scholarly journals Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole embryo imaging

Development ◽  
2021 ◽  
Author(s):  
Corey Bunce ◽  
Jennifer McKey ◽  
Blanche Capel
Keyword(s):  
Time Course ◽  
Body Position ◽  
Gonad Development ◽  
Spatial Relationship ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Context Analysis ◽  
Native Form ◽  
Light Sheet Microscopy ◽  
Mesonephric Duct

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position, and spatial relationship with the mesonephric ducts, kidneys, and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements and delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2020 ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
Early Production ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Minor Role ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals Lineage recording reveals dynamics of cerebral organoid regionalization

2020 ◽  
Author(s):  
Zhisong He ◽  
Tobias Gerber ◽  
Ashley Maynard ◽  
Akanksha Jain ◽  
Rebecca Petri ◽  
...  
Keyword(s):  
Time Course ◽  
Light Sheet ◽  
Brain Regions ◽  
Patient Specific ◽  
Light Sheet Microscopy ◽  
Brain Organoid ◽  
Induced Pluripotent ◽  
Models Of Disease ◽  
Fate Restriction ◽  
Lineage Trees

Diverse regions develop within cerebral organoids generated from human induced pluripotent stem cells (iPSCs), however it has been a challenge to understand the lineage dynamics associated with brain regionalization. Here we establish an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during cerebral organoid development. We infer fate-mapped whole organoid phylogenies over a scarring time course, and reconstruct progenitor-neuron lineage trees within microdissected cerebral organoid regions. We observe increased fate restriction over time, and find that iPSC clones used to initiate organoids tend to accumulate in distinct brain regions. We use lineage-coupled spatial transcriptomics to resolve lineage locations as well as confirm clonal enrichment in distinctly patterned brain regions. Using long term 4-D light sheet microscopy to temporally track nuclei in developing cerebral organoids, we link brain region clone enrichment to positions in the neuroectoderm, followed by local proliferation with limited migration during neuroepithelial formation. Our data sheds light on how lineages are established during brain organoid regionalization, and our techniques can be adapted in any iPSC-derived cell culture system to dissect lineage alterations during perturbation or in patient-specific models of disease.

scholarly journals Ex vivolive cell tracking in kidney organoids using light sheet fluorescence microscopy

2017 ◽  
Author(s):  
Marie Held ◽  
Ilaria Santeramo ◽  
Bettina Wilm ◽  
Patricia Murray ◽  
Raphaël Lévy
Keyword(s):  
Fluorescence Microscopy ◽  
Time Course ◽  
Cell Tracking ◽  
Organic Anion ◽  
Light Sheet ◽  
Differentiation Potential ◽  
Light Sheet Microscopy ◽  
Kidney Organoids

AbstractScreening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregationin vitroassays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect developmentin vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.

scholarly journals Achromatic terahertz Airy beam generation with dielectric metasurfaces

Nanophotonics ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Qingqing Cheng ◽  
Juncheng Wang ◽  
Ling Ma ◽  
Zhixiong Shen ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Biomedical Imaging ◽  
Frequency Dispersion ◽  
Light Sheet ◽  
Photonic Devices ◽  
Self Healing ◽  
Beam Focusing ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Potential Applications ◽  
Airy Beams

AbstractAiry beams exhibit intriguing properties such as nonspreading, self-bending, and self-healing and have attracted considerable recent interest because of their many potential applications in photonics, such as to beam focusing, light-sheet microscopy, and biomedical imaging. However, previous approaches to generate Airy beams using photonic structures have suffered from severe chromatic problems arising from strong frequency dispersion of the scatterers. Here, we design and fabricate a metasurface composed of silicon posts for the frequency range 0.4–0.8 THz in transmission mode, and we experimentally demonstrate achromatic Airy beams exhibiting autofocusing properties. We further show numerically that a generated achromatic Airy-beam-based metalens exhibits self-healing properties that are immune to scattering by particles and that it also possesses a larger depth of focus than a traditional metalens. Our results pave the way to the realization of flat photonic devices for applications to noninvasive biomedical imaging and light-sheet microscopy, and we provide a numerical demonstration of a device protocol.

scholarly journals Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

2021 ◽  
Vol 93 (8) ◽  
pp. 4092-4099
Author(s):  
Bing Li ◽  
Aleks Ponjavic ◽  
Wei-Hsin Chen ◽  
Lee Hopkins ◽  
Craig Hughes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Effect of captopril on post-infarction remodelling visualized by light sheet microscopy and echocardiography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Urmas Roostalu ◽  
Louise Thisted ◽  
Jacob Lercke Skytte ◽  
Casper Gravesen Salinas ◽  
Philip Juhl Pedersen ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Light Sheet ◽  
Automated Image Analysis ◽  
Border Zone ◽  
Vascular Density ◽  
Mouse Heart ◽  
Imaging Method ◽  
Light Sheet Microscopy ◽  
Post Surgery ◽  
Captopril Treatment

AbstractAngiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research.

The Light-Sheet Microscopy

2021 ◽  
Author(s):  
Rolf Theodor Borlinghaus
Keyword(s):  
Light Sheet ◽  
Light Sheet Microscopy
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