JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
ACS Nano. 2014 May 27;8(5):4984-95. doi: 10.1021/nn5010588. Epub 2014 Apr 1.
Enhancing the short-term force precision of atomic force microscopy (AFM) while maintaining excellent long-term force stability would result in improved performance across multiple AFM modalities, including single molecule force spectroscopy (SMFS). SMFS is a powerful method to probe the nanometer-scale dynamics and energetics of biomolecules (DNA, RNA, and proteins). The folding and unfolding rates of such macromolecules are sensitive to sub-pN changes in force. Recently, we demonstrated sub-pN stability over a broad bandwidth (Δf = 0.01-16 Hz) by removing the gold coating from a 100 μm long cantilever. However, this stability came at the cost of increased short-term force noise, decreased temporal response, and poor sensitivity. Here, we avoided these compromises while retaining excellent force stability by modifying a short (L = 40 μm) cantilever with a focused ion beam. Our process led to a ∼10-fold reduction in both a cantilever's stiffness and its hydrodynamic drag near a surface. We also preserved the benefits of a highly reflective cantilever while mitigating gold-coating induced long-term drift. As a result, we extended AFM's sub-pN bandwidth by a factor of ∼50 to span five decades of bandwidth (Δf ≈ 0.01-1000 Hz). Measurements of mechanically stretching individual proteins showed improved force precision coupled with state-of-the-art force stability and no significant loss in temporal resolution compared to the stiffer, unmodified cantilever. Finally, these cantilevers were robust and were reused for SFMS over multiple days. Hence, we expect these responsive, yet stable, cantilevers to broadly benefit diverse AFM-based studies.
提高原子力显微镜（AFM）的短期力精度，同时保持优异的长期力稳定性，将改善多种 AFM 模式的性能，包括单分子力谱学（SMFS）。SMFS 是一种强大的方法，可以探测生物分子（DNA、RNA 和蛋白质）的纳米级动力学和能量学。这些大分子的折叠和展开速率对力的亚皮牛变化敏感。最近，我们通过去除 100 µm 长悬臂上的金涂层，在较宽的带宽（Δf = 0.01-16 Hz）内实现了亚皮牛稳定性。然而，这种稳定性是以增加短期力噪声、降低时间响应和降低灵敏度为代价的。在这里，我们通过使用聚焦离子束修改短（L = 40 µm）悬臂来避免这些折衷，同时保留优异的力稳定性。我们的过程导致悬臂的刚度和其在表面附近的流体动力阻力降低了约 10 倍。我们还保留了高反射悬臂的优势，同时减轻了金涂层引起的长期漂移。结果，我们通过将 AFM 的亚皮牛带宽扩展了约 50 倍，扩展了五个数量级的带宽（Δf ≈ 0.01-1000 Hz）。测量单个蛋白质的机械拉伸表明，与较硬的未修改悬臂相比，力精度提高了，同时保持了最先进的力稳定性，并且时间分辨率没有明显降低。最后，这些悬臂坚固耐用，可以在多天内重复用于 SMFS。因此，我们预计这些响应灵敏但稳定的悬臂将广泛受益于各种基于 AFM 的研究。
