Gavin M. King

Stabilization of an optical microscope to 0.1 nm in three dimensions

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.

Patterned growth of single-walled carbon nanotube arrays from a vapor-deposited Fe catalyst

Single-walled carbon nanotubes have been grown on a variety of substrates by chemical vapor deposition using low-coverage vacuum-deposited iron as a catalyst. Ordered arrays of suspended nanotubes ranging from submicron to several micron lengths have been obtained on Si, SiO 2 , Al 2 O 3 , and Si 3 N 4 substrates that were patterned on hundred nanometer length scales with a focused ion beam machine. Electric fields applied during nanotube growth allow the control of growth direction. Nanotube circuits have been constructed directly on contacting metal electrodes of Pt/Cr patterned with catalysts. Patterning with solid iron catalyst is compatible with modern semiconductor fabrication strategies and may contribute to the integration of nanotubes in complex device architectures.

Ultrastable Atomic Force Microscopy : Atomic-Scale Stability and Registration in Ambient Conditions

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 (Deltaf = 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 ( approximately 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 tip-sample 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.

Routine and Timely Sub-picoNewton Force Stability and Precision for Biological Applications of Atomic Force Microscopy

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 h 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.

Low-operating voltage and stable organic field-effect transistors with poly (methyl methacrylate) gate dielectric solution deposited from a high dipole moment solvent

A low-operating voltage and stable pentacene field-effect transistor (FET) employing thin low-dielectric constant gate layer of poly (methyl methacrylate) (PMMA) dissolved in propylene carbonate (PC) has been realized. This device exhibiting high field-effect mobility, a threshold voltage of −1 V, and a small sub-threshold slope at operating voltages below −3 V is compared with an FET cast from PMMA film dissolved in a low dipole moment solvent. The negligible hysteresis and excellent electrical stability of FETs under gate bias stress with the use of PC are traceable to the low density of trap states in PMMA bulk and at the interfaces.

Back-scattered detection provides atomic-scale localization precision, stability, and registration in 3D.

State-of-the-art microscopy techniques (e.g., atomic force microscopy, scanning-tunneling microscopy, and optical tweezers) are sensitive to atomic-scale (100 pm) displacements. Yet, sample drift limits the ultimate potential of many of these techniques. We demonstrate a general solution for sample control in 3D using back-scattered detection (BSD) in both air and water. BSD off a silicon disk fabricated on a cover slip enabled 19 pm lateral localization precision (Deltaf = 0.1-50 Hz) with low crosstalk between axes (</=3%). We achieved atomic-scale stabilization (88, 79, and 98 pm, in x, y, and z, respectively; Deltaf = 0.1-50 Hz) and registration ( approximately 50 pm (rms), N = 14, Deltat = 90 s) of a sample in 3D that allows for stabilized scanning with uniform steps using low laser power (1 mW). Thus, BSD provides a precise method to locally measure and thereby actively control sample position for diverse applications, especially those with limited optical access such as scanning probe microscopy, and magnetic tweezers.

Quartz tuning forks as sensors for attractive-mode force microscopy under ambient conditions

We present investigations of the frequency versus distance behavior of a quartz tuning-fork-based atomic force microscope. We show that if the amplitude of the motion A of the tip is large, then the apparent shape of the tip–surface interaction curve depends on A. For smaller amplitudes of oscillation (A≲3 nm), we find that the shape of the interaction curve becomes independent of A. In this low amplitude limit, a simple relation between the observed frequency shift and the underlying interaction allows quantitative determination of tip–sample forces. Tuning fork sensors open a window for dynamic-mode force microscopy in a regime where conventional microfabricated sensors are overwhelmed by long range capillary forces.

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer. The active translocase for the precursor of periplasmic galactose-binding protein contains twice the number of heterotrimeric units of SecYEG as does that for the precursor of outer membrane protein A.

Transient Collagen Triple Helix Binding to a Key Metalloproteinase in Invasion and Development

Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic nuclear magnetic resonance (NMR) detection have revealed how the HPX domain docks to collagen I-derived triple helix. Mutations impairing triple-helical peptidase activity corroborate the interface. Saturation transfer difference NMR suggests rotational averaging around the longitudinal axis of the triple-helical peptide. Part of the interface emerges as unique and potentially targetable for selective inhibition. The triple helix crosses the junction of blades I and II at a 45° angle to the symmetry axis of the HPX domain, placing the scissile Gly∼Ile bond near the HPX domain and shifted ∼25 Å from MMP-1 complexes. This raises the question of the MT1-MMP catalytic domain folding over the triple helix during catalysis, a possibility accommodated by the flexibility between domains suggested by atomic force microscopy images.

Dynamic Structure of the Translocon SecYEG in Membrane DIRECT SINGLE MOLECULE OBSERVATIONS

Background: Numerous proteins are exported across membranes by the translocon SecYEG, a highly conserved complex. Results: Multiple structural conformations and oligomeric states of SecYEG observed in lipid bilayers. Conclusion: Cytoplasmic membrane-external segments of SecYEG that orchestrate translocon function are highly dynamic. Significance: Direct visualization of disordered, flexible structures and oligomeric states in lipid bilayers provides a near-native vista of the translocon. Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude (∼1–10 Å) and were attributed to the two large loops of SecY linking transmembrane helices 6–7 and 8–9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.