Faulk J K, Edwards D T, Bull M S, Perkins T T
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States.
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States; University of Colorado, Boulder, CO, United States.
Methods Enzymol. 2017;582:321-351. doi: 10.1016/bs.mie.2016.08.007. Epub 2016 Oct 31.
Atomic force microscopy (AFM) is widely used in biophysics, including force-spectroscopy studies of protein folding and protein-ligand interactions. The precision of such studies increases with improvements in the underlying quality of the data. Currently, data quality is limited by the mechanical properties of the cantilever when using a modern commercial AFM. The key tradeoff is force stability vs short-term force precision and temporal resolution. Here, we present a method that avoids this compromise: efficient focused-ion-beam (FIB) modification of commercially available cantilevers. Force precision is improved by reducing the cantilever's hydrodynamic drag, and force stability is improved by reducing the cantilever stiffness and by retaining a cantilever's gold coating only at its free end. When applied to a commonly used short cantilever (L=40μm), we achieved sub-pN force precision over 5 decades of bandwidth (0.01-1000Hz) without significantly sacrificing temporal resolution (~75μs). Extending FIB modification to an ultrashort cantilever (L=9μm) also improved force precision and stability, while maintaining 1-μs-scale temporal resolution. Moreover, modifying ultrashort cantilevers also eliminated their inherent underdamped high-frequency motion and thereby avoided applying a rapidly oscillating force across the stretched molecule. Importantly, fabrication of FIB-modified cantilevers is accessible after an initial investment in training. Indeed, undergraduate researchers routinely modify 2-4 cantilevers per hour with the protocol detailed here. Furthermore, this protocol offers the individual user the ability to optimize a cantilever for a particular application. Hence, we expect FIB-modified cantilevers to improve AFM-based studies over broad areas of biophysical research.
原子力显微镜（AFM）在生物物理学中被广泛应用，包括蛋白质折叠和蛋白质-配体相互作用的力谱研究。此类研究的精度会随着基础数据质量的提高而提升。目前，在使用现代商用AFM时，数据质量受悬臂梁机械性能的限制。关键的权衡在于力稳定性与短期力精度及时间分辨率之间。在此，我们提出一种避免这种折衷的方法：对商用悬臂梁进行高效聚焦离子束（FIB）修饰。通过降低悬臂梁的流体动力阻力可提高力精度，通过降低悬臂梁刚度并仅在其自由端保留悬臂梁的金涂层可提高力稳定性。当应用于常用的短悬臂梁（L = 40μm）时，我们在5个数量级的带宽（0.01 - 1000Hz）上实现了亚皮牛的力精度，且未显著牺牲时间分辨率（约75μs）。将FIB修饰扩展到超短悬臂梁（L = 9μm）也提高了力精度和稳定性，同时保持了1微秒级的时间分辨率。此外，修饰超短悬臂梁还消除了其固有的欠阻尼高频运动，从而避免在拉伸的分子上施加快速振荡的力。重要的是，在经过初始培训投资后，即可进行FIB修饰悬臂梁的制备。实际上，本科研究人员按照此处详细介绍的方案，通常每小时能修饰2 - 4个悬臂梁。此外，该方案为个人用户提供了针对特定应用优化悬臂梁的能力。因此，我们预计FIB修饰的悬臂梁将在广泛的生物物理研究领域中改善基于AFM的研究。
