Drori, P. et al.
Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy
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bioRxiv (Submitted).at <>
Publisher's VersionAbstractMainstream 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.
Harris, P.D., Ben Eliezer, N., Keren, N. & Lerner, E.
Multiparameter-based photosynthetic state transitions of single phytoplankton cells
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bioRxiv (Submitted).at <>
Publisher's VersionAbstractFluorescence emitted by light-harvesting pigments responds to the physiological state of phytoplankton. We developed a time resolved system to monitor fluorescence from single cells. It captures multiple spectral channels and fluorescence lifetimes, eliminating ensemble averaging of bulk experiments. Tracking the diurnal cycles of three phytoplankton species, we uncover each species segregation into multiple distinct cell states, a feature previously hidden in bulk measurements. We also tested their response to light-intensity perturbations, and determined each species unique acclimation strategy involving transition between distinct cell subpopulations. This approach will be useful for characterizing natural phytoplankton population responses to environmental conditions, aiming for a better understanding of their acclimation strategies and the effects of global climate changes.
Munoz, G.Gabriel Mo et al. Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).
bioRxiv (Submitted).at <>
Publisher's VersionAbstractOver the past decades, single-molecule spectroscopy and super-resolution microscopy have advanced significantly and by now represent important tools for life science research. Despite rapid progress and ongoing development, there is a growing gap between the state-of-the-art and what is accessible to non-optics specialists, e.g., biologists, biochemists, medical researchers, and labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and enables exchange between different modalities within minutes. In this work, we present three variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging. With the three variants, we were able to observe conformational changes and absolute inter-dye distances in single macromolecules and perform single-molecule localization microscopy (STORM and PAINT) of DNA origami nanostructures. Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.
Razvag, Y., Drori, P., Klemfner, S., Meshorer, E. & Lerner, E. FRET-sensitized acceptor emission localization (FRETsael) – nanometer localization of biomolecular interactions using fluorescence lifetime imaging.
bioRxiv (Submitted).at <>
Publisher's VersionAbstractSuper-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.
Joron, K., Zamel, J., Kalisman, N. & Lerner, E. Evidence for a compact σ70 conformation in vitro and in vivo. (Submitted).at <>
Publisher's VersionAbstractInitiation of transcription in Escherichia coli is facilitated by promoter specificity factors, such as σ70, which bind promoter dsDNA when in complex with RNA polymerase (RNAP), in which it is in an extended conformation with solvent-exposed DNA-interacting residues. If so, what in the structure of apo-σ70 prevents binding to promoter dsDNA at high affinity? By performing cross-linking mass spectrometry (CL-MS) and integrative structural modelling we elucidate structure models of apo-σ70 that exhibit burial of almost all DNA-binding residues. In vivo CL-MS detects crosslinks unique to the compact fold of apo-σ70 that occur at stationary growth phase. Conclusively, we provide structural information to show that the high affinity DNA-binding capabilities of apo-σ70 are conformationally-inhibited and can be activated mostly in the context of transcription.