Mechanical drift is a long-standing problem in optical microscopy that occurs in all three dimensions. This drift increasingly limits the resolution of advanced surface-coupled, single-molecule experiments. We overcame this drift and achieved atomic-scale stabilization (0.1 nm) of an optical microscope in 3D. This was accomplished by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. Several significant factors contributed to this experimental realization, including (i) dramatically reducing the low frequency noise in BFP detection, (ii) increasing the sensitivity of BFP detection to vertical motion, and (iii) fabricating a regular array of nanometer-sized fiducial marks that were firmly coupled to the cover slip. With these improvements, we achieved short-term (1 s) stabilities of 0.11, 0.10, and 0.09 nm (rms) and long-term (100 s) stabilities of 0.17, 0.12, and 0.35 nm (rms) in x, y, and z, respectively, as measured by an independent detection laser.

Force drift is a significant, yet unresolved, problem in atomic force microscopy (AFM). We show that the primary source of force drift for a popular class of cantilevers is their gold coating, even though they are coated on both sides to minimize drift. Drift of the zero-force position of the cantilever was reduced from 900 nm for gold-coated cantilevers to 70 nm (N =10; rms) for uncoated cantilevers over the first 2 hours after wetting the tip; a majority of these uncoated cantilevers (60%) showed significantly less drift (12 nm, rms). Removing the gold also led to ~10-fold reduction in reflected light, yet short-term (0.1–10 s) force precision improved. Moreover, improved force precision did not require extended settling; most of the cantilevers tested (9 out of 15) achieved sub-pN force precision (0.54 ± 0.02 pN) over a broad bandwidth (0.01–10 Hz) just 30 min after loading. Finally, this precision was maintained while stretching DNA. Hence, removing gold enables both routine and timely access to sub-pN force precision in liquid over extended periods (100 s). We expect that many current and future applications of AFM can immediately benefit from these improvements in force stability and precision.

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning α-helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

Instrumental drift in atomic force microscopy (AFM) remains a critical, largely unaddressed issue that limits tip-sample stability, registration, and the signal-to-noise ratio during imaging. By scattering a laser off the apex of a commercial AFM tip, we locally measured and thereby actively controlled its three-dimensional position above a sample surface to <40 pm (Δf = 0.01-10 Hz) in air at room temperature. With this enhanced stability, we overcame the traditional need to scan rapidly while imaging and achieved a 5-fold increase in the image signal-to-noise ratio. Finally, we demonstrated atomic-scale (~100 pm) tip-sample stability and registration over tens of minutes with a series of AFM images on transparent substrates. The stabilization technique requires low laser power (<1 mW), imparts a minimal perturbation upon the cantilever, and is independent of the tipsample interaction. This work extends atomic-scale tip-sample control, previously restricted to cryogenic temperatures and ultrahigh vacuum, to a wide range of perturbative operating environments.Atomic force microscopy (AFM) is a crucial tool in diverse fields [1][2][3] and is most commonly applied in ambient conditions (i.e., in air at room temperature). 1 Historically, the AFM community has focused on developing sharper tips and higher-sensitivity force-detection schemes for improved microscope performance. [4][5][6] Yet, imaging and other AFM applications are limited by mechanical drift between the probe tip and the sample. Drift limits both tipsample stability and registration. This stability is the capacity to hold the tip over a precise sample location. Tip-sample registration is the ability to return the tip to a particular feature in an image. While atomic-resolution imaging has been achieved at room temperature in both air 7 and liquid, 8 atomic-scale (~100 pm) stability and registration have not. Precise threedimensional (3D) control of the tip, the sample, and their relative position is needed to fully exploit AFM across a broad array of fields. For example, atomic-scale registration and stability would enable returning an AFM tip to a specific domain of a protein and then monitoring the Supporting Information Available: Detailed description of sample preparation protocol, laser alignment and calibration method, and stabilized imaging technique. Further, we show time-varying drift rates require high-bandwidth control ( Figure S1), backscattered tip signals from AFM tips in air and water through different substrates ( Figure S2), and highly linear and orthogonal stabilized scanning ( Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. Figure S1). Independent measurement of both the tip and sample places no restriction on tip activities. Compatibility with commercial tips enables broad applicability. NIH Public AccessIn optical-trapping applications, laser-based detection of micron-sized beads using forwardscattered light provides high-bandwidth (>1 kHz) detection coupled with picometer-s...