AFM-TIRF combination

AFM-TIRF 组合

• 批准号: 515275267
• 金额: --
• 依托单位: Ruprecht-Karls-Universität Heidelberg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/515275267?language=en
• 关键词: AFM; TIRF; combination

Atomic force microscopy allows the measurement of cellular forces on both molecular and cellular scales. Such measurements are essential for quantification and an in-depth understanding of the interaction of cells with surfaces. A current shortcoming of force microscopy data is that information on the contact area of the cells during the measurement is missing, which affects the analysis of the data. For example, higher cell-surface interaction forces can be caused by both stronger adhesion and larger cell-surface contact area. Therefore, a force measurement system based on an atomic force microscope (AFM) is used here, which is set up in combination with a fluorescence TIRF (Total Internal Reflection Fluorescence Microscopy) device. TIRF will optically detect the contact area of the cells, which will then be correlated with AFM force curves. This correlation of TIRF fluorescence data and AFM data should allow much more accurate data interpretation than with conventional systems. Since living cells are to be studied, special requirements are placed on the atomic force microscope (e.g., heatable liquid cell). To ensure the attachment of cells to the measurement system, a microfluidic cell aspiration device will be used. The combination of an AFM with a TIRF microscope applied for here is unique on campus and will allow completely novel conclusions on the forces acting during cell adhesion. Because of this specialized equipment, the requested atomic force microscope-TIRF system will be used primarily to characterize cell-surface interactions. Since the instrument is to be used by several research groups, an easy-to-use instrument that can also be used stably in liquid at different temperatures must be procured. Furthermore, the laser used in the AFM must not overlap with the excitation or emission wavelengths of the fluorescent dyes used in cells, so an infrared laser is necessary. An extremely critical instrument parameter is the range of the piezo in the z-direction (vertical to the surface). Since cells often reach diameters of 50-100 µm in the adhered state and deform significantly during the detachment process due to their viscoelasticity, a piezo with a minimum range of 100 µm is envisaged.

原子力显微镜可以在分子和细胞尺度上测量细胞力。此类测量对于量化和深入了解细胞与表面的相互作用至关重要。目前力显微镜数据的一个缺点是测量过程中细胞接触面积的信息缺失，影响了数据的分析。例如，更强的粘附力和更大的细胞表面接触面积可以引起更高的细胞表面相互作用力。因此，这里使用基于原子力显微镜（AFM）的力测量系统，该系统与荧光TIRF（全内反射荧光显微镜）装置结合建立。 TIRF 将以光学方式检测细胞的接触面积，然后将其与 AFM 力曲线相关联。 TIRF 荧光数据和 AFM 数据的这种相关性应该可以比传统系统更准确地解释数据。由于要研究活细胞，因此对原子力显微镜有特殊要求（例如可加热液体池）。为了确保细胞附着到测量系统，将使用微流体细胞抽吸装置。这里应用的 AFM 与 TIRF 显微镜的结合在校园中是独一无二的，并且将得出关于细胞粘附过程中作用力的全新结论。由于这种专用设备，所需的原子力显微镜-TIRF 系统将主要用于表征细胞表面相互作用。由于该仪器要供多个研究小组使用，因此必须采购一种易于使用且能够在不同温度的液体中稳定使用的仪器。此外，AFM 中使用的激光不得与细胞中使用的荧光染料的激发或发射波长重叠，因此需要红外激光。 一个极其关键的仪器参数是压电在 z 方向（垂直于表面）的范围。由于细胞在粘附状态下的直径通常达到 50-100 µm，并且由于其粘弹性而在分离过程中显着变形，因此设想了最小范围为 100 µm 的压电。