scholarly journals High-throughput live-imaging of embryos in microwell arrays using a modular, inexpensive specimen mounting system

2017 ◽  
Author(s):  
Seth Donoughe ◽  
Chiyoung Kim ◽  
Cassandra G. Extavour
Keyword(s):  
High Throughput ◽  
Fruit Fly ◽  
Time Lapse ◽  
Live Imaging ◽  
Model Systems ◽  
Wide Range ◽  
Amphipod Crustacean ◽  
Live Image ◽  
Time Lapse Imaging ◽  
Annelid Worm

AbstractLive-imaging embryos in a high-throughput manner is essential for shedding light on a wide range of questions in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device that makes it easy to mount up to hundreds of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be cut from a sheet of acrylic using a laser cutter. Even a novice user will be able to cut the pieces and assemble the device in less than an hour. At the time of writing, the total materials cost is less than five US dollars. We include all device design files in a commonly used format, as well as complete instructions for its fabrication and use. We demonstrate a detailed workflow for designing a custom mold and employing it to simultaneously live-image dozens of embryos at a time for more than five days, using embryos of the cricket Gryllus bimaculatus as an example. Further, we include descriptions, schematics, and design files for molds that can be used with 14 additional vertebrate and invertebrate species, including most major traditional laboratory models and a number of emerging model systems. Molds have been user-tested for embryos including zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), coqui frog (Eleutherodactylus coqui), annelid worm (Capitella teleta), amphipod crustacean (Parhyale hawaiensis), red flour beetle (Tribolium castaneum), and three-banded panther worm (Hofstenia miamia), as well mouse organoids (Mus musculus). Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein.Summary StatementThis Techniques and Resources article describes an inexpensive, customizable device for mounting and live-imaging a wide range of tissues and species; complete design files and instructions for assembly are included.

scholarly journals Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology

PLoS ONE ◽  
2019 ◽  
Vol 14 (11) ◽  
pp. e0224878 ◽  
Author(s):  
Sarah H. Needs ◽  
Tai The Diep ◽  
Stephanie P. Bull ◽  
Anton Lindley-Decaire ◽  
Partha Ray ◽  
...  
Keyword(s):  
Open Source ◽  
High Throughput ◽  
Time Lapse ◽  
3D Printer ◽  
Throughput Time ◽  
Time Lapse Imaging

scholarly journals Automated profiling of individual cell–cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING)

2015 ◽  
Vol 31 (19) ◽  
pp. 3189-3197 ◽  
Author(s):  
Amine Merouane ◽  
Nicolas Rey-Villamizar ◽  
Yanbin Lu ◽  
Ivan Liadi ◽  
Gabrielle Romain ◽  
...  
Keyword(s):  
High Throughput ◽  
Individual Cell ◽  
Time Lapse ◽  
Cell Interactions ◽  
Throughput Time ◽  
Time Lapse Imaging ◽  
Cell Cell

scholarly journals Time-lapse imaging of molecular evolution by high-throughput sequencing

2018 ◽  
Vol 46 (15) ◽  
pp. 7480-7494 ◽  
Author(s):  
Nam Nguyen Quang ◽  
Clément Bouvier ◽  
Adrien Henriques ◽  
Benoit Lelandais ◽  
Frédéric Ducongé
Keyword(s):  
Molecular Evolution ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Time Lapse ◽  
Time Lapse Imaging

scholarly journals Low-Cost Automated Vectors and Modular Environmental Sensors for Plant Phenotyping

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3319
Author(s):  
Stuart A. Bagley ◽  
Jonathan A. Atkinson ◽  
Henry Hunt ◽  
Michael H. Wilson ◽  
Tony P. Pridmore ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Time Lapse ◽  
Plant Phenotyping ◽  
Environmental Sensors ◽  
Controlled Conditions ◽  
Sensor Platform ◽  
High Throughput Phenotyping ◽  
Controlled Environments ◽  
Time Lapse Imaging

High-throughput plant phenotyping in controlled environments (growth chambers and glasshouses) is often delivered via large, expensive installations, leading to limited access and the increased relevance of “affordable phenotyping” solutions. We present two robot vectors for automated plant phenotyping under controlled conditions. Using 3D-printed components and readily-available hardware and electronic components, these designs are inexpensive, flexible and easily modified to multiple tasks. We present a design for a thermal imaging robot for high-precision time-lapse imaging of canopies and a Plate Imager for high-throughput phenotyping of roots and shoots of plants grown on media plates. Phenotyping in controlled conditions requires multi-position spatial and temporal monitoring of environmental conditions. We also present a low-cost sensor platform for environmental monitoring based on inexpensive sensors, microcontrollers and internet-of-things (IoT) protocols.

High-throughput RNAi screening by time-lapse imaging of live human cells

2006 ◽  
Vol 3 (5) ◽  
pp. 385-390 ◽  
Author(s):  
Beate Neumann ◽  
Michael Held ◽  
Urban Liebel ◽  
Holger Erfle ◽  
Phill Rogers ◽  
...  
Keyword(s):  
High Throughput ◽  
Time Lapse ◽  
Human Cells ◽  
Rnai Screening ◽  
Time Lapse Imaging

scholarly journals A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1

2021 ◽  
Author(s):  
Funmilayo O Fagbadebo ◽  
Philipp D Kaiser ◽  
Katharina Zittlau ◽  
Natascha Bartlick ◽  
Teresa R Wagner ◽  
...  
Keyword(s):  
Time Lapse ◽  
Model Systems ◽  
Live Cells ◽  
Mitochondrial Outer Membrane ◽  
Potential Biomarker ◽  
Time Lapse Imaging ◽  
Lateral Sclerosis ◽  
Neuronal Disorders ◽  
Research Tools

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

scholarly journals Macrophage centripetal migration drives spontaneous healing process after spinal cord injury

2019 ◽  
Vol 5 (5) ◽  
pp. eaav5086 ◽  
Author(s):  
Kazu Kobayakawa ◽  
Yasuyuki Ohkawa ◽  
Shingo Yoshizaki ◽  
Tetsuya Tamaru ◽  
Takeyuki Saito ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Healing Process ◽  
Inflammatory Cells ◽  
Time Lapse ◽  
Poor Functional Outcome ◽  
Wide Range ◽  
Cord Injury ◽  
Time Lapse Imaging ◽  
Centripetal Movement

Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury.

scholarly journals Robust colony recognition for high-throughput growth analysis from suboptimal low-magnification brightfield micrographs

2018 ◽  
Author(s):  
Yevgeniy Plavskin ◽  
Shuang Li ◽  
Naomi Ziv ◽  
Sasha F. Levy ◽  
Mark L. Siegal
Keyword(s):  
High Throughput ◽  
Time Lapse ◽  
Manual Curation ◽  
Technological Advances ◽  
Wide Range ◽  
Colony Recognition ◽  
Gradient Based ◽  
High Throughput Phenotyping ◽  
High Resolution Images ◽  
Object Center

AbstractNew technological advances have enabled high-throughput phenotyping at the single-cell level. However, analyzing the large amount of data automatically and accurately is a great challenge. Currently available software achieves cell and colony tracking through the use of either manual curation of images, which is time consuming, or high-resolution images requiring specialized microscopy setups or fluorescence, which limits applicability and results in greatly decreased experimental throughput. Here we introduce a new algorithm, Processing Images Easily (PIE), that automatically tracks colonies of the yeast Saccharomyces cerevisiae in low-magnification brightfield images by combining adaptive object-center detection with gradient-based object-outline detection. We tested the performance of PIE on low-magnification brightfield time-lapse images. PIE recognizes colony outlines very robustly and accurately across a wide range of image brightnesses and focal depths. We show that PIE allows for unbiased and precise measurement of growth rates in a large number (>90,000) of microcolonies in a single time-lapse experiment.

scholarly journals LIVE IMAGING OF ERK ACTIVITY STEPWISE PATTERNING DURING SOMITOGENESIS

2015 ◽  
Vol 2 (1) ◽  
pp. 363
Author(s):  
Dini Wahyu Kartika Sari ◽  
Ryutaro Akiyama ◽  
Takaaki Matsui ◽  
Yasumasa Bessho
Keyword(s):  
Clock Genes ◽  
Time Lapse ◽  
Live Imaging ◽  
Molecular Signature ◽  
Vertebrate Development ◽  
Segmentation Clock ◽  
Somite Formation ◽  
Repetitive Structure ◽  
Time Lapse Imaging ◽  
Erk Activity

<p>Periodical segmentation of the anterior extremity of the presomitic mesoderm (PSM) generates metameric structure of somites during vertebrate development. During somite segmentation in zebrafish, msep determines a future somite boundary at position B-2 within the PSM. However, heat shock experiments suggest that an earlier future of somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Our recent study demonstrated that fibroblast growth factor (FGF) gradient is converted into an ON-OFF boundary of downstream Erk activity, which corresponds to the future B-5 somite boundary. Moreover, we also revealed that the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. To clarify this evidence, here we perform time-lapse imaging of Erk activity in living embryos using a FRET biosensor. We focused on FRET signals within the PSM to observe spatial and temporal changes of Erk activity. Consistent with the data from fixed embryos, we observed ON-OFF boundary of Erk activity within the PSM and the position stepwisely shifted to posterior during somite formation. In order to test the contribution of the segmentation clock to the stepwise movements of the ON-OFF boundary of Erk activity, we disrupted the segmentation clock in zebrafish embryos by knocking down two segmentation clock genes, her1 and her7. Double knockdown of her1 and her7 resulted in the failure of the shift of the Erk ON-OFF boundary during somite segmentation, leading to segmentation defects of somites. These results strongly suggest that the clock-dependent stepwise movement of Erk activity is a key mechanism to generate the perfect repetitive structure of somites.</p><p><br /><strong>Keywords</strong>: Live imaging, Erk, stepwise, somitogenesis</p>

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