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2D and 3D instance segmentation of nuclei from array tomography (AT) and FIB-SEM datasets, respectively. In this deposition, 10 AT and 2 FIB-SEM datasets and paired annotations are included. Note: in several datasets, partial annotations of nuclei at image edges have been deleted. These are indicated in the annotation metadata.

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

Organisms: Homo sapiens Mus musculus

Calcium transients drive cells to discharge prostaglandin E2 (PGE2). The authors visualized PGE2-induced protein kinase A (PKA) activation and quantitated PGE2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, they first prepared PGE2-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 (CRY2clust) or Gq-DREADD, respectively. Second, they prepared reporter cells expressing the Gs-coupled PGE2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Forster resonance FRET. Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE2 to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE2 diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, the authors estimated that a single HeLa cell secretes 0.25 fmol PGE2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE2 discharge rate is comparable to the diffusion rate. Thus, their method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE2.

Imaging Methods: recorded image fluorescence microscopy time lapse microscopy FRET

Organisms: Homo sapiens Canis lupus familiaris

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, the authors develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument- dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI- independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Imaging Methods: recorded image two-photon laser scanning microscopy time lapse microscopy

Organisms: Arabidopsis thaliana

Plants develop along apical-basal and radial axes. In Arabidopsis thaliana, the radial axis becomes evident when the cells of the 8-cell proembryo divide periclinally, forming inner and outer cell layers. Although changes in cell polarity or morphology likely precede this oriented cell division, the initial events and the factors regulating radial axis formation remain elusive. Here, we report that three transcription factors belonging to the class IV homeodomain-leucine zipper (HD-ZIP IV) family redundantly regulate radial pattern formation:HOMEODOMAIN GLABROUS11 (HDG11), HDG12, and PROTODERMAL FACTOR2 (PDF2). The hdg11 hdg12 pdf2 triple mutant failed to undergo periclinal division at the 8-cell stage and cell differentiation along the radial axis. Live-cell imaging revealed that the mutant defect is already evident in the behavior of the embryo’s initial cell (apical cell), which is generated by zygote division. In the wild type, the apical cell grows longitudinally and then radially, and its nucleus remains at the bottom of the cell, where the vertical cell plate emerges. By contrast, the mutant apical cell elongates longitudinally, and its nucleus releases from its basal position, resulting in a transverse division. Computer simulations based on the live-cell imaging data confirmed the importance of the geometric rule (the minimal plane principle and nucleus-passing principle) in determining the cell division plane. We propose that HDG11, HDG12, and PDF2 promote apical cell polarization, i.e., radial cell growth and basal nuclear retention, and set proper radial axis formation during embryogenesis.

Imaging Methods: recorded image spinning disk confocal microscopy spinning disk confocal microscopy

Organisms: NCBITaxon:3702

This dataset contains high-content, live-cell, time-lapse imaging of HeLa cells undergoing apoptosis across a 10-point dose response to the apoptosis inducer staurosporine. Using the ChromaLIVE™ Live Cell Painting assay, cells were imaged every 30 minutes over a six-hour period on the Yokogawa CellVoyager CQ1 spinning-disk confocal platform, capturing multiplexed fluorescence signals (Hoechst, ChromaLIVE 488_yellow, ChromaLIVE 488_red, and ChromaLIVE 561) across three z-planes at four fields of view per well. After live imaging, cells were fixed and stained with AnnexinV to provide a single-cell ground-truth readout of apoptosis. The dataset includes raw and illumination-corrected images, cell segmentation masks, single-cell tracking outputs, and extracted morphological features generated using CellProfiler and scDINO deep-learning embeddings. In total, the dataset comprises over 188,000 single-cell observations and 20,677 cell tracks across time, with 2,336 curated morphology features per cell. These measurements quantify the dynamic morphological signatures preceding apoptosis and capture dose- and time-dependent phenotypic trajectories. This resource enables the study of temporal morphology changes, benchmarking of image-based profiling pipelines, development of machine-learning models for early apoptosis prediction, and exploration of dynamic cell-state transitions. It supports future work such as profiling additional forms of regulated cell death and constructing temporal morphology atlases.

Organisms: Homo sapiens

Plasma membrane calcium ATPases (PMCAs) are believed to function exclusively at the plasma membrane where they expel calcium from the cytosol. We have unexpectedly identified a splice variant-dependent localisation of the PMCA isoform PMCA2 to the lysosome, where it forms an evolutionarily conserved complex with NPC1, the lysosomal membrane protein defective in the rare lysosomal storage disease Niemann-Pick disease type C (NPC). This interaction is required for lysosomal Ca2+ homeostasis and implicates PMCA2 as a mediator of Ca2+ uptake into lysosomes. Disruption of the NPC1-PMCA2 complex contributes to the pathophysiology of both Niemann-Pick disease type C and Parkinson’s disease, revealing an unrecognised intracellular function for PMCA2 and a shared mechanism linking lysosomal Ca2+ and lipid regulation in neurodegeneration.

Imaging Methods: confocal microscopy

Organisms: Rattus norvegicus

We developed a method to enlarge Dictyostelium discoideum cells by partial cytokinesis inhibition, generating multinucleated yet functional giant cells. These cells retained chemotactic signaling, polarity, and motility, enabling high-resolution live-cell imaging. Using fluorescent probes for cAMP and Ca2+, we uncovered a directional, front-to-rear propagation of cAMP signaling and a biphasic Ca2+ response coordinated with actin wave dynamics.

Imaging Methods: spinning disk confocal microscopy recorded image

Organisms: Dictyostelium discoideum

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, the authors develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument- dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI- independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Imaging Methods: recorded image

Organisms: Arabidopsis thaliana

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