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 Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis

2020 ◽  
Vol 30 (3) ◽  
pp. 455-464.e7 ◽  
Author(s):  
Sacha Escamez ◽  
Domenique André ◽  
Bernadette Sztojka ◽  
Benjamin Bollhöner ◽  
Hardy Hall ◽  
...  
Keyword(s):  
Cell Death ◽  
Lateral Root ◽  
Root Primordia ◽  
Lateral Root Primordia ◽  
Organ Growth ◽  
In Cells

scholarly journals Cell Death in Cells Overlying Lateral Root Primordia Contributes to Organ Growth in Arabidopsis

2019 ◽  
Author(s):  
Sacha Escamez ◽  
Domenique André ◽  
Bernadette Sztojka ◽  
Benjamin Bollhöner ◽  
Hardy Hall ◽  
...  
Keyword(s):  
Cell Death ◽  
Lateral Root ◽  
Root Primordia ◽  
Lateral Root Primordia ◽  
Organ Growth ◽  
In Cells

scholarly journals Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lillian K Fritz-Laylin ◽  
Megan Riel-Mehan ◽  
Bi-Chang Chen ◽  
Samuel J Lord ◽  
Thomas D Goddard ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Time Lapse ◽  
Three Dimensions ◽  
Cortical Actin ◽  
Linear Arrangement ◽  
Collagen Matrices ◽  
Flat Surfaces ◽  
Actin Networks ◽  
Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

scholarly journals Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sota Takanezawa ◽  
Takashi Saitou ◽  
Takeshi Imamura
Keyword(s):  
High Speed ◽  
Bessel Beam ◽  
Light Sheet ◽  
Time Lapse ◽  
Whole Body ◽  
Wide Field ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Two Photon Excitation

AbstractTwo-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600–1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2–3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

scholarly journals An orthotopic glioblastoma animal model suitable for high-throughput screenings

2018 ◽  
Vol 20 (11) ◽  
pp. 1475-1484 ◽  
Author(s):  
Linda Pudelko ◽  
Steven Edwards ◽  
Mirela Balan ◽  
Daniel Nyqvist ◽  
Jonathan Al-Saadi ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Large Scale ◽  
Light Sheet ◽  
Time Lapse ◽  
Zebrafish Model ◽  
Light Sheet Microscopy ◽  
Disease Biology ◽  
Vertebrate Model

Abstract Background Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens. Methods We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy. Results We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease. Conclusion We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.

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 Crossbill: an open access single objective light-sheet microscopy platform

2021 ◽  
Author(s):  
Manish Kumar ◽  
Sandeep Kishore ◽  
David McLean ◽  
Yevgenia Kozorovitskiy
Keyword(s):  
Open Access ◽  
Light Sheet ◽  
Time Lapse ◽  
Volumetric Imaging ◽  
Open Hardware ◽  
Light Sheet Microscopy ◽  
Multiple Alternative ◽  
Optical Configuration ◽  
Time Lapse Imaging ◽  
Single Objective

We present an open access scanned oblique plane microscopy platform Crossbill. It combines a new optical configuration, open hardware assembly, a systematic alignment protocol, and dedicated control software to provide a compact, versatile, high resolution single objective light-sheet microscopy platform. The demonstrated configuration yields the most affordable sub-micron resolution oblique plane microscopy system to date. We add galvanometer enabled tilt-invariant lateral scan for multi-plane, multi-Hz volumetric imaging capability. A precision translation stage extends stitched field of view to centimeter scale. The accompanying open software is optimized for Crossbill and can be easily extended to include alternative configurations. Using Crossbill, we demonstrate large volume structural fluorescence imaging with sub-micron lateral resolution in zebrafish and mouse brain sections. Crossbill is also capable of multiplane functional imaging, and time-lapse imaging. We suggest multiple alternative configurations to extend Crossbill to diverse microscopy applications.

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 Same same, but different: growth responses of primary and lateral roots

2020 ◽  
Vol 71 (8) ◽  
pp. 2397-2411 ◽  
Author(s):  
Sascha Waidmann ◽  
Elizabeth Sarkel ◽  
Jürgen Kleine-Vehn
Keyword(s):  
Root System ◽  
Lateral Root ◽  
Growth Regulation ◽  
Lateral Roots ◽  
Primary Root ◽  
Spatial Arrangement ◽  
Root System Architecture ◽  
Growth Responses ◽  
Lateral Root Primordia ◽  
Organ Growth

Abstract The root system architecture describes the shape and spatial arrangement of roots within the soil. Its spatial distribution depends on growth and branching rates as well as directional organ growth. The embryonic primary root gives rise to lateral (secondary) roots, and the ratio of both root types changes over the life span of a plant. Most studies have focused on the growth of primary roots and the development of lateral root primordia. Comparably less is known about the growth regulation of secondary root organs. Here, we review similarities and differences between primary and lateral root organ growth, and emphasize particularly how external stimuli and internal signals differentially integrate root system growth.

scholarly journals Enhanced in vivo-imaging in fish by optimized anaesthesia, fluorescent protein selection and removal of pigmentation

2018 ◽  
Author(s):  
Colin Q. Lischik ◽  
Leonie Adelmann ◽  
Joachim Wittbrodt
Keyword(s):  
Fluorescent Protein ◽  
Light Sheet ◽  
Time Lapse ◽  
Light Sheet Microscopy ◽  
Inner Organs ◽  
Traditional Approaches ◽  
Time Lapse Imaging ◽  
Targeted Inactivation ◽  
Imaging Conditions

AbstractFish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation.We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

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