Biology subjects

Ronceray, N.

Publications and source records attributed to Ronceray, N..

5 recordsLinked to original sources

bioRxiv2025

Single-Photon Single-Particle Tracking

Mobile biological particles, ranging from biomolecules to viral capsids, often diffuse faster than 1 {micro}m2/s, resulting in severe motion blur in conventional millisecond-scale imaging. While shorter exposures may help provide the data needed to capture faster dynamics, quantization of signal intensity per pixel at such exposures eventually interferes with our ability to track. In the extreme case of binary (1-bit-per-pixel) output-- where going from 8-bit conventional grayscale imaging to 1-bit directly corresponds to a 255-fold faster acquisition rate--no existing tracking methods can be used, as these methods fundamentally rely on intensity-based localization, which does not leverage the binary output. For this reason, we introduce single-photon single-particle tracking (SP2T), a framework that bypasses localization and linking by estimating particle numbers and trajectories directly by jointly considering 1-bit image stacks. While cameras capable of microsecond-scale exposures, typically based on scientific CMOS (sCMOS) sensors or single-photon detectors (SPDs), are increasingly central to this effort, in this work, we focus on single-photon avalanche diode (SPAD). Single-photon detector (SPD) arrays offer microsecond exposures over large fields of view (512x512 pixels). SP2T accounts for detector-specific artifacts such as hot and cold pixels and is validated with programmed fluorescent bead trajectories and biological systems (aerolysin and ganglioside). These experiments, in addition to simulations, reveal that analysis performed with longer exposures can bias diffusion coefficient estimates (up to 70% for particles with diffusion coefficients of 5 {micro}m2/s) and distort jump-distance distributions, underscoring the need for photon-by-photon tracking in fast-diffusion regimes. Moreover, SP2T delivers substantial computational gains--achieving more than a 50-fold GPU speedup over CPU-based likelihood tracking methods that assume continuous intensity, when compared on datasets with the same frame size and number of frames. Together, these advances establish SP2T as a robust, data-efficient solution for unbiased particle tracking with millisecond-to-microsecond temporal resolution.

biophysics↗

bioRxiv2024

Lumen charge governs ion transport in β-barrel nanopores

{beta}-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains in its early stages. In this study, we integrate experimental, numerical, and theoretical approaches to elucidate ion transport mechanisms in these biological nanopores. We identify and characterise two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. Additionally, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for the characterisation and understanding of biological ion transport processes, enabling the design of adaptable biosensors. We illustrate this by optimizing an aerolysin mutant for computing applications, paving the way for novel nanofluidic technologies.

biophysics↗

bioRxiv2024

Multiplexed lifetime imaging of single molecules with a gated single-photon camera

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512x512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Forster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

biophysics↗

bioRxiv2023

Label-free imaging of DNA interactions with 2D materials

Two-dimensional (2D) materials offer potential as substrates for biosensing devices, as their properties can be engineered to tune interactions between the surface and biomolecules. Yet, not many methods can measure these interactions in a liquid environment without introducing labeling agents such as fluorophores. In this work, we harness interferometric scattering (iSCAT) microscopy, a label-free imaging technique, to investigate the interactions of single molecules of long dsDNA with 2D materials. The millisecond temporal resolution of iSCAT allows us to capture the transient interactions and to observe the dynamics of unlabeled DNA binding to a hexagonal boron nitride (hBN) surface in solution for extended periods (including a fraction of 10%, of trajectories lasting longer than 110 ms). Using a Focused Ion Beam (FIB) technique to engineer defects, we find that DNA binding affinity is enhanced at defects; when exposed to long lanes, DNA binds preferentially at lane edges. Overall, we demonstrate that iSCAT imaging is a useful tool to study how biomolecules interact with 2D materials, a key component in engineering future biosensors.

biophysics↗

bioRxiv2023

OpenSIM: open source microscope add-on for structured illumination microscopy

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

bioengineering↗