AFM-TIRF combination

原子力显微镜允许在分子和细胞尺度上测量细胞力。这种测量对于定量和深入了解细胞与表面的相互作用是必不可少的。目前力显微镜数据的一个缺点是在测量过程中缺少关于细胞接触面积的信息，这影响了数据的分析。例如，更高的细胞表面相互作用力可以由更强的附着力和更大的细胞表面接触面积引起。因此，本文采用一种基于原子力显微镜（AFM）的力测量系统，该系统与荧光全内反射荧光显微镜（TIRF）装置组合而成。TIRF将光学检测细胞的接触面积，然后将与AFM力曲线相关联。这种TIRF荧光数据和AFM数据的相关性应该允许比传统系统更准确的数据解释。由于要研究活细胞，对原子力显微镜有特殊要求（例如，可加热的液体细胞）。为了确保细胞附着在测量系统上，将使用微流体细胞吸入装置。AFM与TIRF显微镜的结合在这里是独一无二的，将允许在细胞粘附过程中作用的力的全新结论。由于这种专用设备，所要求的原子力显微镜- tirf系统将主要用于表征细胞表面相互作用。由于该仪器将由几个研究小组使用，因此必须采购一种易于使用的仪器，该仪器也可以在不同温度的液体中稳定使用。此外，AFM中使用的激光不能与细胞中使用的荧光染料的激发或发射波长重叠，因此红外激光器是必要的。一个非常关键的仪器参数是压电片在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50-100µm，并且在剥离过程中由于其粘弹性而发生显著变形，因此设想的压电片的最小范围为100µm。

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.