Biology subjects

Razvag, Y.

Publications and source records attributed to Razvag, Y..

3 recordsLinked to original sources

bioRxiv2024

Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/573251v3_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@11781bdorg.highwire.dtl.DTLVardef@46d845org.highwire.dtl.DTLVardef@120d399org.highwire.dtl.DTLVardef@240022_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

FRET-sensitized acceptor emission localization (FRETsael) - nanometer localization of biomolecular interactions using fluorescence lifetime imaging

Super-resolution light microscopy techniques facilitate the observation of nanometer-size biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity, however not necessarily in direct interaction. Using FRET-sensitized acceptor emission localization (FRETsael), we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analyses of the simulation results report the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of Actin-Vinculin and ER-ribosomes interactions, as well as on experimental samples of actin-myosin two-color confocal imaging. Conclusively, the FRETsael approach paves the way towards studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging. SignificanceFRET is used in fluorescence microscopy to report whether dye-labeled biomolecules of choice are close within distances of 10 nm or less, hence typical interaction distances. However, in many cases, using FRET imaging for the study of biomolecular interactions is difficult due to the high density of dye-labeled biomolecules and due to the existence of unbound dye-labeled biomolecules. In addition, the resolution of localizing molecules using light microscopy is diffraction limited. This work presents FRETsael, a new approach for localizing interacting biomolecules undergoing FRET, with improved resolution of 20-30 nm for confocal microscopy using search algorithms for local extrema in contribution to FRET using two-channel fluorescence intensity and lifetime data.

biophysics↗

bioRxiv2019

Abundant α-Synuclein compact dimers

Parkinsons disease is associated with the aggregation of the protein -synuclein. While -synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate that -synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers, we identify one that is compact, stable, abundant, and exhibits partially exposed {beta}-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in proximity that may promote dityrosine covalent linkage upon hydroxyl radicalization, which is implicated in -synuclein amyloid fibrils. We propose that this -synuclein dimer features etiological relevance to Parkinsons disease.

biophysics↗