IDBR: Type A: Development of a Polymer-Probe-Based Scanning Probe Microscope for Noninvasive, High-Speed, Broadband Investigation of Live Mammalian Cell

罗格斯大学的这一奖项是由两个项目联合颁发的-（1）生物基础设施部（生物科学理事会）的生物研究仪器开发，以及（2）化学，生物工程，环境和运输系统部（工程理事会）的生物光子学。扫描探针显微镜（SPM）是一种使用扫描样品的物理探针创建表面图像的技术。它为研究人员提供了在同一平台上成像，测量和操纵单个活细胞和亚细胞生物标本的独特能力，具有纳米级的空间和力分辨率。拟议的研究旨在开发一种扫描探针显微镜，克服商业上可用的设备的局限性，这些设备具有“僵硬”的探针，不利于创建活生物标本的3D图像。该IDBR奖的研究成果将通过以下方式传播给科学界：（1）专利披露和许可，（2）与领先的SPM公司密切合作，以及（3）在主要的细胞生物学，实验生物学和生物物理学会议上发表演讲。该项目的教育活动包括：（1）通过为生物学和工程学课程开发以研究为基础的内容，促进多学科培训;（2）招收和留住生物学和工程学领域研究生和本科生中代表性不足的学生;以及（3）利用罗格斯大学完善的项目，通过开放实验室图尔斯之旅，为初中和高中女生开展外联活动（Rutgers Society for Women Engineers）。在所有市售产品上普遍使用的硅基悬臂梁探针太硬和粗糙，无法避免变形/损坏活的生物表面（例如，细胞膜），特别是对于大体积和柔软且波纹状的膜的哺乳动物细胞。此外，接触模式成像协议（目前用于成像液体中的单个活细胞的最有效模式）和开环性质的纳米机械测量协议两者不仅易于受到液体相关的干扰和对细胞膜的损伤，而且在成像和测量活细胞的纳米机械性质时相当缓慢和窄。该项目旨在通过开发和集成基于软聚合物的悬臂梁探针，最小变形的自适应成像协议和基于控制的纳米机械测量协议来克服这些限制。基于聚合物的悬臂梁将被设计和制造为具有针对SPM成像和哺乳动物细胞上的力相互作用的接触刚度和其他机械特性。最小变形的成像方案将基于实时扫描引起的膜变形的准确量化来开发，以自适应地调整扫描速度和法向力，从而最小化膜变形并最大化整体成像功效。然后，基于控制的纳米力学协议的开发，以完全消除悬臂梁加速度的影响，并大大减少了流体动力学的影响，压痕测量的活细胞。将对开发的仪器进行评估，评估其实时量化细胞骨架黏弹性振荡的能力，以及量化和关联细胞分裂过程中活细胞的形态和机械演变的能力。

This award to Rutgers University is being jointly made by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Biophotonics, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate). Scanning probe microscopy (SPM) is a technique that creates images of surfaces using a physical probe that scans the specimen. It provides researchers with unique capabilities of imaging, measuring, and manipulating single live cells and sub-cellular biological specimen on the same platform, with nanoscale spatial and force resolutions. The proposed research aims to develop a scanning probe microscope that overcomes limitations of commercially available devices that have "stiff" probes which are detrimental to creating 3D images of live biological specimens. The research outcomes of this IDBR award will be disseminated to the scientific community through (1) patent disclosure and licenses, (2) close collaboration with leading SPM companies, and (3) presentations in major cell biology, experimental biology, and biophysics conferences. The educational activities of the project include (1) Fostering multidisciplinary training by developing research based content for the curriculum in biology and engineering courses, (2) Recruitment and retention of under-represented students at the graduate and undergarduate level in the fields of biology and engineering, and (3) Outreach activities for middle- and high- school girls through open lab tours by leveraging the well-established programs at Rutgers (Rutgers Society for Women Engineers).Silicon-based cantilever probes universally used on all commercially available are too stiff and harsh to avoid deforming/damaging live biological surfaces (e.g., cell membrane), particularly for mammalian cells of large volume and soft and corrugated membrane. Moreover, both the contact-mode imaging protocol, currently the most effective mode for imaging single live cells in liquid, and the nanomechanical measurement protocol of open-loop nature, are not only prone to liquid-related disturbances and damage to the cell membrane, but also rather slow and narrow banded in imaging and measuring the nanomechanical properties of live cells. The project aims to overcome these limits through the development and integration of soft polymer-based cantilever probes, an adaptive imaging protocol of minimal deformation, and a control-based nanomechanical measurement protocol. The polymer-based cantilever will be designed and fabricated with contact stiffness and other mechanical properties tailored to SPM imaging and force interaction on mammalian cells. The imaging protocol of minimal-deformation will be developed based on an accurate quantification of the scanning-induced membrane deformation in real-time, to adaptively adjust both the scanning speed and the normal force to minimize membrane deformation and maximize the overall imaging efficacy. Then the control-based nanomechanical protocol is developed to completely remove the cantilever acceleration effect and substantially reduce the hydrodynamic force effect on the indentation measurement of live cells. The developed instrument will be evaluated and for its ability to quantify the viscoelasticity oscillation of cytoskeleton in real-time, and to quantify and correlate the morphological and mechanical evolutions of live cells during the cell division process.