Thermal Noise Microscope for Nanoscale Imaging of the Plasma Membrane

该奖项是为了表彰用于质膜纳米级成像的热噪声成像显微镜的开发。热噪声成像显微镜将能够成像软动态纳米结构而不破坏活细胞膜。对单个扩散膜分子或用胶体纳米粒子标记的分子复合体的精确位置跟踪将用于对膜结构进行成像，并产生膜表面的能量景观。这种新型显微镜将有助于解决生物群落中特定感兴趣的问题，例如质膜中的脂质蛋白簇称为脂筏可以控制细胞反应的可能性。目前，还没有一种方法可以让这种纳米结构直接在活细胞中可视化。热噪声成像显微镜的核心将是一个光学陷阱，配有1 MHz带宽和1 nm精度的3D粒子位置检测系统。光阱用于将探针分子-粒子复合物定位到细胞膜内的一个微小点，并为位置检测器提供照明。荧光和差示干涉对比显微镜将集成到设置观察样品和探针，并量化细胞反应。更大的膜区域将通过精确的压电扫描仪移动样品来探测。由于热噪声成像不同于传统的扫描探针显微镜（它使用随机波动而不是刚性扫描模式），因此需要新的记录和显示图像的方法。具体来说，探测器定位的反馈机制必须基于对在当地环境中获得的数据进行复杂的统计分析。将开发一个数字反馈系统来实现探头定位。该系统将成为界面的一部分，使非专业用户能够通过基本培训进行实验。热噪声成像将产生包含丰富信息的大量数据。将开发一个用户友好的界面，用于热噪声成像数据的离线分析，结合微分干涉和荧光图像的综合图像处理。该软件将量化可接近的膜区域，估计典型结构元素的大小、形状和动力学，并近似膜结构的物理性质。确定质膜中纳米结构的组织对于理解药物、激素和其他分子如何与细胞相互作用至关重要。利用这种新型显微镜，利用探针分子的热位置波动作为自然扫描仪来探测其局部环境。这项研究将有助于理解膜分子的作用方式，从而刺激许多生物医学应用。此外，该项目是整合本科生，研究生和博士后水平的研究和教育的理想选择。

This award is for the development of a Thermal Noise Imaging Microscope for nanoscale imaging of the plasma membrane. The Thermal Noise Imaging Microscope will be capable of imaging soft dynamic nanostructures without damaging living cell membranes. Precise position tracking of an individual diffusing membrane molecule or a molecular complex labeled with a colloidal nanoparticle will be used to image membrane structures and produce an energy landscape of the membrane surface. This novel microscope will be useful for addressing questions of specific interest in the biological community, such as the possibility that lipid-protein clusters in the plasma membrane called lipid rafts can control cell response. Currently, no method allows such nanostructures to be visualized directly in a living cell. The core of the thermal noise imaging microscope will be an optical trap equipped with a 3D particle position detection system with 1 MHz bandwidth and 1 nm precision. The optical trap serves to localize the probe molecule-particle complex to a minute spot within the cell membrane and also to provide illumination for the position detector. Fluorescence and differential interference contrast microscopy will be integrated into the setup to observe the sample and probe and to quantify cellular response. Larger membrane areas will be explored by moving the sample with a precise piezo-scanner. Since thermal noise imaging differs from conventional scanning probe microscopy (it uses stochastic fluctuations instead of rigid scanning patterns) new ways of recording and displaying images are required. Specifically, feedback mechanisms for probe positioning have to be based on a complex statistical analysis of data acquired within the local environment. A digital feedback system will be developed to implement probe positioning. This system will be a part of an interface that enables the non-specialized user to perform experiments with a basic training. Thermal noise imaging will produce large amounts of data that contain a wealth of information. A userfriendly interface will be developed for off-line analysis of thermal noise imaging data with integrated image processing of differential interference and fluorescence images. The software will quantify accessible membrane areas, estimate the size, shape and dynamics of typical structural elements, and approximate physical properties of the membrane structures. Determining the organization of nanostructures in the plasma membrane is crucial to understanding how drugs, hormones, and other molecules interact with the cell. With this new and novel microscope, thermal position fluctuations of probe molecules are used as natural scanners to explore their local environment. This research will contribute significantly to the understanding of the mode of action of membrane molecules, which in turn will stimulate many biomedical applications. In addition, the project is ideal for integrating research and education on the level of undergraduates, graduates, and postdoctoral students.