scholarly journals Cell morphological motif detector for high-resolution 3D microscopy images

2018 ◽  
Author(s):  
Meghan K. Driscoll ◽  
Erik S. Welf ◽  
Kevin M. Dean ◽  
Reto Fiolka ◽  
Gaudenz Danuser
Keyword(s):  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
Automated Analysis ◽  
Light Sheet ◽  
Cell Types ◽  
Light Sheet Microscopy ◽  
Cytoskeletal Dynamics ◽  
Computational Workflow ◽  
Analytical Tools ◽  
Microscopy Images

AbstractRecent advances in light-sheet microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, the analytical tools to systematically measure and visualize the intricate relations between cell morphodynamics, intracellular signaling, and cytoskeletal dynamics have been largely missing. Here, we introduce a set of computer vision and graphics methods to dissect molecular mechanisms underlying 3D cell morphogenesis and to test whether morphogenesis itself affects intracellular signaling. We demonstrate a machine learning based generic morphological motif detector that automatically finds lamellipodia, filopodia, and blebs on various cell types. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This suggests that local morphological cues differentially organize and activate sub-cellular signaling processes. Overall, our computational workflow enables the objective, automated analysis of the 3D coupling of morphodynamics with cytoskeletal dynamics and intracellular signaling.

scholarly journals Cell death in cells overlying lateral root primordia contributes to organ growth in Arabidopsis

2018 ◽  
Author(s):  
Sacha Escamez ◽  
Benjamin Bollhöner ◽  
Hardy Hall ◽  
Domenique André ◽  
Béatrice Berthet ◽  
...  
Keyword(s):  
Cell Death ◽  
Lateral Root ◽  
Light Sheet ◽  
Time Lapse ◽  
Cell Types ◽  
Marker Genes ◽  
Lateral Root Primordia ◽  
Organ Growth ◽  
Light Sheet Microscopy ◽  
In Cells

AbstractUnlike animal development, plant organ growth is widely accepted to be determined by cell division without any contribution of cell elimination. We investigated this paradigm during Arabidopsis lateral root formation when growth of the new primordia (LRP) from pericycle-derived stem cells deep inside the root is reportedly facilitated by remodeling of the walls of overlying cells without apparent cell death. However, we observed the induction of marker genes for cell types undergoing developmental cell death in several cells overlying the growing LRP. Transmission electron microscopy, time-lapse confocal and light sheet microscopy techniques were used to establish that cell death occurred at least in a subset of endodermal LRP-overlying cells during organ emergence. Significantly, organ emergence was retarded in mutants lacking a positive cell death regulator, and restored by inducing cell death in cells overlying LRP. Hence, we conclude that in the case of LRP, cell elimination contributes to organ growth.

scholarly journals Cerebellar contributions to a brainwide network for flexible behavior

2021 ◽  
Author(s):  
Jessica L Verpeut ◽  
Silke Bergeler ◽  
Mikhail Kislin ◽  
F William Townes ◽  
Ugne Klibaite ◽  
...  
Keyword(s):  
Open Field ◽  
Body Movement ◽  
Automated Analysis ◽  
Light Sheet ◽  
Anterior Cingulate ◽  
Whole Body ◽  
Light Sheet Microscopy ◽  
Field Environment ◽  
Flexible Behavior ◽  
Crus I

The cerebellum regulates nonmotor behavior, but the routes by which it exerts its influence are not well characterized. Here we report a necessary role for posterior cerebellum in guiding flexible behavior, acting through a network of diencephalic and neocortical structures. After chemogenetic inhibition of Purkinje cells in lobule VI or crus I, high-throughput automated analysis of complex whole-body movement revealed deficiencies in adaptation across days to an open field environment. Neither perturbation affected gait, within-day open-field adaptation, or location preference. In a Y-maze task, mice could learn but were impaired in their ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic regions and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (thalamus and habenula) and crus I (hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. Taken together, these experiments reveal parts of a brainwide system for cerebellar influence to guide flexible learning.

scholarly journals Molecular Background of Leak K+ Currents: Two-Pore Domain Potassium Channels

2010 ◽  
Vol 90 (2) ◽  
pp. 559-605 ◽  
Author(s):  
Péter Enyedi ◽  
Gábor Czirják
Keyword(s):  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Detailed Examination ◽  
Resting Membrane Potential ◽  
Membrane Stretch ◽  
Physiological Processes ◽  
Pore Domain ◽  
K Currents

Two-pore domain K+ (K2P) channels give rise to leak (also called background) K+ currents. The well-known role of background K+ currents is to stabilize the negative resting membrane potential and counterbalance depolarization. However, it has become apparent in the past decade (during the detailed examination of the cloned and corresponding native K2P channel types) that this primary hyperpolarizing action is not performed passively. The K2P channels are regulated by a wide variety of voltage-independent factors. Basic physicochemical parameters (e.g., pH, temperature, membrane stretch) and also several intracellular signaling pathways substantially and specifically modulate the different members of the six K2P channel subfamilies (TWIK, TREK, TASK, TALK, THIK, and TRESK). The deep implication in diverse physiological processes, the circumscribed expression pattern of the different channels, and the interesting pharmacological profile brought the K2P channel family into the spotlight. In this review, we focus on the physiological roles of K2P channels in the most extensively investigated cell types, with special emphasis on the molecular mechanisms of channel regulation.

scholarly journals Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

2021 ◽  
Vol 17 (2) ◽  
pp. e1008780
Author(s):  
Mark R. Winter ◽  
Miri Morgulis ◽  
Tsvia Gildor ◽  
Andrew R. Cohen ◽  
Smadar Ben-Tabou de-Leon
Keyword(s):  
Sea Urchin ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Growth Factor Receptor ◽  
Light Sheet ◽  
Physical Support ◽  
Light Sheet Microscopy ◽  
Mineral Deposition ◽  
Sea Urchin Embryo ◽  
Diffusion Motion

Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn’t affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cell of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.

scholarly journals Large-scale automated identification of mouse brain cells in confocal light sheet microscopy images

2014 ◽  
Vol 30 (17) ◽  
pp. i587-i593 ◽  
Author(s):  
Paolo Frasconi ◽  
Ludovico Silvestri ◽  
Paolo Soda ◽  
Roberto Cortini ◽  
Francesco S. Pavone ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Large Scale ◽  
Light Sheet ◽  
Brain Cells ◽  
Automated Identification ◽  
Light Sheet Microscopy ◽  
Microscopy Images

scholarly journals Ultrafast phasor-based hyperspectral snapshot microscopy for biomedical imaging

2020 ◽  
Author(s):  
Per Niklas Hedde ◽  
Rachel Cinco ◽  
Leonel Malacrida ◽  
Andrés Kamaid ◽  
Enrico Gratton
Keyword(s):  
Hyperspectral Imaging ◽  
Biomedical Imaging ◽  
Cultured Cells ◽  
Light Sheet ◽  
Cell Types ◽  
Biological Tissues ◽  
Metabolic Imaging ◽  
Imaging Device ◽  
Light Sheet Microscopy ◽  
Mouse Tissues

AbstractHyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters to overcome the limitations of conventional hyperspectral imaging methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold and enabled previously elusive hyperspectral metabolic imaging of live, three-dimensional mouse tissues. As a fit-free method that does not require any a priori information, the phasor approach could also spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging broadly available to researchers and the public.

scholarly journals Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy

2020 ◽  
Author(s):  
Sobhika Agarwala ◽  
Keun-Young Kim ◽  
Sebastien Phan ◽  
Saeyeon Ju ◽  
Ye Eun Kong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Light Sheet ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Light Sheet Microscopy ◽  
Hematopoietic Stem Cell Niche ◽  
Multiple Imaging ◽  
Stem And Progenitor Cells ◽  
Rare Cells

AbstractThe blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently-labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. Our approach allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglia cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

scholarly journals Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

2020 ◽  
Author(s):  
Mark R. Winter ◽  
Miri Morgulis ◽  
Tsvia Gildor ◽  
Andrew R. Cohen ◽  
Smadar Ben-Tabou de-Leon
Keyword(s):  
Sea Urchin ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Growth Factor Receptor ◽  
Light Sheet ◽  
Vascular Endothelial ◽  
Diffusive Motion ◽  
Light Sheet Microscopy ◽  
Vesicle Dynamics ◽  
Endothelial Growth Factor

ABSTRACTBiomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of mineral-vesicle motion and the molecular mechanisms that regulate it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for the analyses of vesicle kinetics. Here we used calcein labeling and lattice light-sheet microscopy to investigate the three-dimensional (3D) vesicle dynamics in control sea urchin embryos and in Vascular Endothelial Growth Factor Receptor (VEGFR) inhibited embryos, where skeletogenesis is blocked. We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics in the different embryonic tissues. Our findings imply that calcium vesicles perform an active diffusion motion in all the cells of the embryo. This mode of diffusion is defined by the mechanical properties of the cells and the dynamic rearrangements of the cytoskeletal network. The diffusion coefficient is larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells, possibly due to the distinct mechanical properties of the two tissues. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. Under VEGFR inhibition, vesicle volume increases and vesicle speed is reduced but the vesicles continue in their diffusive motion. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and illuminate possible molecular mechanisms that control vesicle dynamics and deposition.Authors summaryBiomineralization is a widespread, fundamental process by which organisms use minerals to harden their tissues. Mineral bearing vesicles were observed in biomineralizing cells and believed to play an essential role in biomineralization, yet little is known about their three-dimensional (3D) dynamics. Here we quantify 3D-vesicle-dynamics during skeleton formation in sea urchin larvae, using lattice-light-sheet microscopy. We discover that calcium vesicles perform an active diffusive motion in both calcifying and non-calcifying cells of the embryo. The motion of the vesicles in the calcifying skeletogenic cells, is not directed toward the biomineralization compartment and has a diffusion coefficient of ~0.01μm2/sec and average speed of ~0.09μm/sec. The inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR) that blocks skeletogenesis, increases vesicle volume and decreases vesicle speed but doesn’t change the diffusion mode in the embryo cells. Our studies reveal the diffusive motion of mineral bearing vesicles and have implications on basic and translational research.

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