The effect of integration time on fluctuation measurements: calibrating an optical trap in the presence of motion blur

Dynamical instrument limitations, such as finite detection bandwidth, do not simply add statistical errors to fluctuation measurements, but can create significant systematic biases that affect the measurement of steady-state properties. Such effects must be considered when calibrating ultra-sensitive force probes by analyzing the observed Brownian fluctuations. In this article, we present a novel method for extracting the true spring constant and diffusion coefficient of a harmonically confined Brownian particle that extends the standard equipartition and power spectrum techniques to account for video-image motion blur. These results are confirmed both numerically with a Brownian dynamics simulation, and experimentally with laser optical tweezers.Comment: 12 pages, 6 figures, revtex4; published in Optics Express. http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-25-1251

Beyond the frame rate: Measuring high-frequency fluctuations with light intensity modulation

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 (IMSA) 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.Comment: 4 pages, 2 figure

Massively parallel singlemolecule manipulation using centrifugal force

ABSTRACT 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 singlemolecule 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 5 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. Received for publication 8 January 2010 and in final form 10 March 2010. Single-molecule research has advanced greatly in the last decade, fueled in part by the development of technologies such as the atomic force microscope (AFM) and optical and magnetic tweezers, which enable precise physical manipulation of single molecular constructs (1). Remarkable studies with these instruments have already yielded new insight into such diverse areas as protein folding and unfolding dynamics, motor proteins, dynamic strength of receptor ligand interactions, enzymatic activity, and DNA mechanics (1-5). Widespread use of these powerful techniques, however, has been impeded by the laborious nature of making measurements one molecule at a time, the typically costly equipment, and the requisite technical expertise to perform these measurements. Recently these issues have received some attention with innovations such as multiplexed magnetic tweezer systems (6,7) to increase efficiency and more cost-effective designs for optical tweezers systems (8). We have developed an approach to solve these problems: massively parallel single-molecule force measurements using centrifugal force. The basic concept is that by rapidly rotating a high-resolution detection system, a centrifugal force field can be applied to an ensemble of objects while simultaneously observing their micro-to-nanoscopic motions. This is implemented in a new instrument that we call the centrifuge force microscope (CFM) The centrifugal force applied to each molecular tether can be easily determined using F ¼ mu 2 R, where m is the mass of the bead (minus the mass of the medium displaced to account for buoyancy), u is the magnitude of its angular velocity, and R is its distance from the axis of rotation. Since R is a macroscopic length much larger than the motion of the particles and the region of observation, the force field is conveniently uniform over the sample and as stable as the constancy of u. For monodisperse beads of known size and density (available commercially or by processing (11)) the centrifugal force on each particle is identical and can be calculated directly without calibration. Detection of molecular transitions, such as bond rupture or tether extension, is also straightforward. Since the force is normal to the coverslip and the whole system rotates, the beads appear relatively stationary in the field of view, but are pulled out of focus as a molecular tether stretches or detaches. Although a variety of bead detection schemes are possible, image focus provides the simplest way to determine if a bead is connected to the surface or not. For example, when measuring bond dissociation kinetics under constant force, one simply needs to measure the times at which singly tethered beads abruptly detach from the coverslip and disappear from view. We demonstrate this method by performing thousands of single-molecule measurements in parallel to characterize the force-dependent unbinding kinetics of digoxigenin and its antibody By applying various force clamps, we determined the force-dependent off-rate k off (f) ¼ k 0 exp(f/f b ) (12,13) for the interaction of digoxigenin and its antibody. We found a stress-free off-rate of k 0 ¼ 0.015 5 0.002 s À1 and a force scale of f b ¼ 4.6 5 1.3 p

Massively parallel single-molecule manipulation using centrifugal force

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 with a new approach: massively parallel single-molecule force measurements using centrifugal force. This approach is realized in a novel instrument that we call the Centrifuge Force Microscope (CFM), 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 $\pm$ 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, "single-molecule centrifugation" has the potential to revolutionize single-molecule experimentation, and open access to a wider range of researchers and experimental systems.Comment: 5 pages, 3 figure

Novel venom gene discovery in the platypus

Background: To date, few peptides in the complex mixture of platypus venom have been identified and sequenced, in part due to the limited amounts of platypus venom available to study. We have constructed and sequenced a cDNA library from an active platypus venom gland to identify the remaining components.Results: We identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). A number of these are expressed in tissues other than the venom gland, and at least three of these families (those with homology to toxins from distant invertebrates) may play non-toxin roles. Thus, further functional testing is required to confirm venom activity. However, the presence of similar putative toxins in such widely divergent species provides further evidence for the hypothesis that there are certain protein families that are selected preferentially during evolution to become venom peptides. We have also used homology with known proteins to speculate on the contributions of each venom component to the symptoms of platypus envenomation.Conclusions: This study represents a step towards fully characterizing the first mammal venom transcriptome. We have found similarities between putative platypus toxins and those of a number of unrelated species, providing insight into the evolution of mammalian venom

Topographical distribution of perioperative cerebral infarction associated with transcatheter aortic valve implantation

Transcatheter aortic valve implantation (TAVI) is associated with a high incidence of cerebrovascular injury. As these injuries are thought to be primarily embolic, neuroprotection strategies have focused on embolic protection devices. However, the topographical distribution of cerebral emboli and how this impacts on the effectiveness of these devices have not been thoroughly assessed. Here, we evaluated the anatomical characteristics of magnetic resonance imaging (MRI)-defined cerebral ischemic lesions occurring secondary to TAVI to enhance our understanding of the distribution of cardioembolic phenomena.Forty patients undergoing transfemoral TAVI with an Edwards SAPIEN-XT valve under general anesthesia were enrolled prospectively in this observational study. Participants underwent brain MRI preprocedure, and 3 ± 1 days and 6 ± 1 months postprocedure.Mean ± SD participant age was 82 ± 7 years. Patients had an intermediate to high surgical risk, with a mean Society of Thoracic Surgeons score of 6.3 ± 3.5 and EuroSCORE of 18.1 ± 10.6. Post-TAVI, there were no clinically apparent cerebrovascular events, but MRI assessments identified 83 new lesions across 19 of 31 (61%) participants, with a median ± interquartile range number and volume of 1 ± 2.8 lesions and 20 ± 190 μL per patient. By volume, 80% of the infarcts were cortical, 90% in the posterior circulation and 81% in the right hemisphere.The distribution of lesions that we detected suggests that cortical gray matter, the posterior circulation, and the right hemisphere are all particularly vulnerable to perioperative cerebrovascular injury. This finding has implications for the use of intraoperative cerebral embolic protection devices, particularly those that leave the left subclavian and, therefore, left vertebral artery unprotected

First Earth-based Detection of a Superbolide on Jupiter

Cosmic collisions on planets cause detectable optical flashes that range from terrestrial shooting stars to bright fireballs. On June 3, 2010 a bolide in Jupiter's atmosphere was simultaneously observed from the Earth by two amateur astronomers observing Jupiter in red and blue wavelengths. The bolide appeared as a flash of 2 s duration in video recording data of the planet. The analysis of the light curve of the observations results in an estimated energy of the impact of 0.9-4.0x10^{15} J which corresponds to a colliding body of 8-13 m diameter assuming a mean density of 2 g cm^{-3}. Images acquired a few days later by the Hubble Space Telescope and other large ground-based facilities did not show any signature of aerosol debris, temperature or chemical composition anomaly, confirming that the body was small and destroyed in Jupiter's upper atmosphere. Several collisions of this size may happen on Jupiter on a yearly basis. A systematic study of the impact rate and size of these bolides can enable an empirical determination of the flux of meteoroids in Jupiter with implications for the populations of small bodies in the outer Solar System and may allow a better quantification of the threat of impacting bodies to Earth. The serendipitous recording of this optical flash opens a new window in the observation of Jupiter with small telescopes

Long-Term Evolution of the Aerosol Debris Cloud Produced by the 2009 Impact on Jupiter

We present a study of the long-term evolution of the cloud of aerosols produced in the atmosphere of Jupiter by the impact of an object on 19 July 2009. The work is based on images obtained during 5 months from the impact to 31 December 2009 taken in visible continuum wavelengths and from 20 July 2009 to 28 May 2010 taken in near-infrared deep hydrogen-methane absorption bands at 2.1-2.3 micron. The impact cloud expanded zonally from approximately 5000 km (July 19) to 225,000 km (29 October, about 180 deg in longitude), remaining meridionally localized within a latitude band from 53.5 deg S to 61.5 deg S planetographic latitude. During the first two months after its formation the site showed heterogeneous structure with 500-1000 km sized embedded spots. Later the reflectivity of the debris field became more homogeneous due to clump mergers. The cloud was mainly dispersed in longitude by the dominant zonal winds and their meridional shear, during the initial stages, localized motions may have been induced by thermal perturbation caused by the impact's energy deposition. The tracking of individual spots within the impact cloud shows that the westward jet at 56.5 deg S latitude increases its eastward velocity with altitude above the tropopause by 5- 10 m/s. The corresponding vertical wind shear is low, about 1 m/s per scale height in agreement with previous thermal wind estimations. We found evidence for discrete localized meridional motions with speeds of 1-2 m/s. Two numerical models are used to simulate the observed cloud dispersion. One is a pure advection of the aerosols by the winds and their shears. The other uses the EPIC code, a nonlinear calculation of the evolution of the potential vorticity field generated by a heat pulse that simulates the impact. Both models reproduce the observed global structure of the cloud and the dominant zonal dispersion of the aerosols, but not the details of the cloud morphology. The reflectivity of the impact cloud decreased exponentially with a characteristic timescale of 15 days; we can explain this behavior with a radiative transfer model of the cloud optical depth coupled to an advection model of the cloud dispersion by the wind shears. The expected sedimentation time in the stratosphere (altitude levels 5-100 mbar) for the small aerosol particles forming the cloud is 45-200 days, thus aerosols were removed vertically over the long term following their zonal dispersion. No evidence of the cloud was detected 10 months after the impact