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We use x-ray photon correlation spectroscopy (XPCS) to probe the dynamics of colloidal particles in polycrystalline ice. During freezing, the dendritic ice morphology and rejection of particles from the ice created regions of high particle density, where some of the colloids were forced into contact and formed disordered aggregates. The particles in these high density regions underwent ballistic motion, with a characteristic velocity that increased with temperature. This ballistic motion is coupled with both stretched and compressed exponential decays of the intensity autocorrelation function. We suggest that this behavior could result from ice grain boundary migration. [ABSTRACT FROM AUTHOR]

We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomyces cerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data. [ABSTRACT FROM AUTHOR]

Since 2010, our physics department has offered a re-imagined calculus-based introductory physics sequence for the life sciences. These courses include a selection of biologically and medically relevant topics that we believe are more meaningful to undergraduate premedical and biological science students than those found in a traditional course. In this paper, we highlight new aspects of the first-semester course, and present a comparison of student evaluations of this course versus a more traditional one. We also present the effect on student perception of the relevance of physics to biology and medicine after having taken this course. [ABSTRACT FROM AUTHOR]

We describe an educational module on feedback and gene circuits that constitute the final topic in a new year-long introductory physics sequence aimed at biology and premedical students at Yale University. The overall goals of this sequence are threefold. First to demonstrate the application of physics and mathematics in the life sciences. Second to introduce biological science majors to mathematical and physical tools, principles, and experiences. Third to seed an enduring appreciation of quantitative approaches in biology and medicine. Here, we present a module on feedback and gene circuits that focuses on a genetic toggle switch and a repressilator. The genetic toggle switch consists of two genes, each of whose protein products represses the other's expression, while the repressilator consists of three genes, each of whose protein products represses the next gene's expression. Analytic, numerical, and electronic treatments of the genetic toggle switch show bistability. A similar treatment of the repressilator reveals sustained oscillations. [ABSTRACT FROM AUTHOR]

By monitoring multiple molecular transitions, force-clamp, and trap-position-clamp methods have led to precise determinations of the free energies and free energy landscapes for molecular states populated in equilibrium at the same or similar forces. Here, we present a powerful new elaboration of the force-clamp and force-jump methods, applicable to transitions far from equilibrium. Specifically, we have implemented a live jump detection and force-clamp algorithm that intelligently adjusts and maintains the force on a single molecule in response to the measured state of that molecule. We are able to collect hundreds of individual molecular transitions at different forces, many times faster than previously, permitting us to accurately determine force-dependent lifetime distributions and reaction rates. Application of our method to unwinding and rewinding the nucleosome inner turn, using optical tweezers reveals experimental lifetime distributions that comprise a statistically meaningful number of transitions, and that are accurately single exponential. These measurements significantly reduce the error in the previously measured rates, and demonstrate the existence of a single, dominant free energy barrier at each force studied. A key benefit of the molecular yo-yo method for nucleosomes is that it reduces as far as possible the time spent in the tangentially bound state, which minimizes the loss of nucleosomes by dissociation. [ABSTRACT FROM AUTHOR]

Three interrelated biologically-relevant examples of biased random walks are discussed: DNA melting, modeled as DNA unzipping, which provides a way to illustrate the role of the Boltzmann factor in a context well known to biology and pre-medical students; the activity of helicase motor proteins in unzipping double-stranded DNA, which is an example of a Brownian ratchet; and force generation by actin polymerization, which is also a Brownian ratchet, and for which the force and actin-concentration dependence of the velocity of actin polymerization is determined. [ABSTRACT FROM AUTHOR]

The newly introduced coherence-based technique of X-ray near-field speckle (XNFS) has been implemented at 8-ID-I at the Advanced Photon Source. In the near-field regime of high-brilliance synchrotron X-rays scattered from a sample of interest, it turns out that, when the scattered radiation and the main beam both impinge upon an X-ray area detector, the measured intensity shows low-contrast speckles, resulting from interference between the incident and scattered beams. A micrometer-resolution XNFS detector with a high numerical aperture microscope objective has been built and its capability for studying static structures and dynamics at longer length scales than traditional far-field X-ray scattering techniques is demonstrated. Specifically, the dynamics of dilute silica and polystyrene colloidal samples are characterized. This study reveals certain limitations of the XNFS technique, especially in the characterization of static structures, which is discussed. [ABSTRACT FROM AUTHOR]

An analysis is presented of how to optimize the experimental beamline configuration for achieving the best possible signal-to-noise ratio (SNR) in X-ray photon correlation spectroscopy experiments using area detectors. It is shown that there exists an optimum detector distance; namely, the highest SNR is achieved by matching the angular pixel size with the angular source size. Binning several pixels together can increase the SNR by permitting to match the shape of a detector pixel to the shape of the source. It is also shown that collimating slits several times wider than the effective transverse coherence length are optimal; further, it is demonstrated that the energy dependence of the SNR is dictated by the energy dependence of detector efficiency and source brilliance. Ultimately the effects of focusing and low longitudinal coherence are discussed. [ABSTRACT FROM AUTHOR]

A new, fast x-ray detector system is presented for high-throughput, high-sensitivity, time-resolved, x-ray scattering and imaging experiments, most especially x-ray photon correlation spectroscopy (XPCS). After a review of the architectures of different CCD chips and a critical examination of their suitability for use in a fast x-ray detector, the new detector hardware is described. In brief, its principal component is an inexpensive, commercial camera—the SMD1M60—originally designed for optical applications, and modified for use as a direct-illumination x-ray detector. The remainder of the system consists of two Coreco Imaging PC-DIG frame grabber boards, located inside a Dell Power-edge 6400 server. Each frame grabber sits on its own PCI bus and handles data from 2 of the CCD’s 4 taps. The SMD1M60 is based on a fast, frame-transfer, 4-tap CCD chip, read out at12-bit resolution at frame rates of up to 62 Hz for full frame readout and up to 500 Hz for one-sixteenth frame readout. Experiments to characterize the camera’s suitability for XPCS and small-angle x-ray scattering (SAXS) are presented. These experiments show that single photon events are readily identified, and localized to within a pixel index or so. This is a sufficiently fine spatial resolution to maintain the speckle contrast at an acceptable value for XPCS measurements. The detective quantum efficiency of the SMD1M60 is 49% for directly-detected 6.3 keV x rays. The effects of data acquisition strategies that permit near-real-time data compression are also determined and discussed. Overall, the SMD1M60 detector system represents a major improvement in the technology for time-resolved x-ray experiments, that require an area detector with time-resolutions in few-milliseconds-to-few-seconds range, and it should have wide applications, extending beyond XPCS. [ABSTRACT FROM AUTHOR]

By monitoring stage drift via the normalized cross-correlation of an image of a stuck bead, obtained in real-time, with an out-of-focus 'template' image of a similar immobile bead, stored in memory, we implement a simple approach to actively stabilize drift in all three dimensions for existing video microscopy setups. We demonstrate stability to 0.0062 nm along the Z-axis and 0.0031 nm along the X- and Y-axes for long (100 s) timescales. [ABSTRACT FROM AUTHOR]

We describe a simple-to-construct programmable direct-digital-synthesis-based controller for use with acousto-optic deflectors. Our controller corrects for nonlinear diffraction efficiency versus diffraction angle, provides superior stability, functionality, and configurability, and costs a fraction of commercially available systems. Using this instrument, we move a 1 μm diameter bead by 1-nm-sized steps and resolve these steps. [ABSTRACT FROM AUTHOR]

A correction to the article "Active drift stabilization on three dimensions via image cross-correlation" that was published in the previous issue is presented.

We describe a new method for calibrating optical trapping measurements in which tension is applied in the direction of the laser beam to a molecule tethered between a surface and an optically trapped bead. Specifically, we present a generally-applicable procedure for converting from the measured scattering intensity and the measured stage displacement to applied tension and bead-coverslip separation, using measurements of the light intensity scattered from an untethered, trapped bead. Our calibration accounts for a number of effects, including aberrations and the interference of forward-reflected bead-scattered light with the trapping beam. To demonstrate the accuracy of our method, we show measurements of the DNA force-versus-extension relation using a range of laser intensities, and show that these measurements match the expected extensible wormlike-chain (WLC) behavior. Finally, we also demonstrate a force-clamp, in which the tension in a tether is held fixed while the extension varies as a result of molecular events. [ABSTRACT FROM AUTHOR]

A method for reducing time sequences of raw scattering images to intensity time-autocorrelation functions is presented. The procedure is based on the use of a charge coupled device (CCD) area detector, and optimized for operating in the regime of short data batches. Its application to x-ray photon correlation spectroscopy (XPCS) measurements is described in detail. Using a slow-scan CCD, we explain how to achieve data acquisition on a 30 ms or faster time scale, while simultaneously acquiring data from many coherence areas in parallel. The statistical uncertainties of the acquired XPCS data are quantified experimentally, and compared to the theoretically expected noise levels of the correlation functions. © 2000 American Institute of Physics. [ABSTRACT FROM AUTHOR]