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We present MitoEM 2.0, a curated benchmark resource for training and evaluating three-dimensional (3D) mitochondria instance segmentation in volume electron microscopy (vEM). The collection assembles multiscale vEM datasets (FIB-SEM, SBF-SEM, and ssSEM) spanning diverse tissues and species, with expert-verified instance labels emphasizing biologically difficult scenarios, including dense mitochondrial packing, hyperfused networks, and thin filamentous connections with ambiguous boundaries. All releases include native-resolution volumes and standardized processed versions, per-volume metadata (voxel size, modality, tissue, and data splits), and official train/validation/test partitions to enable reproducible benchmarking. Annotations follow a consistent protocol with quality checks and instance reindexing. Data are provided in NIfTI format with an nnU-Net–compatible layout, alongside machine-readable split files and checksums. MitoEM 2.0 facilitates robust model development and fair comparison across methods while supporting reuse in bioimage analysis, algorithm benchmarking, and teaching.

Imaging Methods: electron microscopy focussed ion beam scanning electron microscopy (FIB-SEM)

Organisms: Homo sapiens Mus musculus Drosophila melanogaster

The chicken embryo has been a long-standing model for studying embryonic development. Its easy access for in vivo experiments, together with the development of ex ovo culture techniques, has made it a choice model system for elaborate experimental manipulations. Here we present a time-lapse imaging dataset of the developing chicken embryo in ex ovo culture covering a range of HH3 to HH14+ embryonic stages, with 3-6 min temporal resolution.

Imaging Methods: bright-field microscopy

Organisms: Gallus gallus

Essential for eukaryotes, multiple copies of the exocyst complex tether each secretory vesicle to the plasma membrane (PM) in constitutive exocytosis. The exocyst higher-order structure (ExHOS) that coordinates the action of these multiple exocysts remains unexplored. We integrated particle tracking, super-resolution microscopy and cryo-electron tomography to time- resolve the continuum conformational landscape of the ExHOS and to functionally annotate its different conformations. We found that 7 exocysts form flexible ring-shaped ExHOS that tether 2 vesicles at <45 nm from the PM. The ExHOS rapidly expands while it pulls the vesicle towards the PM in a stepwise mechanism comprising three metastable states at 27, 18 and 5 nm from the PM. After fusion, Sec18 mediates the disassembly of the stationary ExHOS, an emergent function that controls the rate of exocytosis. By resolving the biophysical principles of tethering we bridged the gap between static isolated structures and the dynamic and multimeric nature of exocytosis. This datset contains in particular the correlative light and electorn microscopy images of lamellae that were subsequently used to target tomogram acqusition.

Organisms: Saccharomyces cerevisiae

Electron microscopy is an important technique for the study of synaptic morphology and its relation to synaptic function. The data analysis for this task requires the segmentation of the relevant synaptic structures, such as synaptic vesicles (SV), active zones, mitochondria, presynaptic densities, synaptic ribbons, and synaptic compartments. Previous studies were predominantly based on manual segmentation, which is very time-consuming and prevented the systematic analysis of large datasets. Here, we introduce SynapseNet, a tool for the automatic segmentation and analysis of synapses in electron micrographs. It can reliably segment SVs and other synaptic structures in a wide range of electron microscopy approaches, thanks to a large annotated dataset, which we assembled, and domain adaptation functionality we developed. We demonstrated its capability for (semi-)automatic biological analysis in two applications and made it available as an easy-to-use tool to enable novel data-driven insights into synapse organization and function.

Imaging Methods: transmission electron microscopy

Organisms: Mus musculus

Microscopy image dataset of ANX A5 in cholestasis

Imaging Methods: ANSOM confocal microscopy

Organisms: Mus musculus

Mitochondrial dysfunction is a critical early driver of retinal ganglion cell (RGC) loss in optic nerve injury. We evaluated whether HDAP2, a mitochondria-targeted aromatic peptide designed to support mitochondrial membrane integrity, could preserve neuronal structure after optic nerve crush (ONC) in C57BL/6 mice (both sexes, n=31). Systemically administered HDAP2 penetrated the blood–retinal barrier and localized to RGCs and mitochondrial-rich retinal layers. Daily treatment significantly improved RGC survival compared to saline-treated ONC animals. RGC densities increased across central, midperipheral, and peripheral regions. Transmission electron microscopy revealed that HDAP2 substantially reduced mitochondrial loss within crushed optic nerve axons. Mitochondrial density in HDAP2-treated nerves approached levels observed in uninjured controls and was nearly 3-fold higher than untreated ONC nerves. Mitochondrial morphology was similar across groups, indicating that HDAP2 prevents mitochondrial loss rather than rescuing damaged organelles. HDAP2-treated nerves also exhibited a numerically higher density of structurally intact axons, consistent with reduced ultrastructural degeneration. These findings demonstrate that HDAP2 limits mitochondrial loss and attenuates neuronal degeneration after ONC. Together, the results support HDAP2 as a promising therapeutic candidate for protecting CNS projection neurons by maintaining mitochondrial stability after axonal injury.

Imaging Methods: confocal microscopy

Organisms: Mus musculus

The orbitofrontal cortex (OFC) is central to cognitive and social functions, yet its presynaptic partners remain incompletely defined. In female mice, the OFC modulates infant-directed caregiving behaviors in an experience-dependent manner. Here, we identify the submedius thalamus (SMT) as a major presynaptic partner of the OFC. Trans-synaptic tracing revealed intensive inputs from both the SMT and mediodorsal thalamus (MD) to OFC layer 5 excitatory neurons. We generated a Tnnt1-Cre mouse line that enables selective targeting of these higher-order thalamic nuclei. Axonal tracing demonstrated complementary projection patterns of the SMT and MD across prefrontal regions. Microendoscopic Ca2+ imaging demonstrated pup retrieval-related activity in both SMT and MD, with SMT exhibiting learning-related plasticity. Projection-specific chemogenetic silencing demonstrated that SMT modulates OFC activity during pup retrieval, although its behavioral relevance appeared limited. Collectively, our study provides a presynaptic map of OFC layer 5 neurons and a proof of concept for selective manipulation of the SMT as a previously understudied thalamic input to the OFC.

Imaging Methods: recorded image

Organisms: Mus musculus

The dataset contains the original confocal fluorescence microscopy images of E. coli BL21(DE3) expressing the iron biosensor MDtxRG149GA (IronSenseR) and its binding deficient mutants used in the publication "A novel biosensor for ferrous iron developed via CoBiSe: A computational method for rapid biosensor design". Images contain a channel for cpsfGFP fluorescence, LSSmApple fluorescence and transmitted light. Prior to imaging, cells were either incubated in PBS buffer only, PBS buffer containing 20 µM 2,2′-Bipyridine (BPD) or 250 µM BPD.

Imaging Methods: fluorescence microscopy

Organisms: Escherichia coli BL21

Prostate cancer (PCa)-related deaths are mainly due to metastasis. The increase in de novo metastatic hormone-naïve prostate cancers (mHNPCs) highlights the urgent need for biomarkers and treatment strategies for PCa dissemination. We report in a cohort of French patients that the levels of vitamin D and of prostate-specific antigen, the progression biomarker used clinically and secreted by prostatic epithelial cells (PECs), are negatively associated. However, the impact of vitamin D receptor (VDR) signaling in PECs on prostate tumorigenesis remains unclear. We show that VDR inactivation in PECs from Pten(i)pe−/− mice, a relevant preclinical model of PCa, promotes tumor aggressiveness. We demonstrate that VDR loss induces oxidative stress that in turn enhances PECs proliferation. Moreover, CXCL5 secretion by PTEN- and VDR-deficient PECs promotes neutrophil infiltration. Importantly, our data highlight elevated circulating neutrophil levels as a biomarker for PCa dissemination and show the potency of targeting neutrophil chemotaxis to reduce liver micrometastases. Overall, this work provides major insight on how vitamin D signaling slows down tumorigenesis and suggests new therapeutic and diagnostic strategies for mHNPC.

Imaging Methods: bright-field microscopy

Organisms: Mus musculus

This dataset contains multichannel fluorescence images from an adapted Cell Painting assay applied to 3D colorectal cancer spheroids. Spheroids were generated in ultra-low attachment microplates, cleared to improve optical transparency, and imaged across multiple channels to capture morphological variation throughout the spheroid volume. The image analysis pipeline includes automated spheroid detection, cell segmentation, and feature extraction, with outputs normalized across plates and imaging depths. It provides a resource for scalable, image-based profiling in physiologically relevant 3D spheroid models.

Imaging Methods: spinning disk confocal microscopy

Organisms: Homo sapiens

Purpose: Retinal ganglion cell (RGC) loss in glaucoma occurs in a large fraction of patients even after intraocular pressure (IOP) is reduced. Mitochondrial dysfunction is a key mechanism that links elevated IOP to RGC degeneration. We tested whether HDAP2, a novel high-density aromatic peptide that binds cardiolipin to stabilize mitochondrial membranes, can protect RGCs in the DBA/2J mouse model. Methods: DBA/2J mice received HDAP2 (3 mg/kg, intraperitoneally, every other day) starting at 4 months of age for 8 months. IOP was measured each month to track pressure exposure. RGC survival was assessed by counting RBPMS-stained cells in retinal wholemounts and optic nerve axons in semithin toludine blue-stained sections. Results: HDAP2-treated retinas had ~49% more RGCs than untreated retinas at similar pressure exposures (p = 0.0063; F(2,59) = 5.524). At mild IOP exposure, HDAP2 preserved 58% more RGCs compared with untreated retinas, and at high IOPs, RGC survival was 180% greater. Kaplan–Meier analysis indicated that HDAP2 increased the threshold for severe RGC loss by 29 mmHg and reduced the chance of developing severe RGC degeneration by a factor of 4.6. Optic nerve axons from treated retinas were also well preserved, with axon morphologies appearing indistinguishable from controls. Axon size distributions did not change significantly among the treatment groups, suggesting that protection by HDAP2 was similar among RGC subtypes. Conclusions: HDAP2 preserved both RGCs and axons in a pressure-dependent manner and increased tolerance to IOP. These results suggest that HDAP2 may complement pressure-lowering therapy, including for normal-tension and treatment-refractory glaucoma.

Imaging Methods: bright-field microscopy confocal microscopy

Organisms: Mus musculus

MERFISH of adult human skin across 114 samples, 22 donors, and 15 anatomic sites. H&E of adult human skin

Imaging Methods: bright-field microscopy microscopy

Organisms: Homo sapiens