Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 +/- 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.
Monodisperse populations of microspheres are desirable for a variety of research and industrial applications, but many desirable sizes and materials can be difficult to synthesize and have limited commercial availability. In this paper, we present an effective, straightforward, and low cost method for sorting polydisperse microspheres into many separate monodisperse samples. The basic approach is to use velocity sedimentation through a density gradient in a long vertical column, followed by carefully targeted extraction. We demonstrate this technique by reducing the coefficient of variation of melamine microspheres from 13% to 1%-4% and glass microspheres from 35% to 3%-8%. This simple and inexpensive method can be used to sort microspheres of many sizes and materials, and is easily scalable, opening the possibility of cheap, monodisperse microspheres.
Much of neurophysiology and vision science relies on careful measurement of a human or animal subject's gaze direction. Video-based eye trackers have emerged as an especially popular option for gaze tracking, because they are easy to use and are completely non-invasive. However, video eye trackers typically require a calibration procedure in which the subject must look at a series of points at known gaze angles. While it is possible to rely on innate orienting behaviors for calibration in some non-human species, other species, such as rodents, do not reliably saccade to visual targets, making this form of calibration impossible. To overcome this problem, we developed a fully automated infrared video eye-tracking system that is able to quickly and accurately calibrate itself without requiring co-operation from the subject. This technique relies on the optical geometry of the cornea and uses computer-controlled motorized stages to rapidly estimate the geometry of the eye relative to the camera. The accuracy and precision of our system was carefully measured using an artificial eye, and its capability to monitor the gaze of rodents was verified by tracking spontaneous saccades and evoked oculomotor reflexes in head-fixed rats (in both cases, we obtained measurements that are consistent with those found in the literature). Overall, given its fully automated nature and its intrinsic robustness against operator errors, we believe that our eye-tracking system enhances the utility of existing approaches to gaze-tracking in rodents and represents a valid tool for rodent vision studies.
The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor's phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time.
The cell-surface-signaling protein Notch, is required for numerous developmental processes and typically specifies which of two adjacent cells will adopt a non-neuronal developmental fate. It has recently been implicated in long-term memory formation in mammals and Drosophila. Here, we investigated whether activity-dependent synaptic plasticity at the neuromuscular junctions (NMJs) of third instar Drosophila larvae depends on Notch signaling. The length and number of axonal branches and number of presynaptic sites (boutons) in NMJ vary with the level of synaptic activity, so we increased activity at the NMJ by two complementary methods: increasing the chronic growth temperature of third instar larvae from 18 to 28 degrees C and using the double-mutant ether-a-gogo,Shaker (eagSh), both of which increase NMJ size and bouton count. Animals homozygous for the functionally null, temperature-sensitive Notch alleles, N(ts1) and N(ts2), displayed no activity-dependent increase in NMJ complexity when reared at the restrictive temperature. Dominant-negative Notch transgenic expression also blocked activity-dependent plasticity. Ectopic expression of wild-type Notch and constitutively active truncated Notch transgenes also reduced activity-dependent plasticity, suggesting that there is a "happy medium" level of Notch activity in mediating NMJ outgrowth. Last, we show that endogenous Notch is primarily expressed in the presynaptic cell bodies where its expression level is positively correlated with motor neuron activity.
We describe an active polymer network in which processive molecular motors control network elasticity. This system consists of actin filaments cross-linked by filamin A (FLNa) and contracted by bipolar filaments of muscle myosin II. The myosin motors stiffen the network by more than two orders of magnitude by pulling on actin filaments anchored in the network by FLNa cross-links, thereby generating internal stress. The stiffening response closely mimics the effects of external stress applied by mechanical shear. Both internal and external stresses can drive the network into a highly nonlinear, stiffened regime. The active stress reaches values that are equivalent to an external stress of 14 Pa, consistent with a 1-pN force per myosin head. This active network mimics many mechanical properties of cells and suggests that adherent cells exert mechanical control by operating in a nonlinear regime where cell stiffness is sensitive to changes in motor activity. This design principle may be applicable to engineering novel biologically inspired, active materials that adjust their own stiffness by internal catalytic control.
Proteins are dynamic molecular machines having structural flexibility that allows conformational changes. Current methods for the determination of protein flexibility rely mainly on the measurement of thermal fluctuations and disorder in protein conformations and tend to be experimentally challenging. Moreover, they reflect atomic fluctuations on picosecond timescales, whereas the large conformational changes in proteins typically happen on micro- to millisecond timescales. Here, we directly determine the flexibility of bacteriorhodopsin -- a protein that uses the energy in light to move protons across cell membranes -- at the microsecond timescale by monitoring force-induced deformations across the protein structure with a technique based on atomic force microscopy. In contrast to existing methods, the deformations we measure involve a collective response of protein residues and operate under physiologically relevant conditions with native proteins.
In the 1970s, deGennes discussed the fundamental geometry of smectic liquid crystals and established an analogy between the smectic A phase and superconductors. It follows that smectic layers expel twist deformations in the same way that superconductors expel magnetic field. We make a direct observation of the penetration of twist at the edge of a single isolated smectic A layer composed of chiral fd virus particles subjected to a depletion interaction. Using the LC-PolScope, we make quantitative measurements of the spatial dependence of the birefringence due to molecular tilt near the layer edges. We match data to theory for the molecular tilt penetration profile and determine the twist penetration length for this system.
Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.
We characterize optical wave propagation along line defects in two-dimensional arrays of air-holes in free-standing silicon slabs. The fabricated waveguides contain random variations in orientation of the photonic lattice elements which perturb the in-plane translational symmetry. The vertical slab symmetry is also broken by a tilt of the etched sidewalls. We discuss how these lattice imperfections affect out-of-plane scattering losses and introduce a mechanism for high-Q cavity excitation related to polarization mixing.
Power-spectral-density measurements of any sampled signal are typically restricted by both acquisition rate and frequency response limitations of instruments, which can be particularly prohibitive for video-based measurements. We have developed a new method called intensity modulation spectral analysis that circumvents these limitations, dramatically extending the effective detection bandwidth. We demonstrate this by video tracking an optically trapped microsphere while oscillating an LED illumination source. This approach allows us to quantify fluctuations of the microsphere at frequencies over 10 times higher than the Nyquist frequency, mimicking a significantly higher frame rate.