Biology subjects

Oheim, M.

Publications and source records attributed to Oheim, M..

9 recordsLinked to original sources

bioRxiv2026

The enlightened entomologist: fast, non-destructive whole-arthropod clearing for three-dimensional imaging

Arthropods are a strikingly diverse phylum of invertebrates with segmented bodies, chitinous exoskeletons, and jointed limbs, many of which are colloquially referred to as insects. Their pigmented and optically dense bodies pose a considerable challenge for microscopy. While early naturalists focused on external features, modern approaches combining tissue clearing and fluorescence microscopy seek to explore internal anatomy in three dimensions. However, both chemical clearing and three-dimensional (3-D) microscopy are specialized techniques and often require considerable adaptation and optimization across species. Here, we introduce a versatile, fast, effective and non-toxic clearing method that renders diverse arthropods transparent within hours to days. Existing upright microscopes or macroscopes can be upgraded with a modular and affordable light-sheet microscope, allowing rapid volumetric imaging of arthropods. Our pipeline is fully compatible with dye staining and immunofluorescence labeling, while endogenous autofluorescence provides valuable anatomical context and facilitates 3-D reconstruction.

zoology↗

bioRxiv2026

Diagnosing Neurodegenerative Diseases by Label-Free 3-D Imaging of Intestinal Samples

Early diagnosis of Parkinsons disease (PD) remains challenging because motor symptoms appear only after extensive neurodegeneration, and a definitive diagnosis still relies on post-mortem neuropathology. Increasing evidence implicates the enteric nervous system (ENS) in prodromal disease stages, but routine ENS-based diagnosis is limited by the complexity of intestinal tissue organization and the need for specific labeling strategies. Here, we developed a label-free autofluorescence (AF) imaging workflow combined with unbiased morphometric analysis to identify neurodegenerative alterations in fixed human colonic tissue. Using a correlative multiscale imaging approach, we generated a database of almost 800 high-resolution confocal images from myenteric and submucosal plexuses of controls, PD, and Alzheimers disease (AD) patients. Blind evaluation by four expert histologists showed reliable identification of control tissue but lower sensitivity for pathological cases, reflecting the heterogeneous distribution of ENS lesions. Semi-quantitative and morphometric image analyses identified a distinct population of enlarged enteric neurons, termed large neural cells (LNCs), strongly enriched in PD and AD compared with controls. LNCs contained autofluorescent cytoplasmic inclusions and frequently prominent nucleoli, both features largely absent from control tissue independent of aging. Co-localization with the amyloid-binding probe Amytracker (AmyT) demonstrated that AF granules correspond to {beta}-sheet-rich protein aggregates rather than merely age-related lipofuscin granules. Similar alterations were detected in intact three-dimensional (3-D) colonic biopsies, demonstrating the feasibility of volumetric ENS imaging without tissue clearing. Together, our results establish label-free AF imaging as a rapid and clinically compatible strategy for detecting enteric neurodegenerative pathology. This approach provides a framework for the future development of ENS-based biomarkers and supports the use of volumetric intestinal imaging for early diagnosis of neurodegenerative diseases.

pathology↗

bioRxiv2025

Morphological heterogeneity of human astrocytes in cerebral organoids

Astrocytes are a type of glial cell in the central nervous system responsible for modulating synaptic transmissions, tissue repair, maintaining homeostasis, and are therefore implicated in many neurological diseases. Human cortical astrocytes are more structurally complex, larger, and have unique subtypes in comparison to the commonly studied rodent cortical astrocytes. As access to human cortical tissue is sparse, cerebral organoids (COs) derived from human pluripotent stem cells have emerged as a promising in vitro model for studying the human cortex. Astrocyte subtypes unique to humans are recapitulated in COs but have not been quantitatively assessed (1, 2). In this study, we characterized human astrocytes in situ in sliced COs cultured at the air-liquid interface (ALI-COs). By 4 months of age, ALI-COs express many mature astrocyte markers and showed increasing levels of glial fibrillary acidic protein (GFAP) with longer culture durations. By employing immunostaining, tissue clearing, morphological reconstruction, and unsupervised clustering analysis, three major GFAP+ astrocyte subtypes were identified in ALI-COs. All subtypes exhibited greater morphological complexity than their mouse counterparts, as revealed by increased branching and longer branch extensions. However, consistent with the mid-gestation fetal stage of the ALI-COs, astrocytes did not fully recapitulate the complexity observed in adult human astrocytes, which are known to continue maturation postnatally.

neuroscience↗

bioRxiv2023

A first morphological and electrophysiological characterization of FCs of the mouse cerebellum.

The "feathered" cells of Fananas (FCs) are cerebellar glia of unknown function. Initially described more than a century ago, they have been practically absent from the scientific literature. Here, we combined whole-cell patch-clamp recordings, dye loading and near UV-laser photolysis for a first characterization of FCs in terms of their morphology, electrophysiological properties and glutamate-evoked currents. FCs were identified in cerebellar slices by their small cell bodies located in the molecular layer and stubby processes. Despite a more compact shape compared to Bergmann glia (BGs) FCs had similar membrane resistances and basal currents, suggesting electrical coupling between neighboring glia. Dye filling and pharmacological experiments confirmed homo- and heterotypic gap junction coupling among FCs and BGs. Parallel-fiber stimulation evoked in FCs a slow inward current partially blocked by NBQX, along with superimposed fast (ms) transients. Repetitive stimulations resulted in a rapid desensitization of this AMPA-receptor mediated current, which recovered for stimulation intervals >0.5s. Laser photolysis of MNI-caged glutamate replicated fast inward currents with highest current densities in the distant process. We conclude that FCs respond with fast AMPA currents to local glutamate release, and that they integrate ambient glutamate to a slow inward current. Interestingly, FCs prevail throughout adulthood with markedly different densities among cerebellar lobes. Thus, FCs are not just displaced BGs as previously suggested, but they may have lobule specific functions, both locally and at the circuit level, yet to be uncovered.

neuroscience↗

bioRxiv2022

Characterizing nanometric thin films with far-field light

Ultra-thin, transparent films are being used as protective layers on semiconductors, solar cells, as well as for nano-composite materials and optical coatings. Nano-sensors, photonic devices and calibration tools for axial super-resolution microscopies, all rely on the controlled fabrication and analysis of ultra-thin layers. Here, we describe a simple, non-invasive, optical technique for simultaneously characterizing the refractive index, thickness, and homogeneity of nanometric transparent films. In our case, these layers are made of the biomimetic polymer, My-133-MC, having a refractive index of 1.33, so as to approach the cytosol for biological applications. Our technique is based on the detection in the far field and the analysis of supercritical angle fluorescence (SAF), i.e., near-field emission from molecular dipoles located very close to the dielectric interface. SAF emanates from a 5-nm J-aggregate emitter layer deposited on and in contact with the inspected polymer film. Our results compare favorably to that obtained through a combination of atomic force and electron microscopy, surface-plasmon resonance spectroscopy and ellipsometry. We illustrate the value of the approach in two applications, (i), the measurement of axial fluorophore distance in a total internal reflection fluorescence geometry; and, (ii), axial super-resolution imaging of organelle dynamics in a living biological sample, cortical astrocytes, an important type of brain cell. In the later case, our approach removes uncertainties in the interpretation of the nanometric axial dynamics of fluorescently labeled vesicles. Our technique is cheap, versatile and it has obvious applications in microscopies, profilometry and optical nano-metrology.

biophysics↗

bioRxiv2022

Label-free, fast, 2-photon volume imaging of the structural organization of peripheral neurons and glia in the enteric ganglia

The enteric nervous system (ENS), sometimes considered as a second brain due to its large autonomy from central circuits is made of interconnected plexuses organized in a mesh-like network lining the gastrointestinal tract. Originally described as a leading actor of the regulation of digestion, bowel advance and intestinal secretion, its implication in various neuropathologies has recently been demonstrated. However, with few exceptions, its morphology and functions have been studied on thin sections of the intestinal wall or in dissected explants. Due to its intricate morphology, precious information on its three-dimensional (3-D) architecture and connectivity is often lost. In this context, we have developed a fast, label-free 3-D imaging method of the ENS, based on intrinsic signals of the tissue. We adapted a fast tissue-clearing protocol based on a high refractive-index aqueous solution, and then characterized the autofluorescence signals arising from the various cellular and sub-cellular components of the ENS. Immunofluorescence and spectral recordings complete this characterization. Finally, we demonstrate the fast acquisition of detailed 3-D image stacks of unlabeled mouse intestine, across the whole intestinal wall and including both the myenteric and submucosal enteric nervous plexuses using a new spinning-disk two-photon microscope. The combination of fast clearing (less than 15min for 73 % transparency), autofluorescence imaging and rapid volumetric imaging (less than a minute for the acquisition of s z-stack of 100 planes (150*150 {micro}m) at 300-nm spatial resolution) paves the way for new applications in fundamental and clinical research.

neuroscience↗

bioRxiv2021

A simple, inexpensive and multi scale 3D fluorescent test sample for optical sectioning microscopies

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-{micro}m to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes. Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective x20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this "primitive" and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/441588v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@13a11dforg.highwire.dtl.DTLVardef@16b818corg.highwire.dtl.DTLVardef@1061c64org.highwire.dtl.DTLVardef@32015a_HPS_FORMAT_FIGEXP M_FIG C_FIG Research highlights- highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies - standard tissue paper presents features ranging from 400 nm to centimetres - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast - it is easy to prepare, versatile, photostable and inexpensive

biophysics↗

bioRxiv2018

Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that - in many aspects - recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that - unlike existing two- or three-lens designs - it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

biophysics↗

bioRxiv2018

Two-photon imaging induces brain heating and calcium microdomain hyper-activity in cortical astrocytes

Unraveling how neural networks process and represent sensory information and how this cellular dynamics instructs behavioral output is a main goal in current neuroscience. Two-photon activation of optogenetic actuators and fluorescence calcium (Ca2+) imaging with genetically encoded Ca2+ indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques expose the brain to high near-infrared light doses raising the concern of light-induced adverse effects on the biological phenomena being studied. Combing Ca2+ imaging of GCaMP6f-expressing cortical astrocytes as a sensitive readout for photodamage and an unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca2+ signals in fine astroglial processes. Illumination conditions routinely being used in biological two-photon microscopy (920-nm excitation, 100-fs regime, ten mW average power) increased the frequency of microdomain Ca2+ events, but left their amplitude, area and duration rather unchanged. This increase in local Ca2+ activity was followed by Ca2+ transients in the otherwise silent soma. Ca2+ hyperactivity occurred without overt morphological damage. Surprisingly, at the same average power, continuous-wave 920-nm illumination was as damaging as fs pulses, indicating a linear, heating-mediated (rather than a highly non-linear) damage mechanism. In an astrocyte-specific IP3-receptor knock-out mouse (IP3R2-KO), Near-infrared light-induced Ca2+ microdomains signals persisted in the small processes, underpinning their resemblance to physiological IP3R2-independent Ca2+ signals, while somatic activity was abolished. Contrary to what has generally been believed in the field, shorter pulses and lower average power are advantageous to alleviate photodamage and allow for longer useful recording windows.\n\nSIGNIFICANCE STATEMENTImaging the fine structure and function of the brain has become possible with two-photon microscopy that uses ultrashort-pulsed infrared laser light for better tissue penetration. The high peak energy of these light pulses has raised concerns about photodamage resulting from multi-photon processes. Here, we show that the time-averaged rather than the peak laser power matters. At wavelengths and with laser powers now commonly used in neuroscience brain damage occurs as a consequence of direct infrared light absorption, i.e., heating. To counteract brain heating we explore a strategy that uses even shorter, more energetic pulses but a lower time-averaged laser power to produce the same image quality while making two-photon microscopy less invasive.

neuroscience↗