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Multi-dielectric coated coverslip can be designed to reach large optical field enhancements when working under proper illumination conditions and in total internal reflection. In an objective-based total internal reflection fluorescence microscopy (TIRF-M), we propose to use the resulting large field enhancement supported in such coverslip to improve TIRF-M sensitivity by amplifying the collected fluorescence signal. We present here the optimization, realization and testing in TIRF-M of resonant coverslips designed to take into account the experimental constraints. The latter's are due to the inverted configuration of TIRF-M together with the use of a high numerical aperture objective. The challenge is therefore to find the best coating design compromising between the angular tolerance and the field enhancement. We will report here enhanced-TIRF-M imaging over model samples but the ultimate goal here is to be able to monitor and quantify dynamics of pathogens at the membrane of living cells., Comment: 2023 23rd International Conference on Transparent Optical Networks (ICTON), Jul 2023, Bucharest, Romania

The life of a cell is governed by highly dynamical microscopic processes. Two notable examples are the diffusion of membrane receptors and the kinetics of transcription factors governing the rates of gene expression. Different fluorescence imaging techniques have emerged to study molecular dynamics. Among them, fluorescence correlation spectroscopy (FCS) and single-particle tracking (SPT) have proven to be instrumental to our understanding of cell dynamics and function. The analysis of SPT and FCS is an ongoing effort, and despite decades of work, much progress remains to be done. In this paper, we give a quick overview of the existing techniques used to analyze anomalous diffusion in cells and propose a collaborative challenge to foster the development of state-of-the-art analysis algorithms. We propose to provide labelled (training) and unlabelled (evaluation) simulated data to competitors all over the world in an open and fair challenge. The goal is to offer unified data benchmarks based on biologically-relevant metrics in order to compare the diffusion analysis software available for the community., Comment: 10 pages, 1 figure

We devise a method to detect and estimate forces in a heterogeneous environment based on experimentally recorded stochastic trajectories. In particular, we focus on systems modeled by the heterogeneous overdamped Langevin equation. Here, the observed drift includes a "spurious" force term when the diffusivity varies in space. We show how Bayesian inference can be leveraged to reliably infer forces by taking into account such spurious forces of unknown amplitude as well as experimental sources of error. The method is based on marginalizing the force posterior over all possible spurious force contributions. The approach is combined with a Bayes factor statistical test for the presence of forces. The performance of our method is investigated analytically, numerically and tested on experimental data sets. The main results are obtained in a closed form allowing for direct exploration of their properties and fast computation. The method is incorporated into TRamWAy, an open-source software platform for automated analysis of biomolecule trajectories., Comment: Keywords: overdamped Langevin equation, Bayesian inference, inverse problems, biomolecule dynamics, It\^o-Stratonovich dilemma, random walks

Electropermeabilization is a method by which molecules can be introduced in living cells through the plasma membrane. Here, we show that electropermeabilization affects the lateral mobility of Rae-1, a GPi anchored protein. Our results shows that 10 to 20 % of the membrane surface is occupied by defaults or pores. Gene electrotransfert also affects the lateral mobility of Rae-1. Furthermore, we clearly show that once inserted into the plasma membrane, DNA totaly excludes Rae-1, indicating that DNA molecules are tightly packed together to forms huge aggregates.

Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2 ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is proposed to explain the appearance of distinct interaction sites, the quantitative increase in DNA and also their immobility leading to a tentative explanation for the success of electro-mediated gene delivery., Comment: Accepted for publication in BBA - Biomembranes

We present experimental results regarding the electrotransfer of plasmid DNA into phosphatidylcholine giant unilamellar vesicles (GUVs). Our observations indicate that a direct entry is the predominant mechanism of electrotransfer. A quantitative analysis of the DNA concentration increments inside the GUVs is also performed, and we find that our experimental data are very well described by a simple theoretical model in which DNA entry is mostly driven by electrophoresis. Our theoretical framework allows for the prediction of the amount of transfered DNA as a function of the electric field parameters, and thus paves the way towards a novel method for encapsulating with high efficiency not only DNA, but any negatively charged macromolecule into GUVs., Comment: Accepted for publication in Soft Matter