)评估蛋白质和肽序列如何改变膜结合和破坏的动力学；(2)询问线粒体膜重塑过程中原位复合体的形成；(3)跟踪细胞骨架蛋白的自组装和组织；***这个高度跨学科的研究项目包括光子学、生物物理学、化学、细胞和分子生物学以及工程学。HQP直接参与独特和创新成像平台的设计、优化、实施和修改，特别关注开放的硬件和软件设计。通过充分参与将这些成像平台应用于复杂的生物学问题和现象，HQP不仅仅是“使用”黑匣子成像策略，而是提供了一个独特而强大的机会，可以充分参与研究的各个方面。**

Understanding and ultimately controlling how molecules assemble into functional structures is critical in fields ranging from materials science and chemistry to structural and cell biology. We have a long-standing interest in understanding the fundamental mechanisms of self-assembly, ranging from the self-association of dye molecules into excitonic structures, to protein and peptide aggregation. Our most recent work has focused on the dynamics of molecular assembly in live cells and specifically how the hetero- and homo-geneous association of membrane receptors impacts cell signaling and cell-cell interactions. Determining the structural and conformational requirements for these complex assembly processes, and the dynamics thereof, is best accomplished by examining these phenomena on the molecular scale, in real-time, and under nominally real-world conditions. We are focused on the design and optimization of combinatorial microscopies for the direct interrogation of molecular and biomolecular self-assembly. ***Our technical objectives are to implement:*** Hyperspectral super-resolution 3-D optical microscopy for tracking sub-cellular dynamics, organelle remodeling, and protein assembly in live cells;*** Super-resolution light sheet microscopy for probing cell-cell and tissue dynamics and structures with sub-micron spatial resolution and high temporal resolution;***These platforms, and other systems in our lab, have unique capabilities for assessing the role of orientation, conformation, and structure on self-assembly over several orders of magnitude in both length and time. Previously, they have allowed us to identify the critical dimensions associated with exciton generation in two-dimensional molecular solids, and determine the spatial, conformational, and dynamic heterogeneity of membrane receptors in live cells. In the present proposal, the unique super-resolution capabilities of the platforms will allow us to (1) assess how protein and peptide sequence alter the dynamics of membrane association and disruption; (2) interrogate in situ complex formation during mitochondrial membrane remodeling; (3) track cytoskeletal protein self-assembly and organization;***This highly interdisciplinary research program embraces photonics, biophysics, chemistry, cell and molecular biology, and engineering. HQP are directly involved in the design, optimization, implementation, and modification of unique and innovative imaging platforms, with a specific focus on open hardware and software design. By being fully involved in the application of these imaging platforms to complex biological questions and phenomena, HQP are not simply "using" black box imaging strategies but  are afforded a unique and powerful opportunity to be fully engaged in all aspects of the research. **