Biology subjects

Schueder, F.

Publications and source records attributed to Schueder, F..

13 recordsLinked to original sources

bioRxiv2026

Microscope control with a natural language agent

Microscopy underpins modern biology, but acquiring high-quality data from complex experiments at scale remains challenging. Smart microscopy can standardize and automate data collection, but each experiment-specific solution requires coding expertise and development time. To address this, we present MicroClaw, an AI agent that advises on experiment- and system-specific parameters and approaches and collaboratively plans and executes diverse, complex, and reusable imaging workflows across microscope platforms, without requiring expert knowledge.

biophysics↗

bioRxiv2026

Fluorogenic speed-optimized DNA-PAINT probes enable super-resolution imaging of whole cells

Super-resolution microscopy with DNA-PAINT enables molecular-scale, multiplexed, and quantitative imaging, but its throughput is limited by slow binding kinetics and elevated background at high probe concentrations. Recent speed-optimized and fluorogenic probes improve performance but impose strong constraints on sequence design, revealing a fundamental tradeoff between fast binding and efficient quenching. Here, we introduce a modular probe architecture that spatially decouples binding kinetics from fluorophore-quencher interactions by integrating speed-optimized sequence motifs with PEG spacers. Using DNA origami nanostructures, we demonstrate enhanced localization rates, signal-to-background ratios, and imaging efficiency compared to state-of-the-art probes. We validate our approach in cells, demonstrating its capability to image nuclear targets and enabling three-dimensional imaging of the endoplasmic reticulum using standard widefield illumination. Our work establishes a general framework for fast, multiplexed, and low-background super-resolution imaging.

bioengineering↗

bioRxiv2025

A correlative quantitative phase contrast and fluorescence super-resolution microscope for imaging molecules in their cellular context

Fluorescence microscopy has been widely used to reveal the spatial distribution of specifically labeled molecules, but it is blind to cellular context. Quantitative phase contrast microscopy (QPC) provides such complementary information. Here we have developed a platform that combines the QPC technique of correlative orientation-independent differential interference contrast (OI-DIC) microscopy with single-molecule super-resolution fluorescence microscopy. We demonstrate a detection sensitivity of 0.05 nm optical path difference, sufficient to detect single microtubules, and show its capability of 3D super-resolution fluorescence imaging in the cellular context. Additionally, we report deep-learning enabled digital staining, identifying nuclei, mitochondria and lipid droplets from OI-DIC data and demonstrate the potential of this approach for long-term live-cell imaging of organelles of interest without the need for fluorescence. OI-DIC can be easily integrated into most fluorescence microscopes and is readily adoptable by microscopy labs. ONE-SENTENCE TEASERA highly sensitive technique to visualize sub-cellular structures, dynamics, and molecules in their cellular context.

cell biology↗

bioRxiv2025

ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis

Autophagosomes form from seed membranes that expand through bulk-lipid transport via the bridge-like lipid transporter ATG2. The origins of the seed membranes and their relationship to the lipid transport machinery are poorly understood. Using proximity labeling and a variety of fluorescence microscopy techniques, we show that ATG2A localizes to extra-Golgi ARFGAP1 puncta during autophagosome biogenesis. ARFGAP1 itself is dispensable during macroautophagy, but among other proteins associating to these membranes, we find that RAB1 is essential. ATG2A co-immunoprecipitates strongly, albeit indirectly, with RAB1A, and siRNA-mediated depletion of RAB1A/B blocks autophagy downstream of LC3B lipidation, similar to ATG2A depletion. Further, when either autophagosome formation or the early secretory pathway is perturbed, ARFGAP1 and RAB1A accumulate at ectopic locations with autophagic machinery. Our results indicate that ATG2A engages a RAB1A complex on select early secretory membranes in support of autophagosome biogenesis. Significance StatementThis study expounds upon the role of early secretory membranes in autophagosome biogenesis. The authors demonstrate that RAB1/ARFGAP1-positive membranes are essential to autophagy and are recruited to the phagophore assembly site at an early step of autophagosome biogenesis. These membranes interact with the bridge-like lipid transport protein ATG2A and are positive for LC3B and WIPI2, suggesting that RAB1 membranes are a direct source for autophagosome formation.

cell biology↗

bioRxiv2023

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

cell biology↗

bioRxiv2023

Unraveling cellular complexity with unlimited multiplexed super-resolution imaging

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here we present a novel imaging method for rapid multiplexed super-resolution microscopy of a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with Transient Adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the cell biological versatility of FLASH-PAINT in mammalian cells in four applications: i) mapping nine proteins in a single mammalian cell, ii) elucidating the functional organization of primary cilia by nine-target imaging, iii) revealing the changes in proximity of twelve different targets in unperturbed and dissociated Golgi stacks and iv) investigating inter-organelle contacts at 3D super-resolution.

cell biology↗

bioRxiv2022

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles

Sarcomeres are the force producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super- resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A- band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

developmental biology↗

bioRxiv2021

Visualization of loop extrusion by DNA nanoscale tracing in single cells

The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of chromatin compartments and topologically associating domains, respectively. However, whether the actual 3D-fold of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a workflow for nanoscale 3D imaging of the genome sequence in structurally well-preserved nuclei in single human cells. Tracing the in situ structure of DNA in thousands of individual cells reveals that genomic DNA folds as a flexible random coil in the absence of loop extruding enzymes such as Cohesin. In the presence of Cohesin and its boundary factor CTCF, reproducibly positioned loop structures dominate the folds, while Cohesin alone leads to randomly positioned loops. The 3D structure and size variability of DNA loops we observe in a large number of single cells allow us to formulate a data-constrained computational model of genomic DNA folding that explains how sparse and dynamic loops in single cells lead to the emergence of compact topological domains in averages of cell populations.

cell biology↗

bioRxiv2021

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

Rotaviruses transcribe eleven distinct protein-coding RNAs that must be stoichiometrically co-packaged prior to their replication to make an infectious virion. During infection, rotavirus transcripts accumulate in cytoplasmic ribonucleoprotein (RNP) condensates, termed viroplasms. Understanding the mechanisms of viroplasm assembly and RNA enrichment within is crucial to gaining greater insight into their function and stoichiometric assortment of individual transcripts. We analysed the subcellular distribution of individual RV transcripts and viroplasm transcriptome by combining multiplexed DNA-barcoded single-molecule RNA FISH of infected cells. Using DNA-PAINT microscopy, we provide evidence of the early onset of viral transcript oligomerisation that occurs prior to the formation of viroplasms. We demonstrate that viral sequences lacking the conserved terminal regions fail to undergo enrichment in rotavirus RNP condensates. We show that individual viral transcripts exhibit variable propensities to partition into viroplasms, irrespective of their absolute numbers in cells, suggesting a selective RNA enrichment mechanism distinct from other known cellular RNP granules. We suggest that rotavirus replication factories represent unique RNP condensates enriched in eleven types of cognate transcripts that may facilitate the assembly of a multi-segmented RNA genome.

biochemistry↗

bioRxiv2019

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

molecular biology↗

bioRxiv2019

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

The Nuclear Pore Complex (NPC) is one of the largest and most complex protein assemblies in the cell and - among other functions - serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

cell biology↗

bioRxiv2019

Localization microscopy at doubled precision with patterned illumination

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field-of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.

molecular biology↗

bioRxiv2019

Bayesian Multiple Emitter Fitting using Reversible Jump Markov Chain Monte Carlo

In single molecule localization-based super-resolution imaging, high labeling density or the desire for greater data collection speed can lead to clusters of overlapping emitter images in the raw super-resolution image data. We describe a Bayesian inference approach to multiple-emitter fitting that uses Reversible Jump Markov Chain Monte Carlo to identify and localize the emitters in dense regions of data. This formalism can take advantage of any prior information, such as emitter intensity and density. The output is both a posterior probability distribution of emitter locations that includes uncertainty in the number of emitters and the background structure, and a set of coordinates and uncertainties from the most probable model.

biophysics↗