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Original image data of the manuscript by Itakura et al.

Imaging Methods: recorded image

Organisms: Drosophila melanogaster

Loss of pluripotency is an essential step in postimplantation development that facilitates the emergence of somatic cell identities essential for gastrulation. Before implantation, pluripotent cell identity is governed by a gene regulatory network that includes the key transcription factors SOX2 and NANOG. However, it is unclear how the pluripotency gene regulatory network is dissolved to enable lineage restriction. Here we show that SOX2 is required for postimplantation pluripotent identity and cells that lose SOX2 expression in the posterior epiblast are no longer pluripotent. Using in vitro and in vivo analyses we demonstrate anticorrelated expression of Nanog and Sox2 preceding gastrulation, culminating in an early disappearance of pluripotent identity from posterior NANOGhigh/SOX2low epiblast. Surprisingly, Sox2 expression is repressed by NANOG and embryos with post-implantation deletion of Nanog maintain posterior SOX2 expression. Our results demonstrate that the distinctive features of post-implantation pluripotency are underpinned by altered functionality of pluripotency transcription factors, ensuring correct spatio-temporal loss of embryonic pluripotency.

Imaging Methods: confocal microscopy

Organisms: Mus musculus

Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes are physically limited by objective-to-specimen distance, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed a photochemical strategy for spatially precise sectioning of specimens. By combining serial photochemical sectioning with lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelin sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory-bulb-wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach.

Imaging Methods: fluorescent label

Organisms: Mus musculus

The honeybee gut microbiota plays a key role in shaping host health and susceptibility to disease. Yet, the nutrient environment it experiences within the gut remains poorly characterized. In particular, little is known about the spatial distribution of nutrients across this community, as resolving such fine gradients in vivo has been technically challenging. Here, we engineer the native honeybee symbiont Snodgrassella alvi as a living biosensor to quantify the bioavailability of the dietary sugar arabinose within the gut. By expanding the genetic toolkit for S. alvi through chromosomal integration of high-burden genes and a suite of low-strength promoters, we achieve stable multi-gene expression without compromising host colonization. The resulting biosensor generates a specific, dose-dependent response to arabinose in situ, enabling visualization of sugar gradients across gut-associated bacterial biofilms at micron-scale resolution. Using this system, we show that diet-derived arabinose distribution is highly heterogeneous and can be influenced by the metabolic activity of co-colonizing Gilliamella species. These findings highlight how diet composition and microbial specialization generate fine-scale microenvironments within the gut. More broadly, this work further establishes S. alvi as a genetically tractable platform for in situ probing of microbial metabolism and nutrient distribution.

Imaging Methods: confocal microscopy

Organisms: Apis mellifera

All imaging regards the study evaluating the role of GBP1 in phagolysosomal response of macrophages to mycobacteria. Folders correspond to each specific figure.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens

RTX toxins (Repeat in ToXins) are pore-forming toxins secreted by gram-negative bacteria. They are known for their ability to disrupt host cell membranes, among which various human cells. The acylation of specific lysine residues in these toxins is crucial for their hemolytic activity, but the precise mechanisms underlying this enhancement remain unclear. By comparing the lytic activities of acylated MbxA and its non-acylated form, we explored the role of acylation in the pore-forming behaviour of this RTX toxin. Our findings demonstrate that acylation specific interactions of MbxA with cholesterol promote membrane disruption, both in vitro and in living cells. More specifically, acylation is not necessary for initial membrane binding, but markedly enhances pore formation. Overall, our results provide detailed insights into the molecular determinants that regulate MbxA toxin activity. We highlight a complex interplay between lipid composition (sterols), acylation, and membrane disruption, thereby advancing our general understanding of RTX toxin pathogenesis.

Organisms: Homo sapiens

Video files (.avi) of 10-20 worms moving in a 2D arena without any stimulus. Each video is an independent replicate of a strain that's either wild-type or mutant for genes in the hedgehog pathway.

Imaging Methods: macroscopy

Organisms: Caenorhabditis elegans

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications, yet photophysical enhancements achieved with NIR and SWIR chromophores observed in solution often fail to translate to complex biological environments. Fluorous-soluble fluorophores, fluorofluorophores, face additional challenges, exhibiting poor brightness and photostability when dissolved in perfluorocarbons (PFCs) due to unfavorable interactions with the fluorous phase. Here, we report counterion exchange as a strategy to enhance the photophysical properties of two heptamethine cyanine fluorofluorophore for NIR and SWIR imaging. Exchanging the small chloride counterion with a large, fluorinated aryl borate counterions significantly improved the brightness (10-fold) and photostability (57-fold) in PFCs. These enhancements were successfully translated across multiple biological systems from macrophage cells to NIR imaging zebrafish retinal tissue and finally to SWIR imaging in mice. These results demonstrate that strategic counterion modification provides a straightforward approach to optimize fluorofluorophores, with solution-phase improvements that translate to in vivo NIR and SWIR imaging.

Organisms: Mus musculus

This repository includes a single slice of human thalamus tissue, stained with various neuronal, vascular, and glial markers. 9 ROIs were imaged over the medio-lateral axis of the thalamus. Refer to included .ppt for details on the locations of each ROI and the included .xlsx for details on the performed stainings.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Original image data of the manuscript by Inagaki and Hayashi

Imaging Methods: recorded image

Organisms: Drosophila melanogaster

Understanding protein distribution patterns across tissue architecture is crucial for deciphering organ function in health and disease. Here, we show the application of single-cell Deep Visual Proteomics to perform spatially-resolved proteome analysis of individual cells in native liver tissue. We built a robust framework comprising strategic cell selection and continuous protein gradient mapping, allowing the investigation of larger clinical cohorts. We generated a comprehensive spatial map of the human hepatic proteome by analyzing hundreds of isolated hepatocytes from 18 individuals. Among the 2,500 proteins identified per cell, about half exhibited zonated expression patterns. Cross-species comparison with male mice revealed conserved metabolic functions and human-specific features of liver zonation. Analysis of samples with disrupted liver architecture demonstrated widespread loss of protein zonation, with pericentral proteins being particularly susceptible. Our study provides a comprehensive and open-access resource of human liver organization while establishing a broadly applicable framework for spatial proteomics analyses along tissue gradients.

Imaging Methods: spinning disk confocal microscopy

Organisms: Homo sapiens

Ewing sarcoma (EWS), a rare pediatric bone tumor, poses unique therapeutic challenges due to its distinct microenvironment and limited molecular understanding. To gain a comprehensive molecular and functional view of the tumors in their microenvironment, we performed a deep mass spectrometry-based proteomic analysis of 168 tumors from 72 patients from primary, relapsed, and metastatic tumors. Analysis of more than 10,000 proteins across patients revealed novel insights into cancer prognosis, chemo-resistance, and progression. We found ferroptosis inhibition as a potential mediator of EWS chemo-resistance and identified novel subclasses of EWS that link the tumor immune landscape with DNA damage repair, ubiquitin-related proteins, and patient prognosis. Validation by multiplexed immunofluorescence imaging confirmed the association between patient prognosis and tumor neutrophils, and association of macrophages and T-cells with better prognosis. These results suggest that immuno-oncological treatments might be efficacious for a subset of patients. Altogether, this comprehensive investigation provides valuable insights into the intricate biology of EWS, paving the way for developing novel therapeutic strategies.

Imaging Methods: fluorescence microscopy light microscopy

Organisms: Homo sapiens