Department of Applied Physics, Aalto University, Espoo, Finland.
Max Planck Institute for Dynamics and Self-Organization (MPIDS), Göttingen, Germany.
Nat Protoc. 2019 Feb;14(2):594-615. doi: 10.1038/s41596-018-0110-x.
Measuring forces from the piconewton to millinewton range is of great importance for the study of living systems from a biophysical perspective. The use of flexible micropipettes as highly sensitive force probes has become established in the biophysical community, advancing our understanding of cellular processes and microbial behavior. The micropipette force sensor (MFS) technique relies on measurement of the forces acting on a force-calibrated, hollow glass micropipette by optically detecting its deflections. The MFS technique covers a wide micro- and mesoscopic regime of detectable forces (tens of piconewtons to millinewtons) and sample sizes (micrometers to millimeters), does not require gluing of the sample to the cantilever, and allows simultaneous optical imaging of the sample throughout the experiment. Here, we provide a detailed protocol describing how to manufacture and calibrate the micropipettes, as well as how to successfully design, perform, and troubleshoot MFS experiments. We exemplify our approach using the model nematode Caenorhabditis elegans, but by following this protocol, a wide variety of living samples, ranging from single cells to multicellular aggregates and millimeter-sized organisms, can be studied in vivo, with a force resolution as low as 10 pN. A skilled (under)graduate student can master the technique in ~1-2 months. The whole protocol takes ~1-2 d to finish.
从皮牛顿到毫牛顿范围的力的测量对于从生物物理角度研究生命系统非常重要。柔性微管作为高灵敏度力探针的使用在生物物理界已经得到确立，这提高了我们对细胞过程和微生物行为的理解。微管力传感器 (MFS) 技术依赖于通过光学检测其挠度来测量作用在力校准的空心玻璃微管上的力。MFS 技术涵盖了可检测力的广泛微观和介观范围（从数十皮牛顿到毫牛顿）和样品尺寸（从微米到毫米），不需要将样品粘接到悬臂梁上，并且允许在整个实验过程中对样品进行同时光学成像。在这里，我们提供了一个详细的协议，描述了如何制造和校准微管，以及如何成功设计、执行和排除 MFS 实验中的故障。我们使用模型线虫秀丽隐杆线虫来说明我们的方法，但通过遵循这个方案，各种各样的活体样本，从单细胞到多细胞聚集体和毫米大小的生物体，都可以在体内进行研究，力分辨率低至 10 pN。一个熟练的（本科）学生可以在 1-2 个月内掌握这项技术。整个协议大约需要 1-2 天完成。
