Biology subjects

Eggeling, C.

Publications and source records attributed to Eggeling, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Overcoming Protein A-driven Nonspecific Antibody Staining of S. aureus in Immunofluorescence Microscopy

The ability to detect host cell factors during Staphylococcus aureus infection in vitro by immunofluorescence microscopy is severely hampered by staphylococcal protein A (SpA), a cell wall-anchored protein that binds the fragment crystallizable (Fc) region of immunoglobulins. This interaction generates strong nonspecific fluorescent signals on the bacterial surface, complicating data interpretation and limiting the accuracy of quantitative image analysis. Several measures have been put forward to overcome this obstacle, most importantly the pre-incubation with an anti-SpA antibody (SpA) and the use of human serum (HS) as blocking agent and antibody diluent. To highlight this feature to general fluorescence microscopy users, we here systematically evaluated these two strategies. Using S. aureus coated on coverslips and S. aureus-infected A549 cells, we highlight the efficiencies of both approaches to markedly reduce nonspecific fluorescence, with HS treatment yielding the most profound suppression. Notably, HS, containing high levels of human immunoglobulins, offered a robust, cost-effective and broadly applicable solution for minimizing SpA-driven artifacts, thereby improving immunofluorescence microscopy in S. aureus infection models in vitro.

microbiology↗

bioRxiv2026

K-Ras controls asymmetric cell divisions from the primary cilium

The Ras-MAPK pathway drives central cellular processes, including cell proliferation and differentiation. How exactly Ras controls differentiation is however not understood. Supported by mathematical modelling and single-cell RNA sequencing we show that K-Ras4B sustains ciliation during differentiation thus restricting commitment of skeletal muscle stem and progenitor cells during asymmetric cell divisions. Modulation of K-Ras4B abundance or expression of oncogenic K-Ras4B-G12C perturb normal differentiation. K-Ras4B, but not N-Ras and H-Ras, localizes to the primary cilium and its abundance there depends on the ciliary trafficking chaperone PDE6D. The presence of B-Raf and active MEK at the base of and active ERK inside the cilium suggests that K-Ras4B is active there. Conditions that localize a K-Ras4B mutant only to the cilium are sufficient to sustain ciliation and normal differentiation. Finally, in vivo modulation of K-Ras4B activity during zebrafish embryogenesis perturbs ciliation-dependent heart-looping. Our results thus imply a novel fundamental role of K-Ras4B in controlling ciliation and differentiation and suggest an explanation for the phenotypic similarities between RASopathies and ciliopathies.

cell biology↗

bioRxiv2025

NanTex enables computational multiplexing and phenotyping of organelles across super-resolution modalities

Super-resolution microscopy (SRM) enables nanoscale visualization of cellular organelles but remains constrained by spectral overlap, labeling requirements, and temporal offsets in multicolor imaging. We present NanTex, a deep learning framework that introduces nanotexture as a universal descriptor of subcellular organization and achieves probabilistic demixing of multiple organelles from single-channel SRM images. Unlike segmentation methods that enforce exclusivity, NanTex preserves overlapping morphologies, enabling faithful reconstruction even in crowded regions. Trained on small curated datasets with augmentation, NanTex generalized across SMLM, MINFLUX, STED, SIM, and live-cell Airyscan, with modality-specific retraining where necessary. Demonstrated on cytoskeletal, endomembrane, and metabolic organelles, NanTex achieved high-fidelity reconstructions and extended uniquely to live-cell imaging, where it eliminated temporal misalignment and enabled quantitative tracking of vesicle-like ER subdomains. Notably, NanTex enables quantitative dynamic readouts directly from single-channel live-cell data, revealing and tracking hidden subdomains in real time without the need for additional labels. Beyond multiplexing, NanTex supported computational phenotyping by distinguishing structurally distinct yet molecularly identical populations, exemplified by nocodazole-induced microtubule depolymerization and its glyoxal-mediated modulation. Together, these results establish nanotexture as a paradigm-shifting descriptor for organelle identity and position NanTex as a modality-agnostic, label-efficient, and live-cell-ready strategy for quantitative nanoscale biology. Significance StatementNanTex reframes multiplexing as texture-based demixing, introducing nanotexture as a universal fingerprint of organelle identity. By enabling quantitative, label-efficient reconstructions across SRM modalities and live-cell phenotyping, including vesicle tracking from single-channel data, NanTex opens a path to studying organelle remodeling and disease progression with unprecedented fidelity across SRM modalities.

biophysics↗

bioRxiv2025

Event-triggered MINFLUX microscopy: smart microscopy to catch and follow rare events

MINFLUX microscopy is a powerful microscopy method allowing for the characterization of molecular organization and dynamics with single nanometer spatial resolution and sub-hundred microseconds temporal resolution. However, acquisition times often span minutes to hours as a single fluorophore is measured at a time. Applying it to study cellular processes in living cells therefore requires careful consideration of where and when to apply MINFLUX data acquisition, a consideration where manual control limits its potential applications. Here, to overcome the limitations of acquisition speed, acquisition initiation, and data throughput, we present a smart microscopy method that uses confocal imaging as a monitoring method, runs real-time image analysis, and only applies MINFLUX data acquisition exactly where and when deemed necessary based on the analysis outcome. The method, event-triggered MINFLUX, is controlled through a custom-written and open source Python widget that automatically controls a commercial MINFLUX microscope. We apply this method to investigate molecular membrane dynamics and organization during three different cellular events: two-dimensional lipid dynamics at caveolae; three-dimensional membrane topography during dynamin-mediated endocytosis; and three-dimensional membrane fluidity and topography during budding site formation of HIV-1 proteins. Thanks to rapid event detection and minimal regions of interest the method provides data that would be unfeasible or impossible to acquire through manual control of the microscope.

biophysics↗

bioRxiv2025

CCB79 is a primate-specific cilium initiation factor essential to maintain neural progenitor diversity in developing brain tissue.

Identifying the genes that regulate the accurate spatiotemporal diversity of neural progenitor cells (NPCs) helps to understand the mechanisms of human neocortex expansion. In primate brains, an additional intermediate progenitor layer, the outer subventricular zone (oSVZ), facilitates the expansion of the neocortex. Here, we identify an uncharacterized gene, KIAA0408, and show that its expression is enriched in intermediate progenitors. Removing KIAA0408 in human-induced pluripotent stem cell (iPSC)-derived brain organoids results in an impaired cortical organization characterized by abnormal cell fate and patterning defects, including the depletion of intermediate and ventral progenitors. Molecularly, KIAA0408 codes for a 79-kilodalton centriolar distal appendage protein (DAP) that controls cilium biogenesis (hereafter CCB79). CCB79 forms a complex with other DAP components and specifically localizes in the DAP at the onset of ciliogenesis, and its absence blocks ciliogenesis. Mechanistically, progenitors in 3D brain tissues are unable to form cilia, which induces aberrant hedgehog signaling and causes premature differentiation. Finally, human CCB79, rather than the mouse ortholog, rescues cilia defects, suggesting that CCB79 has undergone rapid evolution from rodents to primates to fine-tune ciliogenesis for proper brain development.

neuroscience↗

bioRxiv2025

Parameter Optimization for Iterative MINFLUX Microscopy enabled Single Particle Tracking

MINFLUX fluorescence microscopy is a recently introduced super-resolution approach for studying cellular structures and their dynamics with highest detail. Iterative MINFLUX (iMFX) performs Single Particle Tracking (SPT) at runtime. The ad hoc signal interpretation necessary to sustain the method relies on several parameters, which need to be optimized in relation to the sample under study, such as fluorescent lipid analogues in membranes, to ensure the fidelity of the measurement. We propose a parameter optimization strategy, an overview of the most important parameters, present a theoretical upper limit for trackable diffusion rates, and demonstrate iMFX-enabled SPT of fast ([<]DMSD[>] = 2.5m2/s) lateral Brownian motion of lipids in membranes.

biophysics↗

bioRxiv2025

Spectral STED microscopy improves spectral sensitivity with polarity-sensitive probes and enables correlative measurements of membrane order and anomalous lipid diffusion

Molecular plasma membrane organization and dynamics play an important role in cellular signalling. Advances in our understanding of the nanoscale architecture of the plasma membrane heavily rely on the development of non-invasive experimental methods, particularly of advanced fluorescence microscopy and spectroscopy techniques with high spatio-temporal resolution and sensitivity to local molecular properties. However, it remains difficult to combine several of them for a multimodal characterisation that would reduce the possibility of misinterpretations. Here, we integrated a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, achieving three goals. First, we show that compared to the standard ratiometric detection using fixed bandpass filters, the spectrally resolved acquisition together with spectral fitting or phasor analysis improves the accuracy of experiments determining membrane lipid order with polarity-sensitive probes multifold. Secondly, we demonstrate that this acquisition scheme allows the use of such probes in combination with other dyes with overlapping spectra, enabling co-localisation of the membrane order maps with other cellular structures of interest, e.g. fluorescently labelled proteins. Finally, we correlate the obtained membrane lipid order with the anomalous trapped diffusion properties of a fluorescent sphingomyelin lipid analogue in the plasma membrane of living cells, as determined by STED fluorescence correlation spectroscopy, and highlight that some of the most apparent trapping sites locate at the boundaries of local ordered environments discernible by the introduced spectral STED microscopy. With additional measurements in model membranes and Monte-Carlo simulations we conclude that for sub-100 nm ordered environments uneven probe partitioning cannot by itself explain the trapping diffusion of SM in cells.

biophysics↗

bioRxiv2024

Homogeneous large field-of-view and compact iSCAT-TIRF setup for dynamic single molecule measurements

Interferometric Scattering Microscopy (iSCAT) enables prolonged and high frame rate Single Particle Tracking (SPT) for single molecule dynamics studies. Typically, iSCAT setups employ scanning illumination schemes to achieve uniform sample illumination. However, this implementation limits the field of view (FoV) and maximum sampling rate, while increasing hardware requirements and setup size. We demonstrate the realization of a large (60{micro}m x 60{micro}m) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. This scanning-free iSCAT microscopy setup is further combined with an objective based Total Internal Reflection Fluorescence Microscopy (TIRF) channel for a complementary fluorescence readout, a focus-lock system, and a tailored control platform via the open-source ImSwitch software, and has a compact footprint. As a proof-of-principle, we highlight the performance of the setup through the acquisition of iSCAT images with a uniform contrast and a [≤]10 nm localization precision throughout the whole FoV. The performance is further demonstrated through dynamic iSCAT SPT and imaging Fluorescence Correlation Spectroscopy of lipid diffusion in a model membrane system. Our iSCAT setup thus depicts an accurate and improved way of recording fast molecular dynamics in life sciences.

biophysics↗

bioRxiv2023

Neural Network Informed Photon Filtering Reduces Artifacts in Fluorescence Correlation Spectroscopy Data

Fluorescence Correlation Spectroscopy (FCS) techniques are well-established tools to investigate molecular dynamics in confocal and super-resolution microscopy. In practice, users often need to handle a variety of sample or hardware-related artifacts, an example being peak artifacts created by bright, slow-moving clusters. Approaches to address peak artifacts exist, but measurements suffering from severe artifacts are typically non-analyzable. Here, we trained a 1-dimensional U-Net to automatically identify peak artifacts in fluorescence time-series and then analyzed the purified, non-artifactual fluctuations by time-series editing. We show that in samples with peak artifacts, the transit time and particle number distributions can be restored in simulations and validated the approach in two independent biological experiments. We propose that it is adaptable for other FCS artifacts, such as detector dropout, membrane movement, or photobleaching. In conclusion, this simulation-based, automated, open-source pipeline makes measurements analyzable which previously had to be discarded and extends every FCS users experimental toolbox.

biophysics↗

bioRxiv2023

A novel PEX14/PEX5 interface links peroxisomal protein import and receptor recycling

Newly synthesized peroxisomal proteins are recognized in the cytosol by the cycling receptor PEX5 and directed to a docking complex comprising PEX14 and PEX13 at the peroxisomal membrane. After cargo translocation, the unloaded PEX5 is recycled in an ATP-dependent manner. Receptor docking involves the WxxxF-motifs in the N-terminal domain (NTD) of PEX5 that are recognized by the N-terminal domain of PEX14. Here, we combine biochemical methods and NMR spectroscopy to identify a novel binding interface between human PEX5 and PEX14. The interaction involves the PEX5 C-terminal cargo-binding TPR domain and a conserved IPSWQI peptide motif in the C-terminal intrinsically disordered region of PEX14. The three-dimensional structure of the PEX14 IPSWQI peptide bound the PEX5 TPR domain, shows the PEX14 interaction is non-overlapping with PTS1 binding to the TPR domain. Notably, PEX14 IPSWQI motif binding to a hinge region in the TPR domain shows a more open supercoil of the TPR fold that resembles the apo conformation in the absence of PTS1 peptide. Mutation of binding site residues in PEX5 or PEX14 leads to a partial protein import defect and decrease of the steady-state-concentration of PEX5. This resembles the mutant phenotype of cells affected in receptor recycling, suggesting a role in this process.

biochemistry↗

bioRxiv2023

Deep learning enables fast, gentle STED microscopy

STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that denoising STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

bioengineering↗

bioRxiv2021

Lipid tracking at kilohertz sampling rates on live cell membranes through Interferometric Scattering microscopy

The specific details of the lateral diffusion dynamics in cellular plasma membrane are an open topic in modern biophysics. Many studies have documented several different behaviours, including free (Brownian) motion, confined diffusion, transiently confined (hop) diffusion, anomalous diffusion, and combinations thereof. Here we have employed Interferometric Scattering Microscopy (ISCAT) to explore the lateral diffusion dynamics in the plasma membrane of living cells of a biotinylated lipid analogue that had been labelled with streptavidin-coated gold nanoparticles (20 and 40nm in diameter) at a sampling rate of 2kHz. The data was analysed with an unbiased statistics-driven mean squared displacement analysis pipeline that was designed to identify both the most likely diffusion mode for a specific data set, and the best fit parameters of the most likely model. We found that the prevalent diffusion mode of the tracked lipids, independent of the particle size, is compartmentalized diffusion, although the use of the larger tags resulted in tighter confinement and reduced diffusion rates. Through our analysis and comparison with simulated data, we quantify significant physical parameters, such as average compartment size, dynamic localization uncertainty, and the diffusion rates. We hereby further demonstrate the use of a confinement strength metric that makes it possible to compare diffusivity measurements across techniques and experimental conditions. Statement of SignificanceThis work offers new details on the data analysis of lipid diffusion on cellular membranes in vitro, through Interferometric Scattering microscopy. With this technique, we performed single particle tracking (SPT) experiments at 2kHz sampling rate. We analyzed the data through an unbiased statistics-driven protocol. The data shows that the diffusion motion of the tracked lipids follows mainly the "hopping" diffusion behaviour, whereby transient confinement zones hinder the particle dynamics. Matching the experimental data with diffusion simulations, we have been able to verify the physical parameters inferred by the experimental data analysis. Finally, we showcase a framework to compare SPT data with other techniques, to offer a complete overview of plasma membrane dynamics.

biophysics↗

bioRxiv2021

STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution, permitting for long acquisition times, and that allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

biophysics↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗

bioRxiv2021

Controlled Fluorescent Labelling of Metal Oxide Nanoparticles for Artefact-free Live Cell Microscopy

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles - the largest subpopulation of nanoparticles by industrial production and applications - and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials mechanism of action and will foster widespread and safe use of new nanomaterials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/440400v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@358ffborg.highwire.dtl.DTLVardef@7faaecorg.highwire.dtl.DTLVardef@1ae7fb3org.highwire.dtl.DTLVardef@141c4b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data

Single Particle Tracking (SPT) is one of the most widespread techniques to evaluate particle mobility in a variety of situations, such as in cellular and model membrane dynamics. The proposed TRAIT2D Python library is developed to provide object tracking, trajectory analysis and produce simulated datasets with graphical user interface. The tool allows advanced users to customise the analysis to their requirements. Availability and implementation: the software has been coded in Python, and can be accessed from: https://github.com/Eggeling-Lab-Microscope-Software/TRAIT2D, or the pypi and condaforge repositories. A comprehensive user guide is provided at https://eggeling-lab-microscope-software.github.io/TRAIT2D/.

bioinformatics↗

bioRxiv2020

Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins - in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.

biophysics↗

bioRxiv2020

Nanoscopy on the Chea(i)p

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

microbiology↗