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Epidermal growth factor receptor (EGFR)-mediated signal transduction controls cell growth and proliferation. The signaling pathway is regulated so that it is activated only by external EGF stimuli, but the mechanisms that prevent EGF-independent spontaneous activation of EGFR-mediated signaling are unknown. Here the authors report cholesterol depletion activates EGFR-mediated signaling without EGF. They applied automated single-molecule imaging to EGFR and characterized the lateral diffusion and cluster formation on cholesterol-depleted and cholesterol-supplemented membranes. In cells in which cholesterol was depleted by methyl-beta-cyclodextrin treatment, EGFR exhibited a reduction in lateral diffusion, an acceleration of cluster formation, and autophosphorylation without EGF. Concurrently, extracellular signal-regulated kinase (ERK), which is regulated by EGFR-mediated signaling, exhibited phosphorylation and nuclear translocation without EGF. These cholesterol depletion-induced changes were similar, albeit less efficient, to those that occurred with EGF stimulation in normal cells without methyl-beta-cyclodextrin, indicating the spontaneous activation of EGFR signaling. The exogenous supplementation of cholesterol suppressed the methyl-beta-cyclodextrin-induced spontaneous activation of EGFR and ERK nuclear translocation. Single-molecule imaging of EGFR in a large number of cells revealed cell-to-cell heterogeneity, with a sub-population showing a high ability for spontaneous activation. These results provide evidence that EGFR-mediated signaling is properly regulated by cholesterol metabolism to prevent uncontrolled spontaneous activation.

Imaging Methods: recorded image evanescent wave microscopy

Organisms: Cricetulus griseus

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C−N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey’s method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin- independent manner in tobacco BY-2 cells.

Imaging Methods: recorded image confocal microscopy

Organisms: Nicotiana tabacum

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C−N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey’s method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin- independent manner in tobacco BY-2 cells.

Imaging Methods: confocal microscopy

Organisms: Nicotiana tabacum

This dataset comprises an independent Korean cohort of clear cell renal cell carcinoma (ccRCC) patients collected at Seoul National University Hospital. This cohort comprises patients that have undergone surgical retention with no prior treatment. It includes 300 haematoxylin and eosin (H&E) whole-slide images (WSIs) providing detailed histopathological context, alongside 71 lo-plex immunofluorescence (mIF) WSIs. The mIF panel targets p21, MCM2, and Lamin B1,with Hoechst used as a nuclear counterstain. Additionally, 71 single-plex immunohistochemistry (IHC) images for CD105 (DAB chromogen staining of vasculature) are provided for complementary vascular analyses. All H&E WSIs were acquired using a Zeiss Axio Scan.Z1 scanner at 20× magnification, and automated image analysis of all mIF slides was performed using HALO® software. The principal derived metrics for each tumour are the percentages of p21⁺/MCM2⁻ cells and CD105⁺/p21⁺/MCM2⁻ cells, representing the cell-cycle arrest fractions in tumour and putative endothelial cell populations, respectively. Each case is accompanied by two CSV files containing quantitative metrics (phenotypic proportions and average nuclear/cell parameters), along with a clinical metadata file providing de-identified patient information. The clinical data include recurrence, disease-free survival (DFS), Fuhrman nuclear grade, tumor status, tumor size, lymph node status, tumour necrosis, Leibovich score based on Fuhrman grade (Lscore_Fuhrman), ISUP grade (International Society of Urological Pathology grade), and Leibovich score based on ISUP grade (Lscore_ISUP).

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Homo sapiens

This dataset corresponds to 443 clear-cell and 37 papillary renal cell carcinoma (RCC) cohort of patients from the UK translational arm of the SORCE trial, a phase III randomized study evaluating adjuvant sorafenib versus placebo in renal cancer (ClinicalTrials.gov identifier: NCT00492258). This multi-center cohort encompasses intermediate- and high-risk RCC cases randomized to receive sorafenib adjuvant therapy (1 or 3 years) or placebo. The image collection comprises tumour samples from 480 patients enrolled in the trial’s UK tissue biobank as part of TRANSORCE. For each patient, a formalin-fixed, paraffin-embedded (FFPE) primary tumor section was used to generate a whole-slide haematoxylin and eosin (H&E) image, and lo-multiplex immunofluorescence (mIF) imaging was performed for 382 cases. The mIF panel included p21^CDKN1a, MCM2, and CD105 (endoglin), together with Hoechst nuclear counterstaining. All H&E whole-slide images (WSIs) were acquired using a Zeiss Axio Scan.Z1 scanner at 40× magnification, and mIF images were acquired on the same platform at 20× magnification. Both image types are provided in CZI format. Automated image analysis was performed on all mIF slides using HALO® software. The principal derived metric for each tumour is the percentage of p21⁺/MCM2⁻ and CD105⁺/p21⁺/MCM2⁻ cells, representing the cell-cycle arrest fraction in tumor and endothelial cells. In total, the dataset includes 480 H&E WSIs and 382 multi-channel mIF WSIs. Also, the dataset includes two CSV files containing quantitative metrics (phenotypic proportions and average nuclear/cell parameters), along with a clinical metadata file providing de-identified patient information. The clinical dataset accompanying this cohort includes the following variables: treatment arm ( A: placebo, B: one year sorafenib, C: three year sorafenib), age at randomization, time from diagnosis to randomization, date of randomization, date of operation, gender, WHO performance status, histology at diagnosis, pathological T stage, regional lymph node status, tumor size, ISUP nuclear grade, presence of tumor necrosis, raw Leibovich score, Leibovich risk group, maximum tumor diameter, disease-free survival status (DFS 0/1) and date, and overall survival status (OS 0/1) and date. All data are released under a CC0 license, enabling unrestricted reuse by the research community.

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Homo sapiens

This entry describes a retrospective cohort of clear-cell renal cell carcinoma (ccRCC) patients from Edinburgh (NHS Lothian). A total of 120 archival nephrectomy specimens from patients with no previous treatment were included. Digital whole-slide images are available for all 120 cases stained with haematoxylin and eosin (H&E) and for 88 cases stained using multiplex immunofluorescence (mIF). The mIF panel comprised p21^CDKN1A, MCM2, and Lamin B1, with Hoechst nuclear counterstain. All slides were scanned at 20× magnification using a Zeiss Axio Scan.Z1, generating high-resolution bright-field and fluorescence images in CZI format. The dataset includes the H&E and multi-channel mIF images, derived single-cell phenotype tables, and a de-identified clinical data file. Image analysis was performed using HALO® software with AI-assisted nuclear segmentation. For each cell, fluorescence intensities of p21, MCM2, and Lamin B1 were quantified, allowing classification into proliferative (MCM2⁺) or cell-cycle arrested (p21⁺) phenotypes. Output data for each image include the proportion of p21⁺/MCM2⁻ cells and average morphological measurements such as cell and nuclear size. Quantitative results are provided in CSV format. The accompanying clinical metadata file contains pathological stage (pT), recurrence status, Fuhrman nuclear grade, ISUP grade, tumour size, nodal status, presence or absence of necrosis, derived Leibovich scores based on Fuhrman and ISUP grading (Lscore_Fuhrman and Lscore_ISUP), and disease-free survival (DFS). All data in this cohort are released under a CC0 license to permit unrestricted reuse.

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Homo sapiens

This dataset consists of a paired primary–metastatic cohort comprising 62 patients with clear-cell renal cell carcinoma (ccRCC), yielding a total of 124 formalin-fixed, paraffin-embedded (FFPE) tumour samples. Each patient contributed both a primary and a metastatic sample. Haematoxylin and eosin (H&E) slides were prepared for all 124 samples, and multiplex immunofluorescence (mIF) was successfully performed for 88 of them where tissue was available for study. The mIF panel targeted key cell-cycle and microenvironmental markers — p21^CDKN1A, MCM2, and CD105 (endoglin) — with nuclear counterstaining using Hoechst 33342. Whole-slide images (WSIs) were captured using a Zeiss Axio Scan.Z1 scanner, generating high-resolution CZI files. The dataset includes all H&E and corresponding multi-channel mIF WSIs, along with derived single-cell phenotype data and de-identified clinical metadata. Quantitative outputs for each slide include the proportions of p21⁺/MCM2⁻ and CD105⁺/p21⁺/MCM2⁺ cells, as well as average cellular and nuclear morphometric parameters. The accompanying clinical CSV file provides patient-level variables including follow-up end date, survival status, follow-up duration, overall survival (OS), and progression-free survival (PFS). This cohort represents advanced-stage cases exhibiting vascular invasion or distant metastases at diagnosis. Ethical approval was granted by the Lothian Regional Ethics Committee (Refs 08/S1101/41 and 10/S1402/33). All images, quantitative data, and metadata are distributed under a CC0 license, allowing unrestricted public reuse.

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Homo sapiens

Protein aggregation is a hallmark of neurodegenerative diseases and is also observed in the brains of elderly individuals without such conditions, suggesting that aging drives the accumulation of protein aggregates. However, the comprehensive understanding of age-dependent protein aggregates involved in brain aging remains unclear. Here, we investigated proteins that become sarkosyl-insoluble with age and identified hyaluronan and proteoglycan link protein 2 (HAPLN2), a hyaluronic acid-binding protein of the extracellular matrix at the nodes of Ranvier, as an age-dependent aggregating protein in mouse brains. Elevated hyaluronic acid levels and impaired microglial function reduced the clearance of HAPLN2, leading to its accumulation. HAPLN2 oligomers induced microglial inflammatory responses both in vitro and in vivo. Furthermore, age-associated HAPLN2 aggregation was also observed in the human cerebellum. These findings suggest that HAPLN2 aggregation results from age-related decline in brain homeostasis and may exacerbate the brain environment by activating microglia. This study provides new insights into the mechanisms underlying cerebellar aging and highlights the role of HAPLN2 in age-associated changes in the brain.

Imaging Methods: electron microscopy recorded image

Organisms: Mus musculus Homo sapiens

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, the authors developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical–basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical–basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, they examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. They found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

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

Organisms: Arabidopsis thaliana

Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, the authors engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, they disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. The authors applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. They achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.

Imaging Methods: recorded image spinning disk confocal microscopy single-beam laser-scanning confocal microscopy (LSCM) structured illumination microscopy (SIM) (Fbbi:,00000332) lattice SIM

Organisms: Homo sapiens Chlorocebus aethiops

Functionally mature neural circuits are shaped during postnatal development by eliminating redundant synapses formed during the perinatal period. In the cerebellum of neonatal rodents, each Purkinje cell (PC) receives synaptic inputs from multiple (more than 4) climbing fibers (CFs). During the first 3 postnatal weeks, synaptic inputs from a single CF become markedly larger and those from the other CFs are eliminated in each PC, leading to mono-innervation of each PC by a strong CF in adulthood. While molecules involved in the strengthening and elimination of CF synapses during postnatal development are being elucidated, much less is known about the molecular mechanisms underlying CF synapse formation during the early postnatal period. The authors show experimental evidence that suggests that a synapse organizer, PTPdelta, is required for early postnatal CF synapse formation and the subsequent establishment of CF to PC synaptic wiring. They showed that PTPdelta was localized at CF-PC synapses from postnatal day 0 (P0) irrespective of the expression of Aldolase C (Aldoc), a major marker of PC that distinguishes the cerebellar compartments. They found that the extension of a single strong CF along PC dendrites (CF translocation) was impaired in global PTPdelta knockout (KO) mice from P12 to P29-31 predominantly in PCs that did not express Aldoc [Aldoc (–) PCs].

Imaging Methods: recorded image confocal microscopy

Organisms: Mus musculus

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, the authors developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Imaging Methods: recorded image fluorescence microscopy confocal microscopy

Organisms: Homo sapiens