scholarly journals A robust ex vivo system to study cellular dynamics underlying mouse peri-implantation development

2021 ◽  
Author(s):  
Takafumi Ichikawa ◽  
Hui Ting Zhang ◽  
Laura Panavaite ◽  
Anna Erzberger ◽  
Dimitri Fabrèges ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Light Sheet ◽  
Cell Segmentation ◽  
Mouse Development ◽  
Cellular Dynamics ◽  
Light Sheet Microscopy ◽  
Embryonic Tissues ◽  
Mammalian Embryos ◽  
3D Architecture ◽  
Embryonic Ectoderm

Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to 6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of cases, its compatibility with light-sheet microscopy enables study of cellular dynamics through automatic cell segmentation. We find that upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D-ex vivo system thus offers an unprecedented access to peri-implantation development for in toto monitoring, measurement and spatio-temporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues.

scholarly journals Imaging the dynamic recruitment of monocytes to the blood–brain barrier and specific brain regions during Toxoplasma gondii infection

2019 ◽  
Vol 116 (49) ◽  
pp. 24796-24807 ◽  
Author(s):  
Christine A. Schneider ◽  
Dario X. Figueroa Velez ◽  
Ricardo Azevedo ◽  
Evelyn M. Hoover ◽  
Cuong J. Tran ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Blood Brain Barrier ◽  
Light Sheet ◽  
Brain Regions ◽  
Brain Barrier ◽  
Cell Segmentation ◽  
Cns Infection ◽  
Light Sheet Microscopy ◽  
Blood Brain ◽  
The Brain

Brain infection by the parasite Toxoplasma gondii in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling T. gondii. However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2+Ly6Chi) and patrolling (CX3CR1+Ly6Clo) monocytes into the blood and brain during T. gondii infection of C57BL/6J and CCR2RFP/+CX3CR1GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood–brain barrier (BBB) within 2 wk of T. gondii infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains using iDISCO+ and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of T. gondii and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of T. gondii clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., Nature 445:168–176 (2007)] indicated a consistent pattern of monocyte infiltration during T. gondii infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during T. gondii CNS infection.

scholarly journals 3D Cell Neighbour Dynamics in Growing Pseudostratified Epithelia

2021 ◽  
Author(s):  
Harold F. Gómez ◽  
Mathilde S. Dumont ◽  
Leonie Hodel ◽  
Roman Vetter ◽  
Dagmar Iber
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Cell Segmentation ◽  
Cross Sectional ◽  
Light Sheet Microscopy ◽  
Cell Organization ◽  
Epithelial Sheets ◽  
Epithelial Layers ◽  
3D Shapes ◽  
Lateral Cell

ABSTRACTDuring morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organize their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organization in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, i.e., Euler’s formula, Lewis’ law, and Aboav-Weaire’s law, at all times and across the entire tissue thickness. This arrangement minimizes the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organization in growing epithelia emerges from simple physical principles.

Advances in imaging feto-placental vasculature: new tools to elucidate the early life origins of health and disease

2020 ◽  
pp. 1-11
Author(s):  
Yutthapong Tongpob ◽  
Caitlin Wyrwoll
Keyword(s):  
Ex Vivo ◽  
Light Sheet ◽  
Experimental Models ◽  
Cardiovascular Development ◽  
Micro Computed Tomography ◽  
Placental Function ◽  
Light Sheet Microscopy ◽  
Health And Disease ◽  
Insight Into

Abstract Optimal placental function is critical for fetal development, and therefore a crucial consideration for understanding the developmental origins of health and disease (DOHaD). The structure of the fetal side of the placental vasculature is an important determinant of fetal growth and cardiovascular development. There are several imaging modalities for assessing feto-placental structure including stereology, electron microscopy, confocal microscopy, micro-computed tomography, light-sheet microscopy, ultrasonography and magnetic resonance imaging. In this review, we present current methodologies for imaging feto-placental vasculature morphology ex vivo and in vivo in human and experimental models, their advantages and limitations and how these provide insight into placental function and fetal outcomes. These imaging approaches add important perspective to our understanding of placental biology and have potential to be new tools to elucidate a deeper understanding of DOHaD.

scholarly journals Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e21303 ◽  
Author(s):  
Giovanni Sena ◽  
Zak Frentz ◽  
Kenneth D. Birnbaum ◽  
Stanislas Leibler
Keyword(s):  
Light Sheet ◽  
Cellular Dynamics ◽  
Light Sheet Microscopy

scholarly journals Open-Top Light-Sheet Microscopy Image Atlas of Prostate Core Needle Biopsies

2019 ◽  
Vol 143 (9) ◽  
pp. 1069-1075 ◽  
Author(s):  
Nicholas P. Reder ◽  
Adam K. Glaser ◽  
Erin F. McCarty ◽  
Ye Chen ◽  
Lawrence D. True ◽  
...  
Keyword(s):  
Immunohistochemical Staining ◽  
Structural Information ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Light Sheet ◽  
Fixed Tissue ◽  
Core Needle ◽  
Light Sheet Microscopy ◽  
Wide Range ◽  
Hematoxylin Eosin

Context.— Ex vivo microscopy encompasses a range of techniques to examine fresh or fixed tissue with microscopic resolution, eliminating the need to embed the tissue in paraffin or produce a glass slide. One such technique is light-sheet microscopy, which enables rapid 3D imaging. Our pathology-engineering collaboration has resulted in an open-top light-sheet (OTLS) microscope that is specifically tailored to the needs of pathology practice. Objective.— To present an image atlas of OTLS images of prostate core needle biopsies. Design.— Core needle biopsies (N = 9) were obtained from fresh radical prostatectomy specimens. Each biopsy was fixed in formalin, dehydrated in ethanol, stained with TO-PRO3 and eosin, optically cleared, and imaged using OTLS microscopy. The biopsies were then processed, paraffin embedded, and sectioned. Hematoxylin-eosin and immunohistochemical staining for cytokeratin 5 and cytokeratin 8 was performed. Results.— Benign and neoplastic histologic structures showed high fidelity between OTLS and traditional light microscopy. OTLS microscopy had no discernible effect on hematoxylin-eosin or immunohistochemical staining in this pilot study. The 3D histology information obtained using OTLS microscopy enabled new structural insights, including the observation of cribriform and well-formed gland morphologies within the same contiguous glandular structures, as well as the continuity of poorly formed glands with well-formed glands. Conclusions.— Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylin-eosin histology images, with the added benefit of useful 3D structural information. Further studies are needed to continue to document the OTLS appearance of a wide range of tissues and to better understand 3D histologic structures.

scholarly journals Segmentation of Tissues and Proliferating Cells in Light-Sheet Microscopy Images using Convolutional Neural Networks

2021 ◽  
Author(s):  
Lucas D. Lo Vercio ◽  
Rebecca M. Green ◽  
Samuel Robertson ◽  
Si Han Guo ◽  
Andreas Dauter ◽  
...  
Keyword(s):  
Neural Networks ◽  
Cell Proliferation ◽  
Convolutional Neural Networks ◽  
Light Sheet ◽  
Cell Segmentation ◽  
Segmentation Method ◽  
Tissue Segmentation ◽  
Proliferating Cells ◽  
Light Sheet Microscopy ◽  
Automated Method

AbstractBackground and ObjectiveA variety of genetic mutations are known to affect cell proliferation and apoptosis during organism development, leading to structural birth defects such as facial clefting. Yet, the mechanisms how these alterations influence the development of the face remain unclear. Cell proliferation and its relation to shape variation can be studied in high detail using Light-Sheet Microscopy (LSM) imaging across a range of developmental time points. However, the large number of LSM images captured at cellular resolution precludes manual analysis. Thus, the aim of this work was to develop and evaluate automatic methods to segment tissues and proliferating cells in these images in an accurate and efficient way.MethodsWe developed, trained, and evaluated convolutional neural networks (CNNs) for segmenting tissues, cells, and specifically proliferating cells in LSM datasets. We compared the automatically extracted tissue and cell annotations to corresponding manual segmentations for three specific applications: (i) tissue segmentation (neural ectoderm and mesenchyme) in nuclear-stained LSM images, (ii) cell segmentation in nuclear-stained LSM images, and (iii) segmentation of proliferating cells in Phospho-Histone H3 (PHH3)-stained LSM images.ResultsThe automatic CNN-based tissue segmentation method achieved a macro-average F-score of 0.84 compared to a macro-average F-score of 0.89 comparing corresponding manual segmentations from two observers. The automatic cell segmentation method in nuclear-stained LSM images achieved an F-score of 0.57, while comparing the manual segmentations resulted in an F-score of 0.39. Finally, the automatic segmentation method of proliferating cells in the PHH3-stained LSM datasets achieved an F-score of 0.56 for the automated method, while comparing the manual segmentations resulted in an F-score of 0.45.ConclusionsThe proposed automatic CNN-based framework for tissue and cell segmentation leads to results comparable to the inter-observer agreement, accelerating the LSM image analysis. The trained CNN models can also be applied for shape or morphological analysis of embryos, and more generally in other areas of cell biology.

scholarly journals CloudReg: Automatic Terabyte-Scale Cross-Modal Brain Volume Registration

2021 ◽  
Author(s):  
Vikram Chandrashekhar ◽  
Daniel J Tward ◽  
Devin Crowley ◽  
Ailey K Crow ◽  
Matthew A Wright ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Brain Magnetic Resonance Imaging ◽  
Light Sheet ◽  
Imaging Data ◽  
Micro Computed Tomography ◽  
Light Sheet Microscopy ◽  
Volume Registration ◽  
Scalable Analysis ◽  
Spatially Varying

AbstractQuantifying terabyte-scale multi-modal human and animal imaging data requires scalable analysis tools. We developed CloudReg, an open-source, automatic, terabyte-scale, cloud-based image analysis pipeline that pre-processes and registers cross-modal volumetric datasets with artifacts via spatially-varying polynomial intensity transform. CloudReg accurately registers the following datasets to their respective atlases: in vivo human and ex vivo macaque brain magnetic resonance imaging, ex vivo mouse brain micro-computed tomography, and cleared murine brain light-sheet microscopy.

scholarly journals Dual spindle formation in zygotes keeps parental genomes apart in early mammalian embryos

2017 ◽  
Author(s):  
Judith Reichmann ◽  
Bianca Nijmeijer ◽  
M. Julius Hossain ◽  
Manuel Eguren ◽  
Isabell Schneider ◽  
...  
Keyword(s):  
Genetic Material ◽  
Spindle Assembly ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Nuclear Compartments ◽  
Assembly Mechanism ◽  
Mammalian Embryos ◽  
Cell Embryo ◽  
Bipolar Spindles ◽  
Fertilized Egg

AbstractAt the beginning of mammalian life the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible to spatially combine the two genomes and then segregate them to create the two-cell embryo. Utilizing light-sheet microscopy, we showed that two bipolar spindles form in the zygote, that independently congress the maternal and paternal genomes. These two spindles aligned their poles prior to anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.One Sentence Summary: After fertilization, two spindles form around pro-nuclei in mammalian zygotes and keep the parental genomes apart during the first division.

scholarly journals 3D Cell neighbour dynamics in growing pseudostratified epithelia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Harold Fernando Gomez ◽  
Mathilde Sabine Dumond ◽  
Leonie Hodel ◽  
Roman Vetter ◽  
Dagmar Iber
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Cell Segmentation ◽  
Cross Sectional ◽  
Light Sheet Microscopy ◽  
Cell Organization ◽  
Epithelial Sheets ◽  
Epithelial Layers ◽  
3D Shapes ◽  
Lateral Cell

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organize their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organization in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, i.e., Euler's formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimizes the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organization in growing epithelia emerges from simple physical principles.

Sign in / Sign up

Export Citation Format

Share Document