Magnetism of isolated Mn12 single-molecule magnets detected by magnetic circular dichroism: Observation of spin tunneling with a magneto-optical technique

We report magnetic and magneto-optical measurements of two Mn12 single-molecule magnet derivatives isolated in organic glasses. Field-dependent magnetic circular dichroism (MCD) intensity curves (hysteresis cycles) are found to be essentially identical to superconducting quantum interference device magnetization results and provide experimental evidence for the potential of the optical technique for magnetic characterization. Optical observation of magnetic tunneling has been achieved by studying the decay of the MCD signal at weak applied magnetic field

Magnetism of isolated Mn12 single-molecule magnets detected by magnetic circular dichroism: Observation of spin tunneling with a magneto-optical technique

We report magnetic and magneto-optical measurements of two Mn12 single-molecule magnet derivatives isolated in organic glasses. Field-dependent magnetic circular dichroism (MCD) intensity curves (hysteresis cycles) are found to be essentially identical to superconducting quantum interference device magnetization results and provide experimental evidence for the potential of the optical technique for magnetic characterization. Optical observation of magnetic tunneling has been achieved by studying the decay of the MCD signal at weak applied magnetic field

Control of optical fields and single photon emitters by advanced nanoantenna structures

The control of optical fields on the nanometre scale is becoming an increasingly important tool in many fields, ranging from channelling light delivery in photovoltaics and light emitting diodes to increasing the sensitivity of chemical sensors to single molecule levels. The ability to design and manipulate light fields with specific frequency and space characteristics is explored in this project. We present an alternative realisation of Extraordinary Optical Transmission (EOT) that requires only a single aperture and a coupled waveguide. We show how this waveguide-resonant EOT improves the transmissivity of single apertures. An important technique in imaging is Near-Field Scanning Optical Microscopy (NSOM); we show how waveguide-resonant EOT and the novel probe design assist in improving the efficiency of NSOM probes by two orders of magnitude, and allow the imaging of single molecules with an optical resolution of as good as 50 nm. We show how optical antennas are fabricated into the apex of sharp tips and can be used in a near-field configuration.

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Counter-propagating dual-trap optical tweezers based on linear momentum conservation

We present a dual-trap optical tweezers setup which directly measures forces using linear momentum conservation. The setup uses a counter-propagating geometry, which allows momentum measurement on each beam separately. The experimental advantages of this setup include low drift due to all-optical manipulation, and a robust calibration (independent of the features of the trapped object or buffer medium) due to the force measurement method. Although this design does not attain the high-resolution of some co-propagating setups, we show that it can be used to perform different single molecule measurements: fluctuation-based molecular stiffness characterization at different forces and hopping experiments on molecular hairpins. Remarkably, in our setup it is possible to manipulate very short tethers (such as molecular hairpins with short handles) down to the limit where beads are almost in contact. The setup is used to illustrate a novel method for measuring the stiffness of optical traps and tethers on the basis of equilibrium force fluctuations, i.e., without the need of measuring the force vs molecular extension curve. This method is of general interest for dual trap optical tweezers setups and can be extended to setups which do not directly measure forces.

Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

ABSTRACT Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrix-matched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities.

Random assignment of intervention points in two phase single-case designs: data-division-specific distributions

The present study explores the statistical properties of a randomization test based on the random assignment of the intervention point in a two-phase (AB) single-case design. The focus is on randomization distributions constructed with the values of the test statistic for all possible random assignments and used to obtain p-values. The shape of those distributions is investigated for each specific data division defined by the moment in which the intervention is introduced. Another aim of the study consisted in testing the detection of inexistent effects (i.e., production of false alarms) in autocorrelated data series, in which the assumption of exchangeability between observations may be untenable. In this way, it was possible to compare nominal and empirical Type I error rates in order to obtain evidence on the statistical validity of the randomization test for each individual data division. The results suggest that when either of the two phases has considerably less measurement times, Type I errors may be too probable and, hence, the decision making process to be carried out by applied researchers may be jeopardized.

A monolithic glass chip for active single-cell sorting based on mechanical phenotyping

The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custombuilt optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.