Cryogenic light microscopy of vitrified samples with angstrom precision
Hisham Mazal,
Franz Wieser,
Daniel Bollschweiler,
Vahid Sandoghdar
Proceedings of the National Academy of Sciences of the United States of America
122
e2513583122
(2025)
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High-resolution studies in structural biology are often limited by the challenges of crystallization and low contrast in the cellular native environment. The exquisite labeling specificity of fluorescence microscopy gets around these issues and allows superresolution microscopy, but to date, these works have used chemically 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 introduce single-particle cryogenic light microscopy (spCryo-LM) as a technique that satisfies these key criteria. We adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing samples and use a high-vacuum cryogenic shuttle system to transfer them in and out of a liquid-helium cryostat that houses a superresolution fluorescence microscope. By exploiting the enhanced photophysics at low temperature, angstrom precision can be achieved in localizing several fluorophores attached to proteins separated by a few hundred nanometers. We present various characterization studies on vitreous ice, single-molecule photoblinking behavior, and the effects of laser intensity and benchmark our method by resolving the heptameric membrane protein alpha-hemolysin in a synthetic lipid membrane. Additionally, we report on the technique’s capability to resolve membrane proteins in their native cellular membrane environment. spCryo-LM enables structural studies of proteins in their native environment without chemical fixation or protein isolation, and can be integrated with other superresolution or spectroscopic techniques. We believe our approach establishes light microscopy as a powerful tool in structural biology and sets the stage for correlative microscopy with Cryo-EM and related techniques.
Red Blood Cell-derived Extracellular Vesicles as biomaterials: the opportunity of freezing-induced accelerated aging
Lucia Paolini,
Miriam Romano,
Valentina Mangolini,
Selene Tassoni,
Shuhan Jiang,
Elena Laura Mazzoldi,
Angelo Musicò,
Andrea Zendrini,
Anna Kashkanova,
Vahid Sandoghdar,
Anna Concetta Berardi,
Silvia Clara Giliani,
Paolo Bergese,
Annalisa Radeghieri
Biomaterials Science
14
122-139
(2026)
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Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, owing to their intrinsic biocompatibility, immune evasion properties, and minimal oncogenic risk. However, their broader application is currently limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage stability. By combining discontinuous sucrose density gradient separation with high-resolution interferometric nanoparticle tracking analysis, we identified a sharp bimodal size distribution of the vesicles in freshly prepared samples. We then tracked how long-term storage at −80 °C drove its conversion into a monomodal distribution. To reproduce these conditions in a shorter time frame, we developed an “accelerated-ageing” protocol based on freeze–thaw cycles that generates RBC-EV samples with homogeneous density, size distribution, and biological activity, effectively replicating the properties of preparations stored for six months at −80 °C. This new vesicle population results stable and retains membrane integrity and cellular internalization capacity, as confirmed by surface-associated enzymatic activity assays and uptake tests in cancer cell lines. These results suggest that freezing-induced “accelerated ageing” represents an effective method for the optimization and standardization of RBC-EVs as building blocks for biomaterial and bioengineering applications.
Nano-electronvolt Fourier-limited transition of a single surface-adsorbed molecule
Masoud Mirzaei,
Alexey Shkarin,
Burak Gurlek,
Johannes Zirkelbach,
Ashley J. Shin,
Irena Deperasińska,
Boleslaw Kozankiewicz,
Tobias Utikal,
Stephan Götzinger,
Vahid Sandoghdar
High-resolution spectroscopy allows one to probe weak interactions and to detect subtle phenomena. While such measurements are routinely performed on atoms and molecules in the gas phase, spectroscopy of adsorbed species on surfaces is faced with challenges. As a result, previous studies of surface-adsorbed molecules have fallen short of the ultimate resolution, where the transition linewidth is determined by the lifetime of the excited state. In this work, we conceive a new approach to surface deposition and report on Fourier-limited electronic transitions in single dibenzoterrylenes adsorbed onto the surface of an anthracene crystal. By performing spectroscopy and super-resolution microscopy at liquid helium temperature, we shed light on various properties of the adsorbed molecules. Our experimental results pave the way for a new class of experiments in surface science, where high spatial and spectral resolution can be combined.
Direct, high-throughput linking of single cell imaging and gene expression
Catherine Xu,
Georg Meisl,
Nikita Moshkov,
Karolis Goda,
Alexey Shkarin,
Maximilian Schlögel,
Tuomas PJ Knowles,
Linas Mazutis,
Jochen Guck
bioRxiv 10.1101/2025.09.01.672798
(2025)
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Single-cell technologies have transformed our ability to dissect cellular heterogeneity by enabling measurements of individual molecular modalities, from genome and transcriptome to proteome and metabolome. However, at the single-cell level, the physical properties of cells, such as size, morphology, and mechanical state, remain largely disconnected from molecular profiling, limiting our understanding of the relationship between these aspects of cellular phenotype and gene expression. We introduce im-seq, a high-throughput, flow-based platform that integrates live-cell imaging with droplet-based mRNA sequencing at the single-cell level. By optically barcoding individual cells, im-seq enables the joint capture of physical and transcriptional profiles from single cells. We demonstrate that this multimodal approach can resolve physical and molecular features across cell lines, to reveal genes associated with phenotypic properties at unprecedented resolution. Our results establish im-seq as a versatile high-throughput framework for linking genetic information to physical properties, providing the large scale, information-dense datasets needed to power the next generation of data-driven discoveries in cell biology.
Bottom-up investigation of spatiotemporal glycocalyx dynamics with interferometric scattering microscopy
Carla M. Brunner,
Lorenz Pietsch,
Ingo vom Sondern,
Michael Röhrl,
Cristian Popov,
Marius F.W. Trollmann,
Richard W. Taylor,
Martin Blessing,
Cornelia Holler,
Karim Almahayni,
Sven Ole Jaeschke,
Vahid Sandoghdar,
Rainer A. Böckmann,
Thisbe K. Lindhorst, et al.
Journal of the American Chemical Society
147
32799-32808
(2025)
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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.
Cryo–light microscopy with angstrom precision deciphers structural conformations of PIEZO1 in its native state
Hisham Mazal,
Franz Wieser,
Daniel Bollschweiler,
Alexandra Schambony,
Vahid Sandoghdar
Science Advances
11
eadw4402
(2025)
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Investigations based on cryo–electron microscopy (cryo-EM), atomic force microscopy, and super-resolution microscopy reveal a symmetric trimer with propeller-like blades for the mechanosensitive ion channel PIEZO. However, a conclusive understanding of its conformations in the cell membrane is lacking. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen cell membranes in and out of a cryostat designed for single-particle cryo–light microscopy (spCryo-LM). By localizing fluorescent labels placed at the extremities of the blades of the mouse PIEZO1 protein in unroofed cell membranes, we ascertain three configurations with radii of 6, 12, and 20 nanometers as projected onto the membrane plane. We elaborate on the correspondence of these data with previous reports in the literature. The combination of spCryo-LM with cryo-EM promises to provide quantitative insights into the structure and function of biomolecular complexes in their native environments without the need for chemical fixation or protein isolation, ushering in a new regime of correlative studies in structural biology.
Imaging single ion channels via their Rayleigh scattering
The fast and convenient study of ion channels in cells continues to pose challenges. Interferometric scattering microscopy delivers robust signals from single channels, paving the way for label-free investigation of their function in live cells.
Hybridization of molecules via a common photonic mode
Jahangir Nobakht,
André Pscherer,
Jan Renger,
Stephan Götzinger,
Vahid Sandoghdar
Proceedings of the National Academy of Sciences of the United States of America
122
e2505161122
(2025)
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Atoms and molecules usually hybridize and form bonds when they come in very close<br>proximity of each other. In this work, we show that molecules can hybridize even<br>through far-field electromagnetic interactions mediated by the shared mode of an<br>optical microcavity. We discuss a collective enhancement of the vacuum Rabi splitting<br>and study super- and subradiant states that arise from the cavity-mediated coupling<br>both in the resonant and dispersive regimes. Moreover, we demonstrate a two-photon<br>transition that emerges between the ground and excited states of the new optical<br>compound. Our experimental data are in excellent agreement with the predictions<br>of the Tavis–Cummings Hamiltonian and open the door to the realization of hybrid<br>light–matter materials.
Bioengineered Bacterial Vesicles and Biomimetic Hybrids Eliminate Biofilms and Balance the Gut Microbiome
Leila Pourtalebi Jahromi,
Benedikt Kronast,
Jennifer Munkert,
Lorenzo Sana,
Marcus Koch,
Heike Danzer,
Sirka Dormeyer,
Shuhan Jiang,
Fabian Herrmann,
Matthias Weiler,
Anna D. Kashkanova,
Vahid Sandoghdar,
Mario M. Zaiss,
Gregor Fuhrmann
Antibiotic-resistant pathogens are a global health challenge, necessitating innovative solutions beyond conventional antibiotics. This study introduces biomimetic nanocarriers - hybrids of bacteriomimetic liposomes and biocompatible Myxobacteria outer-membrane vesicles (OMVs) - as tunable platforms for targeted antibiotic delivery. Comparative analyses of their physicochemical properties and interactions with immune cells, intestinal epithelium, and biofilm-forming pathogens reveal distinct advantages. Hybrids excel at delivering antibiotics to intracellular targets, while Myxobacteria OMVs, particularly those of strain SBSr 073, evade immune clearance and prolong extracellular drug exposure. To support clinical translation, this study optimizes antibiotic encapsulation methods for SBSr 073 OMVs and evaluates the short- and long-term impact of Cystobacter ferrugineus 23 strain OMVs on the gut microbiome in mice. Summing up, this study highlights the promise of Myxobacteria OMVs and their biomimetic hybrids as versatile tools for treating Gram-negative biofilm-forming pathogens. These findings underscore the potential of bioengineered and biomimetic drug carriers for combating antimicrobial resistance and pave the way for their translation toward difficult-to-treat infections.
Interferometric scattering microscopy
Naomi S. Ginsberg,
Chia-Lung Hsieh,
Philipp Kukura,
Marek Piliarik,
Vahid Sandoghdar
Over the past two decades, interferometric scattering (iSCAT) microscopy has become a powerful label-free imaging method with a range of applications in fundamental science and technology. iSCAT detects the scattering of subwavelength entities through interference with a reference beam of light. Performed in a variety of illumination and detection schemes, iSCAT has exploited both amplitude and phase information to reach single-molecule detection sensitivity; to determine the size, mass and refractive index of nanoparticles; to achieve high spatiotemporal precision in 3D tracking of nanoparticles; to image subcellular nanostructures; and to quantify ultrafast diffusion and transport of energy in solids. In this Primer, we describe the basic principles of iSCAT detection and imaging from theoretical and practical points of view. We discuss various factors that affect the attainable signal-to-noise ratio, which in turn determines crucial performance features such as sensitivity and speed. We survey selected applications in which iSCAT has been instrumental in providing new insights. Finally, we discuss some of the current challenges and potential avenues for advancing the technique further.
Electrostatic All-Passive Force Clamping of Charged Nanoparticles
Yazgan Tuna,
Amer Al-Hiyasat,
Anna D. Kashkanova,
Andreas Dechant,
Eric Lutz,
Vahid Sandoghdar
In the past decades, many techniques have been explored for trapping microscopic and nanoscopic objects, but the investigation of nano-objects under arbitrary forces and conditions remains nontrivial. One fundamental case concerns the motion of a particle under a constant force, known as force clamping. Here, we employ metallic nanoribbons embedded in a glass substrate in a capacitor configuration to generate a constant electric field on a charged nanoparticle in a water-filled glass nanochannel. We estimate the force fields from Brownian trajectories over several micrometers and confirm the constant behavior of the forces both numerically and experimentally. Furthermore, we manipulate the diffusion and relaxation times of the nanoparticles by tuning the charge density on the electrode. Our highly compact and controllable setting allows for the trapping and force-clamping of charged nanoparticles in a solution, providing a platform for investigating nanoscopic diffusion phenomena.
Lipidic folding pathway of α-Synuclein via a toxic oligomer
Vrinda Sant,
Dirk Matthes,
Hisham Mazal,
Leif Antonschmidt,
Franz Wieser,
Kumar Tekwani Movellan,
Kai Xue,
Evgeny Nimerovsky,
Marianna Stampolaki,
Magdeline Nathan,
Dietmar Riedel,
Stefan Becker,
Vahid Sandoghdar,
Bert L. de Groot, et al.
Nature Communications
16
760
(2025)
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Aggregation intermediates play a pivotal role in the assembly of amyloid fibrils, which are central to the pathogenesis of neurodegenerative diseases. The structures of filamentous intermediates and mature fibrils are now efficiently determined by single-particle cryo-electron microscopy. By contrast, smaller pre-fibrillar α-Synuclein (αS) oligomers, crucial for initiating amyloidogenesis, remain largely uncharacterized. We report an atomic-resolution structural characterization of a toxic pre-fibrillar aggregation intermediate (I1) on pathway to the formation of lipidic fibrils, which incorporate lipid molecules on protofilament surfaces during fibril growth on membranes. Super-resolution microscopy reveals a tetrameric state, providing insights into the early oligomeric assembly. Time-resolved nuclear magnetic resonance (NMR) measurements uncover a structural reorganization essential for the transition of I1 to mature lipidic L2 fibrils. The reorganization involves the transformation of anti-parallel β-strands during the pre-fibrillar I1 state into a β-arc characteristic of amyloid fibrils. This structural reconfiguration occurs in a conserved structural kernel shared by a vast number of αS-fibril polymorphs, including extracted fibrils from Parkinson’s and Lewy Body Dementia patients. Consistent with reports of anti-parallel β-strands being a defining feature of toxic αS pre-fibrillar intermediates, I1 impacts the viability of neuroblasts and disrupts cell membranes, resulting in increased calcium influx. Our results integrate the occurrence of anti-parallel β-strands as salient features of toxic oligomers with their significant role in the amyloid fibril assembly pathway. These structural insights have implications for the development of therapies and biomarkers.
Contact
Nano-Optics Division Prof. Vahid Sandoghdar
Max Planck Institute for the Science of Light Staudtstr. 2 91058 Erlangen, Germany
