Biology subjects

Bethge, P.

Publications and source records attributed to Bethge, P..

10 recordsLinked to original sources

bioRxiv2026

Water Kefir as a Model for Aggregative Development of Microbial Multicellularity within a Multispecies Biofilm

Water kefir is a traditional fermented beverage and a non-dairy alternative to milk kefir. Water kefir grains are cauliflower-like aggregates, ranging in size from 1-20 mm. They are composed of an exopolysaccharide matrix derived from the fermentation substrate sucrose. The microbial community on and within the grains is a complex, highly stable multicellular aggregate typically consisting of 10-15 core species that primarily belong to three major groups: lactic acid bacteria, acetic acid bacteria, and yeasts. Remarkably, the structural organisation, stability, and growth of kefir grains cannot be readily explained by their constituent species alone, and attempts to reconstitute them from the isolated species have so far been unsuccessful. Here, we combined a comprehensive structural investigation of the macro- and micromorphology of water kefir grains with culture-dependent and -independent analyses of its microbial biodiversity. The dynamics and interactions within this complex microbial community were studied by combining flow cytometry, fluorescence-activated cell sorting, and amplicon sequencing of selected subpopulations. Furthermore, the growth of water kefir grains was spatiotemporally resolved and quantitatively characterised, thereby unravelling an emergent multicellular growth function, documented for the first time. Our combined evidence provides an unprecedented understanding of a highly structured and metabolically synergistic multicellular microbial aggregate and lets us propose water kefir as a model system for the aggregative development of microbial multicellularity from multiple species. It should lay the foundation for future mechanistic studies aimed at understanding how multispecies microbial communities self-organise into stable, spatially structured multicellular aggregates.

microbiology↗

bioRxiv2026

PURELIGHT: a quantitative photon-counting framework unifying intensity and lifetime imaging at video rate across detector technologies

Quantitative fluorescence microscopy requires photon-efficiency, speed and accurate intensity and lifetime measurements. Time-correlated single-photon counting (TCSPC) simultaneously captures intensity and lifetime, but photon pile-up distorts both signals at high count rates, preventing fast acquisitions. Existing corrections discard photons, distort intensity, or require specialized detectors. Here we introduce PURELIGHT, an integrated hardware and software framework that simultaneously recovers undistorted intensities and lifetimes at count rates far beyond conventional pile-up limits. PURELIGHT works with hybrid photodetectors, silicon photomultipliers and photomultiplier tubes while retaining over three times more photons than alternative approaches. Using two-photon imaging, we showcase PURELIGHT's superior accuracy and spatial contrast, demonstrating video-rate subcellular lifetime imaging in awake mice, a unique lifetime-calibrated ratiometric modality and crosstalk-free temporal multiplexing. By removing the limits that have confined TCSPC to low-signal applications, PURELIGHT promotes the adoption of quantitative, photon-efficient microscopy across the life sciences.

neuroscience↗

bioRxiv2025

aDISCO: A Broadly Applicable Method for 3D Microscopy of Archival Paraffin-Embedded Human Tissues

Human surgery and autopsy specimens are routinely stored as formalin-fixed paraffin-embedded (FFPE) tissue blocks. Diagnoses are based on microscopic examination of two-dimensional sections. Good clinical practice requires that samples be retained for decades, thus giving rise to vast archives of healthy and diseased tissues. While tissue clearing and whole-mount microscopy enable 3D analysis, FFPE human tissue blocks are often unsuitable for clearing and immunolabeling due to their large size, extensive cross-linking, and wax embedding. Here, we introduce archival DISCO (aDISCO), a versatile and robust clearing method designed to overcome these challenges. aDISCO achieves complete clearing and immunolabeling of large samples stored for 15 years or more. We applied aDISCO to human brain, spinal cord, peripheral nerve, skin, muscle, heart, kidney, liver, spleen, colon, and lung, using a broad range of antibodies. To investigate the clinical usefulness of quantitative 3D histology by aDISCO, we performed a case study of focal cortical dysplasia, a neurodevelopmental disease causing epilepsy. Using deep-learning-based analysis, we found disrupted cortical layering and both focal and global neuronal density variations in FCD, features that are likely to be overlooked by conventional histology. In summary, aDISCO delivers large datasets suitable for deep-learning-based processing, enabling the detection of subtle and sparse pathologies in large archival human tissue specimens.

bioengineering↗

bioRxiv2024

Pythia: Non-random DNA repair allows predictable CRISPR/Cas9 integration and gene editing

CRISPR-based genome engineering holds enormous promise for basic science and therapeutic applications. Integrating and editing DNA sequences is still challenging in many cellular contexts, largely due to insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep-learning models and adheres to sequence context specific rules. Based on in silico predictions, we devised a strategy of triplet base-pair repeat repair arms that correspond to microhomologies at double-strand breaks (trimologies), which facilitated integration of large cargo (>2 kb) and protected the targeted locus and transgene from excessive damage. Successful integrations occurred in >30 loci in human cells and in in vivo models. Germline transmissible transgene integration in Xenopus, and endogenous tagging of tubulin in adult mice brains demonstrated integration during early embryonic cleavage and in non-dividing differentiated cells. Further, optimal repair arms for single- or double nucleotide edits were predictable, and facilitated small edits in vitro and in vivo using oligonucleotide templates. We provide a design-tool (Pythia, pythia-editing.org) to optimize custom integration, tagging or editing strategies. Pythia will facilitate genomic integration and editing for experimental and therapeutic purposes for a wider range of target cell types and applications.

molecular biology↗

bioRxiv2024

Investigating microscopic angioarchitecture in the human visual cortex in 3D with angioMASH tissue clearing and labelling

Non-invasive imaging techniques, such as ultra-high field fMRI, are intricately connected to the underlying vasculature and are approaching ever higher resolutions. For the analysis of fMRI signals over cortical depth at such high resolutions, microvascular differences might have to be taken into account. Therefore, a better understanding of the laminar distribution and interareal differences in the cortical vasculature is becoming more important. However, in comparison to cyto- and myeloarchitecture, the study of angioarchitecture has received far less attention and relatively few methods have been described to visualise the vascular network in the human brain. Here we present angioMASH, a method for double labelling angioarchitecture and cytoarchitecture in archival human brain tissue, based on the recently published MASH protocol. The double labelling and optical clearing of thick human brain slices can be accomplished within 16 days. We use this method to acquire multi-resolution 3D datasets of combined cyto- and angioarchitecture in large ([~]30 mm x 10 mm x 3 mm) volumes of human samples covering visual areas V1 and V2. We demonstrate for the first time, that classical angioarchitectonic features can be visualised in the human cortex and in 3D using tissue clearing and light-sheet microscopy. Lastly, we show differences in the vessel density and orientation over cortical depth within and between the two areas. Especially in V1, the vascular density is not homogeneous over cortical depth but shows distinct layering. These layers are also determined by changes in the orientation of the blood vessels from a predominantly radial to a more tangential distribution. In V2, differences in vascular density are less pronounced, but orientation profiles follow a similar trend over cortical depth. We discuss potential consequences of these differences for the interpretation of non-invasive functional imaging modalities such as fMRI or fNIRS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/609648v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@f9f841org.highwire.dtl.DTLVardef@1197e29org.highwire.dtl.DTLVardef@a9c9ccorg.highwire.dtl.DTLVardef@ffaefe_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 {micro}m laterally, 3.3 {micro}m axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

neuroscience↗

bioRxiv2023

Leptomeningeal collaterals regulate reperfusion in ischemic stroke

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet unknown functions. Utilizing a thrombin-based mouse model of stroke and the gold standard fibrinolytic treatment rt-PA, we here show that LMCs play a critical role in preserving vascular function in ischemic territories. We applied laser speckle contrast imaging, ultrafast ultrasound, and two-photon microscopy, to show that after thrombolysis, LMCs allow for gradual reperfusion resulting in small infarcts. On the contrary, in mice with poor LMCs, distal segments of recanalized arteries collapse and deleterious hyperemia causes hemorrhage and mortality. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for gradual reperfusion of ischemic tissues after stroke.

neuroscience↗

bioRxiv2021

Dendritic integration of sensory and reward information facilitates learning

Learning goal-directed behavior requires association of pertinent sensory stimuli with behaviorally relevant outcomes. In the mammalian neocortex, dendrites of pyramidal neurons are suitable association sites1-3 but how their activities adapt during learning remains elusive. Here, we track calcium signals in apical dendrites of layer 5 (L5) pyramidal neurons in mouse barrel cortex during texture discrimination learning4. We observe diverse task-related responses, either localized to branches or widespread throughout the apical tuft. However, even in expert mice, the tufts capability to discriminate go/no-go stimuli remains poor. Yet, we identify two prevailing response types in dendritic branches: 1) responses to unexpected outcome (reward) in naive mice that decrease with growing task proficiency, and 2) responses associated with salient sensory stimuli, especially the outcome-predicting texture touch, that strengthen upon learning. We demonstrate that these response types match distinct unsigned components of the temporal difference error5 by replicating our results with a reinforcement learning model. Moreover, optogenetic apical inhibition of L5 neurons during the outcome time window prevents naive animals from learning the task, consistent with the effect of equivalent model perturbation. Our findings suggest that salience signals in L5 apical dendrites facilitate the recruitment of task-relevant neurons via dendritic gain modulation.

neuroscience↗

bioRxiv2019

Context-dependent limb movement encoding in neuronal populations of motor cortex

Neuronal networks of the mammalian motor cortex (M1) are important for dexterous control of limb joints. Yet it remains unclear how encoding of joint movement in M1 networks depends on varying environmental contexts. Using calcium imaging we measured neuronal activity in layer 2/3 of the mouse M1 forelimb region while mice grasped either regularly or irregularly spaced ladder rungs during locomotion. We found that population coding of forelimb joint movements is sparse and varies according to the flexibility demanded from them in the regular and irregular context, even for equivalent grasping actions across conditions. This context-dependence of M1 network encoding emerged during learning of the locomotion task, fostered more precise grasping actions, but broke apart upon silencing of projections from secondary motor cortex (M2). These findings suggest that M2 reconfigures M1 neuronal circuits to adapt joint processing to the flexibility demands in specific familiar contexts, thereby increasing the accuracy of motor output.

neuroscience↗

bioRxiv2019

The mesoSPIM initiative: open-source light-sheet mesoscopes for imaging in cleared tissue

Over the course of the past decade, tissue clearing methods have reached a high level of sophistication with a wide variety of approaches now available1. To image large cleared samples, light-sheet microscopes have proven to be ideal due to their excellent optical sectioning capability in transparent tissue2. Such instruments have recently seen extensive technological and commercial development. However, despite this progress, the community is lacking instruments capable of exploring large samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy (mesoSPIM) initiative, an open-hardware project that provides researchers with instructions and software to easily build and operate light-sheet microscopes for centimeter-sized cleared samples (http://www.mesospim.org).\n\nA wide range of commercial light-sheet microscopes have ...

neuroscience↗