Antoine Le Gall

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.

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.