Probing and imaging cellular and molecular event dynamics at the interface using atomic force microscopy

原子力显微镜（AFM）在其多种配置是纳米科学和纳米技术的一个不可或缺的工具和一个重要的系统，为研究人员提供定性和定量的数据，在纳米和亚纳米尺度上的样品，包括其表面粗糙度，形态和机械性能。这些能力使AFM成为申请人研究计划的首选技术，其目的是设计表面并开发表面改性技术，以增强和促进生物材料与各种生物靶标的相互作用能力，并将其应用于再生医学和纳米医学以及芯片实验室设备。 PI在17年前购买了一台AFM，从那时起，这台设备不仅支持PI的研究计划，还支持合作者和许多外部用户的研究计划。在过去的3年里，该集团在获得一致的结果和图像分析方面面临着显微镜的主要问题，自去年以来，该系统根本无法运行。这对我们的研究是非常有害的，因为我们对校外AFM的访问非常有限（在校园内没有，在大流行期间也没有），特别是在正常工作时间，这对测量产生了不利影响，其中大多数测量需要不同的时间点，因此经常丢失宝贵的样本。迫切需要通过更换有缺陷的部件和更新数据采集和图像分析软件以及升级来恢复我们的AFM，以使申请人能够充分利用其发展中的研究计划的潜力。在本提案中，我们提供了申请人研究的示例，即用于细胞治疗、组织工程和监测细胞活性的纳米结构生物界面、纳米复合物和生物材料”;使用猪主动脉脱细胞基质作为支架的个性化移植物生物力学研究”;用于免疫工程的非病毒纳米制剂”;病理性钙化对软组织表面的影响”;和骨膜内细胞的机械生物学“，其中AFM是达到我们的研究目标的关键。通过这项RTI 1提案，申请人要求提供资金，以修复，校准和升级现有的非功能性AFM。这种方法比购买新的显微镜更具成本效益，因为设备的大多数硬件部件仍然处于良好状态。在这个阶段，申请人可以从最新的AFM中受益，以保持和提高其在再生医学，纳米医学和诊断工具方面的创新研究能力，这一点至关重要。除了提高我们的研究能力，AFM在我们的实验室的可用性将提供一个跨学科的培训，以小组成员和学生，其中基于问题的学习和团队合作是脚手架，以帮助他们，首先学会跨越一个单一的学科边界，然后跨越多个边界，从而更好地培养他们的多元化未来的职业生涯。

Atomic Force Microscopy (AFM) in its multiple configurations is an integral tool of nanoscience and nanotechnology and a crucial system to provide researchers with qualitative and quantitative data, at nano and sub-nano scale on a sample, including its surface roughness, morphology and mechanical properties. These capabilities make the AFM a technique of choice for the applicants' research program where the aim is to design surfaces and develop surface modification techniques that enhance and promote the interaction capabilities of biomaterials with various biological targets, and this, for their application in regenerative medicine and nanomedicine and lab-on-a chip devices. The PI has acquired an AFM 17 years ago, and since, this equipment has supported not only the PI's research program but also those of the collaborators and many external users. In the last 3 years, the group has faced major problems with the microscope, in terms of acquiring consistent results and image analysis, and since last year the system is not operational at all. This is very harmful to our research as our access is extremely limited to out-campus AFM (nil on-campus et nil during pandemic), especially during regular work hours, adversely affecting measurements, most of which require different time points thus very often losing precious samples. It is urgent to claim our AFM to back in order by replacing defective parts and updating software for data acquisition and image analysis as well as upgrading to enable the applicants to use its full potential for their evolving research program. In this proposal, we have provided examples of applicants' research namely Nanostructured biointerfaces, nanoplexes and biomaterials for cell therapy, tissue engineering and monitoring cellular activities'; Investigation of biomechanics of personalized graft using decellularized matrix of porcine aorta as scaffold'; Non-viral nanoformulations for immunoengineering'; Effect of pathological calcification on soft tissue surfaces'; and Mechanobiology of cells within the periosteum', for which the AFM is critical to reach our research objectives. Through this RTI1 proposal, the applicants request funding to repair, calibrate and upgrade the existing non-functional AFM. This approach is more cost-effective than buying a new microscope, since most hardware parts of the equipment are still in good condition. At this stage, it is crucial that the applicants could benefit from an up-to-date AFM to maintain and enhance its innovative research capacity in regenerative medicine, nanomedicine and diagnostic tools. In addition to enhance our research capacity, the availability of the AFM in our laboratory will provide a transdisciplinary training to the group members and students in which problem-based learning and teamwork are scaffolded to help them, first learn to cross a single disciplinary border and then to cross multiple borders, thus better training them for diverse future careers.