Thomas T. Perkins

Gold nanoparticles: enhanced optical trapping and sensitivity coupled with significant heating

Gold nanoparticles appear to be superior handles in optical trapping assays. We demonstrate that relatively large gold particles (R(b)=50 nm) indeed yield a sixfold enhancement in trapping efficiency and detection sensitivity as compared to similar-sized polystyrene particles. However, optical absorption by gold at the most common trapping wavelength (1064 nm) induces dramatic heating (266 degrees C/W). We determined this heating by comparing trap stiffness from three different methods in conjunction with detailed modeling. Due to this heating, gold nanoparticles are not useful for temperature-sensitive optical-trapping experiments, but may serve as local molecular heaters. Also, such particles, with their increased detection sensitivity, make excellent probes for certain zero-force biophysical assays.

Forward and Reverse Motion of Single RecBCD Molecules on DNA

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

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.

Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection

Back-focal-plane detection of micrometer-sized beads offers subnanometer resolution for single-molecule, optical trapping experiments. However, laser beam-pointing instability and mechanical drift of the microscope limit the resolution of optical-trapping experiments. By combining two infrared lasers with improved differential beam-pointing stability (< or = 0.05 microrad), we simultaneously measure and subtract the motion of the microscope stage, leading to a resolution of <0.1 nm in 1 ms and stability of 0.5 nm over 60 s. Repeated steps of 0.4 nm at 1 Hz are resolved with a signal-to-noise ratio of 25.

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.

Precision Surface-Coupled Optical-Trapping Assay with One-Basepair Resolution

The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale ( approximately 0.1 nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (<0.05 nm) of bead detection over a broad range of trap stiffness (k(T) = 0.05-0.5 pN/nm) and frequency (Deltaf = 0.03-100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision ( approximately 0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.

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.

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.

Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins

Pulling apart protein unfolding Elucidating the details of how complex proteins fold is a longstanding challenge. Key insights into the unfolding pathways of diverse proteins have come from single-molecule force spectroscopy (SMFS) experiments in which proteins are literally pulled apart. Yu et al. developed a SMFS technique that could unfold individual bacteriorhodopsin molecules in a native lipid bilayer with 1-µs temporal resolution (see the Perspective by Müller and Gaub). The technique delivered a 100-fold improvement over earlier studies of bacteriorhodopsin and revealed many intermediates not seen before. The authors also observed unfolding and refolding transitions between intermediate states. Science, this issue p. 945; see also p. 907 Mechanical unfolding of a membrane protein reveals previously undetected intermediates and equilibrium refolding. Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as two or three amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

Nano-chemical infrared imaging of membrane proteins in lipid bilayers.

The spectroscopic characterization of biomolecular structures requires nanometer spatial resolution and chemical specificity. We perform full spatio-spectral imaging of dried purple membrane patches purified from Halobacterium salinarum with infrared vibrational scattering-type scanning near-field optical microscopy (s-SNOM). Using near-field spectral phase contrast based on the Amide I resonance of the protein backbone, we identify the protein distribution with 20 nm spatial resolution and few-protein sensitivity. This demonstrates the general applicability of s-SNOM vibrational nanospectroscopy, with potential extension to a wide range of biomolecular systems.