RESUMO
Bioimage data are generated in diverse research fields throughout the life and biomedical sciences. Its potential for advancing scientific progress via modern, data-driven discovery approaches reaches beyond disciplinary borders. To fully exploit this potential, it is necessary to make bioimaging data, in general, multidimensional microscopy images and image series, FAIR, that is, findable, accessible, interoperable and reusable. These FAIR principles for research data management are now widely accepted in the scientific community and have been adopted by funding agencies, policymakers and publishers. To remain competitive and at the forefront of research, implementing the FAIR principles into daily routines is an essential but challenging task for researchers and research infrastructures. Imaging core facilities, well-established providers of access to imaging equipment and expertise, are in an excellent position to lead this transformation in bioimaging research data management. They are positioned at the intersection of research groups, IT infrastructure providers, the institution´s administration, and microscope vendors. In the frame of German BioImaging - Society for Microscopy and Image Analysis (GerBI-GMB), cross-institutional working groups and third-party funded projects were initiated in recent years to advance the bioimaging community's capability and capacity for FAIR bioimage data management. Here, we provide an imaging-core-facility-centric perspective outlining the experience and current strategies in Germany to facilitate the practical adoption of the FAIR principles closely aligned with the international bioimaging community. We highlight which tools and services are ready to be implemented and what the future directions for FAIR bioimage data have to offer.
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Microscopia , Pesquisa Biomédica/métodos , Gerenciamento de Dados/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodosRESUMO
The discovery of the amphibian gastrula organizer and its publication by Hans Spemann and Hilde Mangold in 1924 is a foundation of experimental embryology, and has shaped our understanding of embryonic induction and pattern formation in vertebrates until today. The original publication is a piece of scientific art, characterized by the meticulous hand drawings by Hilde Mangold, as well as the text that develops mechanistic concepts of modern embryology. While historic microphotographs of specimens got lost, the original microscope slides and Hilde Mangold's laboratory notebook have been secured in embryological collections until today. Here, we make the original data of the six embryonic specimens reported in 1924, as well as the laboratory notebook, available in an accessible digital format. Together, these data shed light on the scientific process that led to the discovery, and should help to make the experiments on the most important signalling center in early vertebrate development transparent for generations of embryologists to come.
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Gástrula , Animais , Microscopia/métodos , Organizadores Embrionários , História do Século XX , Embriologia/história , Embriologia/métodosRESUMO
Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.
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Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.
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Lista de Checagem , Editoração , Reprodutibilidade dos Testes , Processamento de Imagem Assistida por Computador , MicroscopiaRESUMO
Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by â¢OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.
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Dano ao DNA , Elétrons , Dímeros de Pirimidina , Reparo do DNA , LasersRESUMO
Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.
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Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.
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Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Reprodutibilidade dos Testes , SoftwareAssuntos
Metadados , Microscopia/instrumentação , Microscopia/métodos , Microscopia/normas , Animais , Pesquisa Biomédica/organização & administração , Calibragem , Coleta de Dados , Mineração de Dados/normas , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Sociedades Científicas , Software , Integração de Sistemas , Interface Usuário-ComputadorRESUMO
For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
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Metadados , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Aplicativos Móveis , Linguagens de Programação , Software , Animais , Linhagem Celular , Biologia Computacional/métodos , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Reconhecimento Automatizado de Padrão , Controle de Qualidade , Reprodutibilidade dos Testes , Interface Usuário-Computador , Fluxo de TrabalhoRESUMO
The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.
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Membrana Celular/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Cinesinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Corpos Basais/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Citoesqueleto/metabolismo , Cães , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/metabolismo , Transdução de SinaisRESUMO
Glycogen storage disease subtypes I and III (GSD I and GSD III) are monogenic inherited disorders of metabolism that disrupt glycogen metabolism. Unavailability of glucose in GSD I and induction of gluconeogenesis in GSD III modify energy sources and possibly, mitochondrial function. Abnormal mitochondrial structure and function were described in mice with GSD Ia, yet significantly less research is available in human cells and ketotic forms of the disease. We hypothesized that impaired glycogen storage results in distinct metabolic phenotypes in the extra- and intracellular compartments that may contribute to pathogenesis. Herein, we examined mitochondrial organization in live cells by spinning-disk confocal microscopy and profiled extra- and intracellular metabolites by targeted LC-MS/MS in cultured fibroblasts from healthy controls and from patients with GSD Ia, GSD Ib, and GSD III. Results from live imaging revealed that mitochondrial content and network morphology of GSD cells are comparable to that of healthy controls. Likewise, healthy controls and GSD cells exhibited comparable basal oxygen consumption rates. Targeted metabolomics followed by principal component analysis (PCA) and hierarchical clustering (HC) uncovered metabolically distinct poises of healthy controls and GSD subtypes. Assessment of individual metabolites recapitulated dysfunctional energy production (glycolysis, Krebs cycle, succinate), reduced creatinine export in GSD Ia and GSD III, and reduced antioxidant defense of the cysteine and glutathione systems. Our study serves as proof-of-concept that extra- and intracellular metabolite profiles distinguish glycogen storage disease subtypes from healthy controls. We posit that metabolite profiles provide hints to disease mechanisms as well as to nutritional and pharmacological elements that may optimize current treatment strategies.
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Fibroblastos/patologia , Doença de Depósito de Glicogênio/classificação , Metaboloma , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromatografia Líquida , Ciclo do Ácido Cítrico , Fibroblastos/metabolismo , Doença de Depósito de Glicogênio/metabolismo , Doença de Depósito de Glicogênio/patologia , Glicólise , Humanos , Lactente , Masculino , Espectrometria de Massas em TandemRESUMO
Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.
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Betacoronavirus/patogenicidade , Pesquisa Biomédica/organização & administração , Infecções por Coronavirus/prevenção & controle , Controle de Infecções , Laboratórios/organização & administração , Microscopia , Saúde Ocupacional , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , COVID-19 , Comportamento Cooperativo , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Descontaminação , Contaminação de Equipamentos/prevenção & controle , Alemanha , Humanos , Exposição Ocupacional/prevenção & controle , Equipamento de Proteção Individual , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Fatores de Proteção , Pesquisadores/organização & administração , Medição de Risco , Fatores de Risco , SARS-CoV-2 , Fluxo de TrabalhoRESUMO
PC12 cells are a popular model system to study changes driving and accompanying neuronal differentiation. While attention has been paid to changes in transcriptional regulation and protein signaling, much less is known about the changes in organization that accompany PC12 differentiation. Fluorescence microscopy can provide extensive information about these changes, although it is difficult to continuously observe changes over many days of differentiation. We describe a generative model of differentiation-associated changes in cell and nuclear shape and their relationship to mitochondrial distribution constructed from images of different cells at discrete time points. We show that the model accurately represents complex cell and nuclear shapes and learn a regression model that relates cell and nuclear shape to mitochondrial distribution; the predictive accuracy of the model increases during differentiation. Most importantly, we propose a method, based on cell matching and interpolation, to produce realistic simulations of the dynamics of cell differentiation from only static images. We also found that the distribution of cell shapes is hollow: most shapes are very different from the average shape. Finally, we show how the method can be used to model nuclear shape changes of human-induced pluripotent stem cells resulting from drug treatments.
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Diferenciação Celular , Imageamento Tridimensional , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Animais , Forma do Núcleo Celular , Forma Celular , Tamanho Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Cinética , Mitocôndrias/metabolismo , Fator de Crescimento Neural/farmacologia , Células PC12 , Probabilidade , RatosRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC1 and PC2 localize to the primary cilium and form a protein complex, which is thought to regulate signaling events. PKD1 mutations are associated with a stronger phenotype than PKD2, suggesting the existence of PC1 specific functions in renal tubular cells. However, the evidence for diverging molecular functions is scant. The bending of cilia by fluid flow induces a reduction in cell size through a mechanism that involves the kinase LKB1 but not PC2. Here, using different in vitro approaches, we show that contrary to PC2, PC1 regulates cell size under flow and thus phenocopies the loss of cilia. PC1 is required to couple mechanical deflection of cilia to mTOR in tubular cells. This study pinpoints divergent functions of the polycystins in renal tubular cells that may be relevant to disease severity in ADPKD.
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Tamanho Celular/efeitos dos fármacos , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/fisiologia , Animais , Fenômenos Biomecânicos , Células Cultivadas , Cílios/metabolismo , Humanos , Túbulos Renais/citologia , Mutação , Serina-Treonina Quinases TOR , Canais de Cátion TRPP/genéticaRESUMO
Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments.
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Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.