Ashley R. Carter

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.

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.

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.

Improving the quantification of Brownian motion

Brownian motion experiments have become a staple of the undergraduate advanced laboratory, yet quantification of these experiments is difficult, typically producing errors of 10%–15% or more. Here, we discuss the individual sources of error in the experiment: sampling error, uncertainty in the diffusion coefficient, tracking error, vibration, and microscope drift. We model each source of error using theoretical and computational methods and compare the model to our experimental data. Finally, we describe various ways to reduce each source of error to less than 1%, improving the quantification of Brownian motion.

Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2–4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD–DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5′ strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.

Back-scattered detection yields viable signals in many conditions

Precision position-sensing is required for many microscopy techniques. One promising method, back-scattered detection (BSD), is incredibly sensitive, allowing for position measurements at the level of tens of picometers in three dimensions. In BSD the position of a micron-sized bead is measured by back-scattering a focused laser beam off the bead and imaging the resulting interference pattern onto a detector. Since the detection system geometry is confined to one side of the objective, the technique is compatible with platforms that have restricted optical access (e.g. magnetic tweezers, atomic force microscopy, and microfluidics). However, general adoption of BSD may be limited according to a recent theory [Volpe et al., J. Appl. Phys. 102, 084701, 2007] that predicts diminished signals under certain conditions. We directly measured the BSD response while varying the experimental conditions, including bead radius, numerical aperture, and relative index. Contrary to the proposed theory, we find that all experimental conditions tested produced a viable signal for atomic-scale measurements.

Improved performance of an ultrastable measurement platform using a field-programmable gate array for real-time deterministic control

Many precision measurement techniques (e.g. scanning probe microscopy, optical tweezers) are limited by sample drift. This is particularly true at room temperature in air or in liquid. Previously, we developed a general solution for sample control in three dimensions (3D) by first measuring the position of the sample and then using this position in a feedback loop to move a piezo-electric stage accordingly (Carter et al., Optics Express, 2007). In that work, feedback was performed using a software-based data acquisition program with limited bandwidth (≤ 100 Hz). By implementing feedback through a field programmable gate array (FPGA), we achieved real-time, deterministic control and increased the feedback rate to 500 Hz - half the resonance frequency of the piezo-electric stage in the feedback loop. This better control led to a three-fold improvement in lateral stability to 10 pm (Δf = 0.01-10 Hz). Furthermore, we exploited the rapid signal processing of FPGA to achieve fast stepping rates coupled with highly accurate and orthogonal scanning.

Intensity‐dependent timing and precision of startle response latency in larval zebrafish

Using high‐speed videos time‐locked with whole‐animal electrical recordings, simultaneous measurement of behavioural kinematics and field potential parameters of C‐start startle responses allowed for discrimination between short‐latency and long‐latency C‐starts (SLCs vs. LLCs) in larval zebrafish. Apart from their latencies, SLC kinematics and SLC field potential parameters were intensity independent. Increasing stimulus intensity increased the probability of evoking an SLC and decreased mean SLC latencies while increasing their precision; subtraction of field potential latencies from SLC latencies revealed a fixed time delay between the two measurements that was intensity independent. The latency and the precision in the latency of the SLC field potentials were linearly correlated to the latencies and precision of the first evoked action potentials (spikes) in hair‐cell afferent neurons of the lateral line. Together, these findings indicate that first spike latency (FSL) is a fast encoding mechanism that can serve to precisely initiate startle responses when speed is critical for survival.

Progress on an optical trapping assay to measure DNA folding pathways in sperm

DNA undergoes a dramatic condensation in sperm nuclei. During this condensation, the DNA rapidly folds into a series of toroids when protamine proteins replace histone proteins. Measuring the mechanics and folding pathway for this incredible condensation is an important goal. Here, we report on progress to use an in vitro, optical trapping assay to measure the DNA folding dynamics for this process. In this assay, a single DNA molecule with its associated histone proteins is attached to a cover slip and to an optically trapped bead. Movement of the optical trap applies a force on the bead, stretching the DNA to a particular extension. When protamine is added, the extension changes, allowing us to measure the preliminary folding dynamics for the process.