Cellular & Molecular Imaging

The files here are the source data for 4 main figures of Ma et al., Regional Ca2+ Wave, Hormone Responses upon Laser Ablation in Arabidopsis.

We here report an aberration-corrected 25-plane camera array Multifocus microscope (M25) for high-speed, high-resolution wide-field optical microscopy in three spatial dimensions (3D). We demonstrate live imaging of 25-plane 3D volumes of up to 180x180x50um at >100 volumes per second. 3D data is recorded simultaneously by an array of 25 small, sensitive, synchronized machine-vision cameras. M25 employs aberration-corrected Multifocus microscopy—an optical method where diffractive Fourier optics are used for multiplexing and refocusing light—with a simplified design for chromatic dispersion correction where a corrective diffractive gratings is placed on each camera in the array. This elegant architecture for chromatic correction will be applicable in a broad range of diffractive imaging applications. M25 is a powerful optical tool for high-speed 3D microscopy in that it allows both non-invasive, label-free bright-field and highly sensitive fluorescence microscopy. We showcase M25 capabilities in 3D particle tracking, bright-field, and fluorescence imaging in D. melanogaster, and locomotion and neural activity studies in C. elegans.

Our recent research, published in PNAS in 2024, highlights the significant role of the endoplasmic reticulum (ER) in modulating activity-driven postsynaptic signaling events. This signaling is associated with long-term potentiation of dendritic spine size, known as structural long-term potentiation (sLTP), which underpins the functional strengthening of glutamatergic synaptic transmission. Our findings demonstrate that high-frequency optical glutamate uncaging (HFGU) induces long-lasting sLTP in most ER-containing (ER+) spines, resulting in a sustained increase in spine ER content. This process is driven by a signaling cascade initiated by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter of which are activated by stromal interaction molecule 1 (STIM1) in response to Ca2+ release from the ER. In contrast, HFGU stimulation of ER-lacking (ER−) spines results in only transient sLTP and diminished Ca2+ signals, notably lacking contributions from Orai1 and the ER. Supporting the notion that spine ER governs structural metaplasticity, delivering a well-timed second HFGU stimulus to ER− spines recruited ER and produced persistent sLTP. However, repeated stimulation of previously potentiated ER+ spines failed to further enhance Ca2+ signaling, spine size, or spine ER content, potentially serving as a protective mechanism against runaway potentiation. Through our exploration of the distinct inhibitory effects of Orai1 inhibitors, AnCoA4 and Synta66, we found that the physical interaction between STIM1 and Orai1 at ER-plasma membrane junctions, rather than the subsequent Ca2+ entry, is essential for the persistence of sLTP.

SUMMARY. Here, we provide the image datasets supporting the development of CellCycleNet, a cell cycle stage classifier tool. We imaged thousands of fixed interphase mouse fibroblast cells containing a Fluorescent Ubiquitination-based Cell Cycle Indicator-2a (Fucci-2a) transgene, stained with DAPI, from two common fluorescent microscope modalities: widefield epifluorescence and spinning-disk confocal microscopy. ABSTRACT. The cell cycle governs proliferation of all eukaryotic cells. Profiling cell cycle dynamics is therefore central to basic and biomedical research. However, current approaches to cell cycle profiling involve complex interventions that may confound experimental interpretation. We developed CellCycleNet, a machine learning (ML) workflow, to simplify cell cycle staging from fluorescent microscopy data with minimal experimenter intervention and cost. CellCycleNet accurately predicts cell cycle phase using only a fluorescent nuclear stain (DAPI) in fixed interphase cells. Using the Fucci2a cell cycle reporter system as ground truth, we collected two benchmarking image datasets and trained 2D and 3D ML models—of support vector machine (SVM) and deep neural network architecture—to classify nuclei in the G1 or S/G2 phases. Our results show that 3D CellCycleNet outperforms SVM models on each dataset. When trained on two image datasets simultaneously, CellCycleNet achieves the highest classification accuracy (AUROC of 0.94-0.95). Overall, we found that using 3D features, rather than 2D features alone, significantly improves classification performance for all model architectures. We released our image data, models, and software as a community resource.

Fluorescent images of nuclear RelA and cRel dynamics upon CD40 and BCR activation. This is the data set behind figures 3L, 3M, 4F, and 4H.

The Darwin Tree of Life project has the goal to sequence the genomes of 70,000 species of eukaryotic organisms in Britain and Ireland. This is a collection of photographs of the samples included in the study, provided by the National History Museum (NHM).

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labelling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labelled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labelling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed in-creased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labelling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labelled peptides was not increased suggesting a reduced spatial overlap.

TrypTag genome-wide protein localisation project data. Widefield epifluorescence microscope images of protein subcellular localisation in the unicellular eukaryotic pathogen Trypanosoma brucei by endogenous tagging with mNeonGreen (mNG). This deposition includes the localisations, ontology and microscopy data used to build the TrypTag database. Data can also be browsed at TrypTag.org. If you use this data resource please cite Billington et al. 2023 Nature Microbiology (doi:10.1038/s41564-022-01295-6). We recommend including this citation in the results or methods if TrypTag was used as part of a discovery process. If directly using TrypTag images, please also indicate in the figure legend or similar which images are from TrypTag. If carrying out a large-scale data analysis, please also cite this BioStudies deposition. Data can be mined via the cellular localization imaging or cellular component GO term searches at the genome database TriTrypDB.org (part of VEuPathDB). If you do, please also cite the genome database. You may also find the following papers informative: Dean et al. 2016 Trends in Parasitology (doi:10.1016/j.pt.2016.10.009), which describes the original project aims and workflow. Halliday et al. 2019 Molecular and Biochemical Parasitology (doi:10.1016/j.molbiopara.2018.12.003), which describes the localisation ontology with example images and comparison to Leishmania.

Sagittal section images showing cross-section of the stomodeal valve at point of greatest opening of 15 sand flies (Lutzomyia longipalpis) at various stages of infection with Leishmania mexicana. The images show the effects of an infection and of a second blood meal on the morphology of the foregut and midgut (especially thoracic midgut) compared to uninfected, control flies. Three flies were imaged by micro-CT scan in each of five groups: T7CON flies were 7 days after a non-infected blood meal. T9INF and T12INF flies were 9 and 12 days, respectively, after an infected blood meal. T6INF+ and T9INF+ flies were 6 and 9 days, respectively after an infected bloodmeal and 1 and 4 days, respectively, after a second, non-infected blood meal. The data are discussed in a paper published on 27th August 2021 in PLoS NTDs, which is accessible through the following link: https://doi.org/10.1371/journal.pntd.0009682