Biology subjects

Sandoghdar, V.

Publications and source records attributed to Sandoghdar, V..

14 recordsLinked to original sources

bioRxiv2026

Characterization of Nanoparticles in Suspension by Simultaneous iNTA and Fluorescence Detection with Single-Molecule Sensitivity

Quantitative knowledge of nanoparticle properties is desirable in a large number of scientific and technological applications, but measurements with a high degree of precision usually prove to be challenging. Among a range of available methodologies, optical techniques with single particle sensitivity are especially interesting because they can reveal intrinsic hetero-geneities in a fast non-invasive manner. Recently, we presented interferometric nanoparticle tracking analysis (iNTA) as a highly sensitive label-free technique that is capable of determining the size, concentration and index of refraction of different subpopulations in a suspension mixture. Here, we enhance this method with biochemical specificity through multicolor fluorescence detection at the single-molecule sensitivity limit. We benchmark the performance of the combined technique, which we name iNTA-F, by distinguishing populations of fluorescent and non-fluorescent nanoparticles of different material, size, and fluorescence intensity, with an emphasis on the characterization of lipid vesicles and biological extracellular vesicles (EVs).

biophysics↗

bioRxiv2025

Red blood Cell-derived Extracellular Vesicles as biomaterials: the opportunity of freezing-induced accelerated aging

Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, due to their inherent biocompatibility, immune evasion capabilities, and minimal oncogenic risk. However, their clinical translation remains limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage. This study proposes a method that leverages freezing-induced ageing for obtaining highly homogeneous RBC-EV batches, an important step towards using RBC-EVs as healthcare biomaterials and advancing their clinical translation in EV-based nanomedicine. This method was made possible thanks to the analytical support of discontinuous sucrose density gradient and high-resolution interferometric nanoparticle tracking analysis, which allowed the identification of a bimodal subpopulation distribution, in terms of vesicle size, interferometric contrast, and subpopulation profiles, in freshly prepared samples, and then tracked how long-term cold storage at -80 {degrees}C channeled this heterogeneity into a monomodal population. Finally, we evaluated the functionality of homogenized RBC-EV samples by assessing surface-associated enzymatic activity and uptake in cancer cell lines, demonstrating that freeze-thaw-induced accelerated-aging provides a viable strategy for producing RBC-EV preparations that retain membrane integrity and remain readily internalized by cells. These findings offer valuable insights into the optimization and standardization of RBC-EV handling and storage protocols, providing a foundation for their reliable integration into EV-based therapeutic applications.

cell biology↗

bioRxiv2025

Cryogenic light microscopy of vitrified samples with Angstrom precision

High-resolution studies in structural biology are commonly based on diffraction methods and on electron microscopy. However, these approaches are limited by the difficulty in crystallization of biomolecules or by a low contrast that makes high-resolution measurements very challenging in crowded samples such as a cell membrane. The exquisite labeling specificity of fluorescence microscopy gets around these issues. Indeed, several recent reports have reached resolutions down to the [A]ngstrom level in super-resolution microscopy, but to date, these works used fixed samples. To establish light microscopy as a workhorse in structural biology, two main requirements must be fulfilled: near-native sample preservation and near-atomic optical resolution. Here, we demonstrate a technique that satisfies these key criteria with particular promise for conformational studies on membrane proteins and their complexes. To prepare cell membranes in their near-native state, we adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing and transfer of samples. We developed a high-vacuum cryogenic shuttle system that allows us to transfer vitrified samples in and out of a liquid-helium cryostat that houses a super-resolution fluorescence microscope. Sample temperatures below 10 K help dissipate the heat from laser illumination, thus maintaining intact vitreous ice. We utilize the photoblinking of organic dye molecules attached to well-defined positions of a protein to localize one label fluorophore at a time. We present various characterization studies of the vitreous ice, photoblinking behavior, and the effects of the laser intensity. Moreover, we benchmark our method by demonstrating [A]ngstrom precision in resolving the full assembled configuration of the heptameric membrane protein alpha-hemolysin (HL) in a synthetic lipid membrane as a model system. Additionally, we report on the techniques capability to resolve membrane proteins in their native cellular membrane environment. Our method, which we term single-particle cryogenic light microscopy (spCryo-LM), enables structural studies of membrane protein tertiary and quaternary conformations without the need for chemical fixation or protein isolation. The approach can also integrate other super-resolution or spectroscopic techniques with particular promise in correlative microscopy with images from Cryo-EM and related techniques.

biophysics↗

bioRxiv2025

Bottom-up investigation of spatiotemporal glycocalyx dynamics with interferometric scattering microscopy

Over recent decades, the glycocalyx, an extracellular organelle comprised of a multitude of glycolipids, glycoproteins, proteoglycans and glycoRNA, has gained considerable interest in cellular biology. While research in this field has revealed its tremendous importance in evermore aspects of physiological and pathological cellular processes, many of the principles that govern the role of the glycocalyx in these processes on a molecular level are still unknown. In order to unravel the fundamental laws underlying glycocalyx function, new technologies are required that enable the distinction between individual subprocesses within the intricate environment of the glycocalyx. Here, we establish an experimental platform to investigate the dynamics of the glycocalyx at the nanometer and microsecond length and time scales in a bottom-up fashion. We synthesized defined model glycans and installed them on supported lipid bilayers, assembling glycocalyx model systems with tunable properties. By investigating these tunable model systems with interferometric scattering (iSCAT) microscopy, we gain access to the required spatiotemporal resolution. We found a strong correlation between the molecular structure of several investigated model glycans and global dynamics of the system. Our findings are corroborated by atomistic and coarsegrained molecular dynamics simulations. Our results provide the first direct experimental evidence on the relationship between glycan structure, organization, and dynamics, offering a robust and versatile basis for a quantitative understanding of glycocalyx biology and physics at the molecular level.

biophysics↗

bioRxiv2024

Cryogenic light microscopy with Angstrom precision deciphers structural conformations of PIEZO1

Despite the impressive progress in molecular biochemistry and biophysics, many questions regarding the conformational states of large (transmembrane) protein complexes persist. In the case of the PIEZO protein, investigations by cryogenic electron microscopy (Cryo-EM) and atomic force microscopy (AFM) have established a symmetric trimer structure with three long-bladed domains in a propeller-like configuration. A transition of PIEZO protein from curved to flat conformation is hypothesized to actuate closed and open channels for the flow of ions. However, conclusive high-resolution data on the molecular organization of PIEZO in its native form are lacking. To address this shortcoming, we exploit single-particle cryogenic light microscopy (spCryo-LM) to decipher the conformational states of the mouse PIEZO1 protein (mPIEZO1) in the cell membrane. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen unroofed cell membranes in and out of a cryostat for super-resolution microscopy at liquid helium temperature. By localizing fluorescent labels placed at the extremities of the three blades with [A]ngstrom precision, we ascertain three configurations of the protein with radii of 6, 12, and 20 nm as projected onto the membrane plane. Our data suggest that in the smallest configuration, the blades form a nano-dome structure that is more strongly curved than previously observed and predicted by AlphaFold-3. In the largest conformation, we believe the structure must fully unbend in an anticlockwise manner to form a flat extended state. We attribute the 12 nm conformation, the most frequently occupied state, to an intermediate state and discuss our results in the context of the findings from other groups. Combination of spCryo-LM and Cryo-EM measurements together with in situ photothermal stimulation promises to provide quantitative insight into the interplay between structure and function of PIEZO and other biomolecular complexes in their native environments.

biophysics↗

bioRxiv2024

Measuring concentration of nanoparticles in polydisperse mixtures using interferometric nanoparticle tracking analysis (iNTA)

Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) as a new method for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.

biophysics↗

bioRxiv2023

Mechanism of sensor kinase CitA transmembrane signaling

Membrane bound histidine kinases (HKs) are ubiquitous sensors of extracellular stimuli in bacteria. Here, we used solid-state NMR in conjunction with crystallography, solution NMR and distance measurements to investigate the transmembrane signaling mechanism of a paradigmatic citrate sensing membrane embedded HK, CitA. Citrate binding in the sensory extracytoplasmic PAS domain (PASp) causes the linker to transmembrane helix 2 (TM2) to adopt a helical conformation. This triggers a piston-like pulling of TM2 and a quaternary structure rearrangement in the cytosolic PAS domain (PASc). Crystal structures of PASc reveal both anti-parallel and parallel dimer conformations. An anti-parallel to parallel transition upon citrate binding agrees with interdimer distances measured in the lipid embedded protein using a site-specific 19F label in PASc. These data show how Angstrom scale structural changes in the sensor domain are transmitted across the membrane to be converted and amplified into a nm scale shift in the linker to the phosphorylation subdomain of the kinase. One-Sentence SummaryTransmembrane signal transduction of a PAS-domain containing histidine kinase occurs via a piston-like pulling of a transmembrane helix, and amplification by cytoplasmic PAS domain dimer rearrangement.

molecular biology↗

bioRxiv2022

Interferometric nanoparticle tracking analysis enables label-free discrimination of extracellular vesicles from large lipoproteins

Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometer size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish between EVs and lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension.

biophysics↗

bioRxiv2021

Deciphering a hexameric protein complex with Angstrom optical resolution

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases, where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and the hexamer geometry of Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic, environmental and dynamic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology. Significance statementFluorescence super-resolution microscopy has witnessed many clever innovations in the last two decades. Here, we advance the frontiers of this field of research by combining partial labeling and 2D image classification schemes with polarization-encoded single-molecule localization at liquid helium temperature to reach Angstrom resolution in three dimensions. We demonstrate the performance of the method by applying it to trimer and hexamer protein complexes. Our approach holds great promise for examining membrane protein structural assemblies and conformations in challenging native environments. The methodology closes the gap between electron and optical microscopy and offers an ideal ground for correlating the two modalities at the single-particle level. Indeed, correlative light and electron microscopy is an emerging technique that will provide new insight into cell biology.

biophysics↗

bioRxiv2021

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions.

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, e.g., for the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, the existing methods are limited in deciphering the constituents of realistic samples. Here, we introduce iNTA as a new method, which combines interferometric detection of scattering with nanoparticle tracking analysis, to reach an unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multi-component and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and nanoparticles in human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

biophysics↗

bioRxiv2021

Optimized analysis for sensitive detection and analysis of single proteins via interferometric scattering microscopy

It has been shown that interferometric detection of Rayleigh scattering (iSCAT) can reach an exquisite sensitivity for label-free detection of nano-matter, down to single proteins. The sensitivity of iSCAT detection is intrinsically limited by shot noise, which can be indefinitely improved by employing higher illumination power or longer integration times. In practice, however, a large speckle-like background and technical issues in the experimental setup limit the attainable signal-to-noise ratio. Strategies and algorithms in data analysis are, thus, crucial for extracting quantitative results from weak signals, e.g. regarding the mass (size) of the detected nano-objects or their positions. In this article, we elaborate on some algorithms for processing iSCAT data and identify some key technical as well as conceptual issues that have to be considered when recording and interpreting the data. The discussed methods and analyses are made available in the extensive python-based platform, PiSCAT[§].

biophysics↗

bioRxiv2020

High-precision protein-tracking with interferometric scattering microscopy

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organisation of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behaviour. Interferometric scattering (iSCAT) microscopy is an emerging technique well suited for visualising the diffusion of gold nanoparticle-labelled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualising the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.

biophysics↗

bioRxiv2020

Differential diffusional properties in loose and tight docking prior to membrane fusion

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study due to their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.

biophysics↗

bioRxiv2018

Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane

Much of the biological functions of a cell are dictated by the intricate motion of proteins within its membrane over a spatial range of nanometers to tens of micrometers and time intervals of microseconds to minutes. While this rich parameter space is not accessible to fluorescence microscopy, it can be within reach of interferometric scattering (iSCAT) particle tracking. Being sensitive even to single unlabeled proteins, however, iSCAT is easily accompanied by a large speckle-like background, which poses a substantial challenge for its application to cellular imaging. Here, we show that these difficulties can be overcome and demonstrate tracking of transmembrane epidermal growth factor receptors (EGFR) with nanometer precision in all three dimensions at up to microsecond speeds and tens of minutes duration. We provide unprecedented examples of nanoscale motion and confinement in ubiquitous processes such as diffusion in the plasma membrane, transport on filopodia, and endocytosis.

biophysics↗