A BioAFM for Intracellular, Cellular, and Tissue Measurement and Manipulation

为了加深我们对细胞、胞内细胞器和组织的力学和功能的理解，需要新的技术，包括直接的机械测量和操作。******这个RTI应用程序需要一个生物原子力显微镜（bioAFM）来支持这个研究团队探索细胞、细胞内结构和组织的力学和力学生物学。与以前的生物afm模型不同，该生物afm包含以下独特的功能，这对我们提出的研究计划至关重要。(1) FluidFM模块，用于提取皮升和纳升亚细胞物质（例如，线粒体、mRNA、非核酸等的活检）。(2)快速扫描和压痕，能够捕捉细胞活动的动态，如细胞膜的密封，机械压痕前后核孔复合物上蛋白质结构的变化，以及机械敏感离子通道的激活（如PIEZO1）。(3)对于扫描粗糙表面（如振动原子切片组织切片）至关重要的大Z范围。(4)开放式培养室，控制和维持细胞培养环境（如温度和二氧化碳），同时允许操作工具（如原子力显微镜探针和玻璃微移液管）进入样品。******所要求的生物afm将安装在我们现有的共聚焦显微镜（用于3D高分辨率荧光成像）上，并与我们的精密细胞烧蚀激光器（用于在细胞膜上开孔或烧蚀细胞中的细丝）集成，形成一个强大的系统。我们的HQP将使用该设备进行细胞内、细胞内和组织内力学和力学生物学实验的研究，并取得令人兴奋的发现。这些设备将用于培养大量的博士后和研究生，他们将获得细胞和组织力学、细胞力学转导、细胞操作、发育生物学、显微镜成像以及精密仪器控制和测量方面的技能。

To deepen our understanding of the mechanics and functions of cells, intracellular organelles and tissues, new techniques are required, including direct mechanical measurement and manipulation.******This RTI application requests a biological atomic force microscope (bioAFM) to support this team of researchers to explore the mechanics and mechanobiology of cells, intracellular structures, and tissues. Different from previous models of bioAFMs, this requested bioAFM contains the following unique capabilities, which are essential to our proposed research programs. (1) A FluidFM module for the extraction of picolitre and nanoliter subcellular substances (e.g., biopsy of mitochondria, mRNA, nnucleic acids etc.). (2) Fast scanning and indention that enable capturing dynamics of cellular activities, such as the sealing of the cell membrane, the protein structure change on the nuclear pore complex before and after mechanical indentation, and the activation of mechanosensitive ion channels (e.g., PIEZO1). (3) A large Z range that is critical for scanning rough surfaces such as vibratome-sectioned tissue slices. (4) An open-stage incubation chamber that controls and maintains the cell incubation environment (e.g., temperature and CO2) while permitting manipulation tools (e.g., AFM probe and glass micropipette) for sample access. ******The requested bioAFM will be mounted on our existing confocal microscope (for 3D high-resolution fluorescence imaging) and integrated with our precision cell ablation laser (for opening a hole on cell membrane or ablate a filament in a cell) to form a powerful system. Our HQP will use the equipment to conduct research on intracellular, cellular and intra-tissue mechanics and mechanobiology experiments and make exciting discoveries. The equipment will be used to train a high number of postdocs and graduate students who will gain skills in cell and tissue mechanics, cell mechanotransduction, cell manipulation, development biology, microscopy imaging, and precision instrument control and measurement.