Far-Field Optical Nanoscopy of Cellular Dynamics

描述(申请人提供)：在本申请中，我们建议证明光谱域相位显微镜(SDPM)作为一种新工具的可行性，用于非侵入性、非接触性、远场光学表征静态和动态纳米结构，包括活细胞膜。SDPM是光谱域光学相干层析成像的功能扩展，它可以实时检测结构表面轮廓、运动和动力学，在轴向维度上具有纳米级的灵敏度。通过使用一种新开发的精密相位稳定的自参考干涉术，在保持在光学远场工作的非接触式优势的同时，获得了轴向的纳米级灵敏度。这种技术的横向分辨率受到物理光学的限制，只能达到微米级。虽然我们提出的技术在生物和非生物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，但在这个R21应用中，我们主要集中在SDPM技术的初步开发和其在细胞生物学中应用的可行性演示。我们建议用这项技术解决的特殊细胞生物学问题包括测量发育中的心肌细胞的机械活动，以及评估神经脊细胞中细胞迁移的大小和动力学。该方案的具体目标如下：1.开发利用谱域相位显微镜(SDPM)进行高速时间和空间分辨率纳米尺度轴向位移测量的新技术实现；2.开发信号和图像处理方法以提高SDPM位移定量的精度；3.将时间和空间分辨率的SDPM应用于活细胞中的静态和动态纳米尺度测量。

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.