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)生物基础设施部门(生物科学局)的生物研究仪器开发，以及(2)化学、生物工程、环境和运输系统部门(工程局)的生物光子学。扫描探针显微镜(SPM)是一种使用扫描样本的物理探针来创建表面图像的技术。它为研究人员提供了在同一平台上对单个活细胞和亚细胞生物标本进行成像、测量和操作的独特能力，并具有纳米级的空间和力分辨率。这项拟议的研究旨在开发一种扫描探针显微镜，它克服了商业设备的局限性，这些设备具有“僵硬”的探针，不利于创建活体生物样本的3D图像。该IDBR奖的研究成果将通过(1)专利披露和许可证，(2)与领先的SPM公司的密切合作，以及(3)在主要细胞生物学、实验生物学和生物物理学会议上的陈述，向科学界传播。该项目的教育活动包括：(1)通过为生物和工程课程的课程开发基于研究的内容，促进多学科培训；(2)招募和保留生物和工程领域的研究生和本科生，以及(3)利用罗格斯大学(罗格斯女工程师学会)成熟的计划，通过开放实验室访问为初中和高中女孩开展外联活动。所有商用的硅基悬臂探头都太硬、太苛刻，无法避免变形/破坏活的生物表面(如细胞膜)，特别适用于哺乳动物细胞的大体积和柔软的波纹膜。此外，无论是目前最有效的液体中单个活细胞成像方式的接触式成像方案，还是开环性质的纳米力学测量方案，都不仅容易受到与液体相关的干扰和细胞膜的损伤，而且成像和测量活细胞的纳米力学特性时也存在较慢和较窄的条带。该项目旨在通过开发和集成软聚合物悬臂探头、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。这种基于聚合物的悬臂将被设计和制造，具有接触刚度和其他机械性能，以适应扫描电子显微镜成像和对哺乳动物细胞的力相互作用。最小变形成像协议将基于对扫描引起的薄膜变形的实时准确量化来开发，以自适应地调整扫描速度和法向力，以最小化薄膜变形，最大化整体成像效果。然后，开发了基于控制的纳米机械方案，以完全消除悬臂梁加速度效应，并大大降低了活细胞压痕测量中的流体动力影响。将对开发的仪器进行评估，以确保其能够实时量化细胞骨架的粘弹性振荡，并量化和关联活细胞在细胞分裂过程中的形态和机械演化。

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.