Far-Field Optical Nanoscopy of Cellular Dynamics

描述（由申请人提供）：在本申请中，我们建议证明谱域相位显微镜（SDPM）作为静态和动态纳米级结构（包括活细胞膜）的非侵入性、非接触性、远场光学表征的新工具的可行性。SDPM是光谱域光学相干层析成像的功能扩展，其允许在真实的时间中以纳米级灵敏度检测结构表面轮廓、运动和动态。纳米尺度的轴向灵敏度是通过使用一个新开发的实现精美的相位稳定的自参考干涉，同时保持在光学远场工作的非接触的优势。这种技术中的横向分辨率受物理光学的限制为微米级。虽然我们提出的技术在活体和非活体纳米材料和机器的静态和动态结构分析中有许多潜在的应用，但在R21应用中，我们主要集中在初始SDPM技术开发及其在细胞生物学中应用的可行性论证上。我们提出用这项技术解决的特定细胞生物学问题包括测量发育中心肌细胞的机械活动和评估神经嵴细胞中细胞迁移的幅度和动力学。本提案的具体目标如下：1.开发新的技术实现高速时间和空间分辨纳米轴向位移测量使用谱域相位显微镜（SDPM）; 2。开发信号和图像处理方法，提高SDPM位移定量的准确性; 3.将时间和空间分辨的SDPM应用于活细胞中的静态和动态纳米级测量。

DESCRIPTION (provided by applicant): In this application, we propose to demonstrate the feasibility of spectral domain phase microscopy (SDPM) as a new tool for non-invasive, non-contact, far-field optical characterization of static and dynamic nanoscale structures including living cell membranes. SDPM is a functional extension of spectral domain optical coherence tomography which allows for the detection of structural surface profiles, motions and dynamics with nanometer-scale sensitivity in the axial dimension in real time. Nanometer-scale sensitivity in the axial direction is obtained by using a newly developed implementation of exquisitely phase stable self-referencing interferometry, while maintaining the non-contact advantages of working in the optical far field. Lateral resolution in this technique is limited by physical optics to the micrometer scale. Although our proposed technique has many potential applications in static and dynamic structural analysis of living and non-living nanoscale materials and machines, in this R21 application we concentrate primarily on initial SDPM technology development and feasibility demonstrations of its applications in cell biology. The particular cell biology questions we propose to address with this technology include measuring mechanical activity in developing cardiomyocytes and assessing the magnitude and kinetics of cell migration in neural crest cells. The specific aims of this proposal are as follows: 1. Develop novel technology implementations for high-speed temporally- and spatially- resolved nanoscale axial displacement measurements using spectral domain phase microscopy (SDPM); 2. Develop signal and image processing approaches for improving the accuracy of SDPM displacement quantitation; 3. Apply temporally and spatially-resolved SDPM for static and dynamic nanoscale measurements in living cells.