RESUMO
In life sciences, tracking objects from movies enables researchers to quantify the behavior of single particles, organelles, bacteria, cells, and even whole animals. While numerous tools now allow automated tracking from video, a significant challenge persists in compiling, analyzing, and exploring the large datasets generated by these approaches. Here, we introduce CellTracksColab, a platform tailored to simplify the exploration and analysis of cell tracking data. CellTracksColab facilitates the compiling and analysis of results across multiple fields of view, conditions, and repeats, ensuring a holistic dataset overview. CellTracksColab also harnesses the power of high-dimensional data reduction and clustering, enabling researchers to identify distinct behavioral patterns and trends without bias. Finally, CellTracksColab also includes specialized analysis modules enabling spatial analyses (clustering, proximity to specific regions of interest). We demonstrate CellTracksColab capabilities with 3 use cases, including T cells and cancer cell migration, as well as filopodia dynamics. CellTracksColab is available for the broader scientific community at https://github.com/CellMigrationLab/CellTracksColab.
Assuntos
Movimento Celular , Rastreamento de Células , Software , Rastreamento de Células/métodos , Humanos , Animais , Processamento de Imagem Assistida por Computador/métodos , Pseudópodes/fisiologia , Linfócitos T , CamundongosRESUMO
In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.
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Pesquisadores , Humanos , Mobilidade Ocupacional , Pesquisa Biomédica/métodos , Escolha da ProfissãoRESUMO
Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results - particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed - AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.
Assuntos
Inteligência Artificial , Software , Microscopia de FluorescênciaRESUMO
Live imaging is a powerful tool, enabling scientists to observe living organisms in real time. In particular, when combined with fluorescence microscopy, live imaging allows the monitoring of cellular components with high sensitivity and specificity. Yet, due to critical challenges (i.e., drift, phototoxicity, dataset size), implementing live imaging and analyzing the resulting datasets is rarely straightforward. Over the past years, the development of bioimage analysis tools, including deep learning, is changing how we perform live imaging. Here we briefly cover important computational methods aiding live imaging and carrying out key tasks such as drift correction, denoising, super-resolution imaging, artificial labeling, tracking, and time series analysis. We also cover recent advances in self-driving microscopy.
Assuntos
Aprendizado Profundo , Microscopia de Fluorescência/métodosRESUMO
Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.
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Imageamento Tridimensional , Microscopia , Imageamento Tridimensional/métodos , Microscopia de VídeoRESUMO
TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.
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Algoritmos , Software , Processamento de Imagem Assistida por Computador/métodosRESUMO
For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Modelos Animais , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA-Seq , Análise Serial de Tecidos , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
Nestin, an intermediate filament protein and marker of undifferentiated cells, is expressed in several cancers. Nestin is important for neuronal survival and is a regulator of myogenesis but its function in malignancy is ambiguous. We show that nestin downregulation leads to a redistribution of phosphorylated focal adhesion kinase (pFAK, also known as PTK2) to focal adhesions and alterations in focal adhesion turnover. Nestin downregulation also leads to an increase in the protein levels of integrin α5ß1 at the cell membrane, activation of integrin ß1 and an increase in integrin clustering. These effects have striking consequences for cell invasion, as nestin downregulation leads to a significant increase in pFAK- and integrin-dependent matrix degradation and cell invasion. Our results indicate that nestin regulates the localisation and functions of FAK and integrin. Because nestin has been shown to be prevalent in a number of specific cancers, our observations have broad ramifications for the roles of nestin in malignant transformation.