Edwards Devin T, Perkins Thomas T
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA.
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA; Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
J Struct Biol. 2017 Jan;197(1):13-25. doi: 10.1016/j.jsb.2016.01.009. Epub 2016 Feb 1.
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100μm) cantilever's gold coating. Next, this sub-pN bandwidth is extended by a factor of ∼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40μm) cantilever. Finally, FIB-modifying an ultrashort (L=9μm) cantilever improves its force stability and precision while maintaining 1-μs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.
基于原子力显微镜（AFM）的单分子力谱（SMFS）能够进行广泛的研究，从测量配体-受体键的强度到阐明单个膜蛋白的复杂折叠途径。通过提高力稳定性、力精度和时间分辨率来提升数据质量，可改善此类SMFS研究，更广泛地说，还能改善AFM在生物物理学和纳米技术中的各种应用。对于一款先进的小型商用AFM，我们阐述了这三个指标如何受悬臂本身而非更大的显微镜结构限制，然后描述了三种日益复杂的悬臂修饰方法，这些方法可提高数据质量。首先，通过去除长（L = 100μm）悬臂的金涂层，通常可在较宽带宽（Δf = 0.01 - 20Hz）内实现亚皮牛力精度和稳定性。其次，使用聚焦离子束（FIB）对较短（L = 40μm）的悬臂进行修饰，可将此亚皮牛带宽扩展约50倍，跨越五个数量级的带宽（Δf = 0.01 - 1000Hz）。最后，对超短（L = 9μm）悬臂进行FIB修饰可提高其力稳定性和精度，同时保持1微秒的时间分辨率。这些经过修饰的超短悬臂具有降低的品质因数（Q≈0.5），因此不会对分子施加大量（30 - 90皮牛）的高频力调制，这一现象在传统SMFS分析中未得到考虑。目前，没有适用于所有应用的完美悬臂。优化基于AFM的SMFS需要了解使用特定悬臂所固有的权衡，并选择最适合特定应用的悬臂。
