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1.
bioRxiv ; 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38645246

RESUMO

Cell growth and division must be coordinated to maintain a stable cell size, but how this coordination is implemented in multicellular tissues remains unclear. In unicellular eukaryotes, autonomous cell size control mechanisms couple cell growth and division with little extracellular input. However, in multicellular tissues we do not know if autonomous cell size control mechanisms operate the same way or whether cell growth and cell cycle progression are separately controlled by cell-extrinsic signals. Here, we address this question by tracking single epidermal stem cells growing in adult mice. We find that a cell-autonomous size control mechanism, dependent on the RB pathway, sets the timing of S phase entry based on the cell's current size. Cell-extrinsic variations in the cellular microenvironment affect cell growth rates but not this autonomous coupling. Our work reassesses long-standing models of cell cycle regulation within complex metazoan tissues and identifies cell-autonomous size control as a critical mechanism regulating cell divisions in vivo and thereby a major contributor to stem cell heterogeneity.

2.
Nat Methods ; 21(5): 798-803, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38509326

RESUMO

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids.


Assuntos
Organoides , Animais , Organoides/citologia , Humanos , Análise de Célula Única/métodos , Microscopia/métodos , Microscopia/instrumentação , Camundongos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/instrumentação
3.
Histochem Cell Biol ; 160(3): 223-251, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37428210

RESUMO

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself-OME-Zarr-along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain-the file format that underlies so many personal, institutional, and global data management and analysis tasks.


Assuntos
Microscopia , Software , Humanos , Apoio Comunitário
4.
bioRxiv ; 2023 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-36865282

RESUMO

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself -- OME-Zarr -- along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain -- the file format that underlies so many personal, institutional, and global data management and analysis tasks.

5.
Nat Commun ; 13(1): 4864, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982061

RESUMO

Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based "Digital Organoid Viewer" tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity.


Assuntos
Intestinos , Organoides , Processamento de Imagem Assistida por Computador
6.
Cancer Res ; 82(10): 1953-1968, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-35570706

RESUMO

Micrometastases of colorectal cancer can remain dormant for years prior to the formation of actively growing, clinically detectable lesions (i.e., colonization). A better understanding of this step in the metastatic cascade could help improve metastasis prevention and treatment. Here we analyzed liver specimens of patients with colorectal cancer and monitored real-time metastasis formation in mouse livers using intravital microscopy to reveal that micrometastatic lesions are devoid of cancer stem cells (CSC). However, lesions that grow into overt metastases demonstrated appearance of de novo CSCs through cellular plasticity at a multicellular stage. Clonal outgrowth of patient-derived colorectal cancer organoids phenocopied the cellular and transcriptomic changes observed during in vivo metastasis formation. First, formation of mature CSCs occurred at a multicellular stage and promoted growth. Conversely, failure of immature CSCs to generate more differentiated cells arrested growth, implying that cellular heterogeneity is required for continuous growth. Second, early-stage YAP activity was required for the survival of organoid-forming cells. However, subsequent attenuation of early-stage YAP activity was essential to allow for the formation of cell type heterogeneity, while persistent YAP signaling locked micro-organoids in a cellularly homogenous and growth-stalled state. Analysis of metastasis formation in mouse livers using single-cell RNA sequencing confirmed the transient presence of early-stage YAP activity, followed by emergence of CSC and non-CSC phenotypes, irrespective of the initial phenotype of the metastatic cell of origin. Thus, establishment of cellular heterogeneity after an initial YAP-controlled outgrowth phase marks the transition to continuously growing macrometastases. SIGNIFICANCE: Characterization of the cell type dynamics, composition, and transcriptome of early colorectal cancer liver metastases reveals that failure to establish cellular heterogeneity through YAP-controlled epithelial self-organization prohibits the outgrowth of micrometastases. See related commentary by LeBleu, p. 1870.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Micrometástase de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia
7.
Sci Rep ; 10(1): 1942, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029815

RESUMO

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.


Assuntos
Microscopia , Animais , Drosophila melanogaster/fisiologia , Imageamento Tridimensional/métodos , Raios Infravermelhos , Lasers , Peixe-Zebra/fisiologia
8.
Nat Methods ; 16(6): 497-500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31036959

RESUMO

To capture highly dynamic biological processes at cellular resolution is a recurring challenge in biology. Here we show that combining selective-volume illumination with simultaneous acquisition of orthogonal light fields yields three-dimensional images with high, isotropic spatial resolution and a significant reduction of reconstruction artefacts, thereby overcoming current limitations of light-field microscopy implementations. We demonstrate medaka heart and blood flow imaging at single-cell resolution and free of motion artefacts at volume rates of up to 200 Hz.


Assuntos
Coração/diagnóstico por imagem , Coração/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Análise de Célula Única/métodos , Algoritmos , Animais , Animais Geneticamente Modificados , Artefatos , Velocidade do Fluxo Sanguíneo , Humanos , Imageamento Tridimensional/métodos , Oryzias
9.
Semin Cell Dev Biol ; 55: 148-55, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27288888

RESUMO

Tackling modern cell and developmental biology questions requires fast 3D imaging with sub-cellular resolution over extended periods of time. Fluorescence microscopy has emerged as a powerful tool to image biological samples with high spatial and temporal resolution with molecular specificity. In particular, the highly efficient illumination and detection scheme of light-sheet fluorescence microscopy is starting to revolutionize the way we can monitor cellular and developmental processes in vivo. Here we summarize the state-of-the art of light-sheet imaging with a focus on mammalian development - from instrumentation, mounting and sample handling to data processing.


Assuntos
Mamíferos/embriologia , Microscopia de Fluorescência/métodos , Envelhecimento/fisiologia , Animais , Desenvolvimento Embrionário , Imageamento Tridimensional
10.
Proc Natl Acad Sci U S A ; 113(13): 3442-6, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26984498

RESUMO

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.

11.
Nat Methods ; 13(2): 139-42, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26657559

RESUMO

Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.


Assuntos
Blastocisto/citologia , Microscopia/instrumentação , Microscopia/métodos , Animais , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Camundongos , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos
12.
Nat Commun ; 6: 8881, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26602977

RESUMO

Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage.

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