Ultrastable AFM: Improved Stability, Precision, and Bandwidth for Bio-AFM

Abstract

Enhancing the short-term force precision and temporal resolution of atomic force microscopy (AFM) while maintaining excellent long-term stability would result in improved performance across multiple AFM modalities, including single molecule force spectroscopy. However, instrumental drift in AFM remains a critical issue that limits the precision and duration of experiments. Previously, we developed an active optical stabilization technique to improve tip-sample stability at ambient conditions. However, force drift also occurs via uncontrolled deflection of the zero-force position of the cantilever. We found that the primary source of force drift in liquid for a popular class of soft cantilevers is their gold coating, even though they are coated on both sides to minimize drift. While removing the gold led to ∼10-fold reduction in reflected light, we nonetheless achieved a 10-fold improvement in force stability of bioAFM, with a sub-pN force precision over a broad bandwidth (0.01-20 Hz) just 30 minutes after loading. We subsequently extended AFM's sub-pN bandwidth by a factor of ∼50 to span five decades of bandwidth (Δf ≈ 0.01-1,000 Hz by developing an efficient process to modify a short (L = 40 μm) commercially available cantilever (BioLever Mini) with a focused ion beam (FIB). 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 stiffer, unmodified cantilevers. Ongoing work in our lab extends this concept down to ultrashort cantilevers (L =10 μm) along with instrumental development to detect these cantilevers in a commercial AFM. Such cantilevers enable sensitive detection of protein unfolding and refolding with ∼1 μs time resolution. Importantly, 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.