Karen Perronet

Simultaneous calibration of optical tweezers spring constant and position detector response

We demonstrate a fast and direct calibration method for systems using a single laser for optical tweezers and particle position detection. The method takes direct advantage of back-focal-plane interferometry measuring not an absolute but a differential position, i.e. the position of the trapped particle relative to the center of the optical tweezers. Therefore, a fast step-wise motion of the optical tweezers yields the impulse response of the trapped particle. Calibration parameters such as the detectors spatial and temporal response and the spring constant of the optical tweezers then follow readily from fitting the measured impulse response.

Improved Photon Yield from a Green Dye with a Reducing and Oxidizing System

An optimized chemistry turns a small green dye into a useful label for single- molecule experiments where steric hin- drance is an issue. Thanks to a reducing and oxidizing system combined to oxygen depletion, a single Bodipy (boron-dipyrromethene) FL fluorophore emits, on average, 20 times more pho- tons around 510 nm (see picture) and its lifetime before photobleaching is in- creased by the same amount, reaching several seconds.

Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination

Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.

Photon emission from polycrystalline Ag induced by scanning tunneling microscopy: comparison of different tip materials

Abstract Photon emission from polycrystalline silver induced by scanning tunneling microscopy is studied for three different tip materials (Au, PtIr and W). Photon emission intensity curves as a function of the tip voltage are observed to be almost identical for platinum–iridium alloy and gold tips (and more than 10 times enhanced as compared with the tungsten tip). An evolution in topography and photon map for different applied voltages is investigated along with the study of the spatial distribution of photon emission in dependence upon the surface local differential height. It turns out that no clear correlation between a local curvature and enhancement of light emission can be found. Simultaneous measurements of tunneling current and photon intensity as a function of vertical tip displacement confirm the earlier observation, namely, that similar apparent barrier heights exist for both elastic and inelastic tunneling channels. The role of the tip material as well as its shape is discussed.

Calibrating optical tweezers with Bayesian inference

We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup. It is based on an inference approach that uses Bayesian probability to infer the diffusion coefficient and the potential felt by a bead trapped in an optical or magnetic trap. It exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy.

Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy

Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

Optical tweezers calibration with Bayesian inference

We present a new method for calibrating an optical-tweezer setup that is based on Bayesian inference1. This method employs an algorithm previously used to analyze the confined trajectories of receptors within lipid rafts2,3. The main advantages of this method are that it does not require input parameters and is insensitive to systematic errors like the drift of the setup. Additionally, it exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. The additional information can be used to detect deviations from the perfect harmonic potential or detect environmental influences on the bead. The algorithm infers the diffusion coefficient and the potential felt by a trapped bead, and only requires the bead trajectory as input. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy. Furthermore, by inferring a higher order potential our method can reveal deviations from the assumed second-order potential. More generally, this method can also be used for magnetic-tweezer calibration.

STM-INDUCED PHOTOEMISSION AT SOLID-LIQUID INTERFACE

We extended STM-induced photoemission from metal surface to solid-liquid interface. A Au(111)-Au junction immersed in a liquid droplet was studied. We observed a decrease of the voltage threshold for photoemission when adding liquid compared to air medium, a strong decrease of the apparent tunnel barrier height and red-shifted emission spectra with two distinct modes of emission. This is attributed to the refractive index of the liquid. Our observations permit to investigate the influence on STM-induced photoemission of a self-assembled molecule monolayer formed on a Au(111) substrate.

STM-induced photon emission at solid–liquid interface

Abstract We extended Scanning Tunnelling Microscopy (STM)-induced photon emission from metal surface to solid–liquid interface. A Au(111)–Au junction immersed in a liquid droplet was studied. We observed a decrease of the voltage threshold for photon emission when adding liquid compared to air medium and a strong decrease of the apparent tunnel barrier height and a parallel behavior for photon emission. This is attributed to the refractive index of the liquid. Our observations permit to investigate the influence on STM-induced photon emission of a self-assembled molecule monolayer formed on a Au(111) substrate.