scholarly journals High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells

Lab on a Chip ◽  
2019 ◽  
Vol 19 (21) ◽  
pp. 3652-3663 ◽  
Author(s):  
Patricia M. Davidson ◽  
Gregory R. Fedorchak ◽  
Solenne Mondésert-Deveraux ◽  
Emily S. Bell ◽  
Philipp Isermann ◽  
...  
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Time Scale ◽  
Viscoelastic Properties ◽  
Micropipette Aspiration ◽  
Automated Image Analysis ◽  
Cell Nuclei ◽  
Intact Cells ◽  
Nuclear Mechanics ◽  
Analysis Platform

We report the development, validation, and application of an easy-to-use microfluidic micropipette aspiration device and automated image analysis platform that enables high-throughput measurements of the viscoelastic properties of cell nuclei.

scholarly journals High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells

2019 ◽  
Author(s):  
Patricia M. Davidson ◽  
Gregory R. Fedorchak ◽  
Solenne Mondésert-Deveraux ◽  
Emily S. Bell ◽  
Philipp Isermann ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Image Analysis ◽  
Microfluidic Device ◽  
High Throughput ◽  
Stem Cell Differentiation ◽  
Micropipette Aspiration ◽  
Model Systems ◽  
Automated Image Analysis ◽  
Time Dependent ◽  
Lamin A

AbstractThe mechanical properties of the cell nucleus are increasingly recognized as critical in many biological processes. The deformability of the nucleus determines the ability of immune and cancer cells to migrate through tissues and across endothelial cell layers, and changes to the mechanical properties of the nucleus can serve as novel biomarkers in processes such as cancer progression and stem cell differentiation. However, current techniques to measure the viscoelastic nuclear mechanical properties are often time consuming, limited to probing one cell at a time, or require expensive, highly specialized equipment. Furthermore, many current assays do not measure time-dependent properties, which are characteristic of viscoelastic materials. Here, we present an easy-to-use microfluidic device that applies the well-established approach of micropipette aspiration, adapted to measure many cells in parallel. The device design allows rapid loading and purging of cells for measurements, and minimizes clogging by large particles or clusters of cells. Combined with a semi-automated image analysis pipeline, the microfluidic device approach enables significantly increased experimental throughput. We validated the experimental platform by comparing computational models of the fluid mechanics in the device with experimental measurements of fluid flow. In addition, we conducted experiments on cells lacking the nuclear envelope protein lamin A/C and wild-type controls, which have well-characterized nuclear mechanical properties. Fitting time-dependent nuclear deformation data to power law and different viscoelastic models revealed that loss of lamin A/C significantly altered the elastic and viscous properties of the nucleus, resulting in substantially increased nuclear deformability. Lastly, to demonstrate the versatility of the devices, we characterized the viscoelastic nuclear mechanical properties in a variety of cell lines and experimental model systems, including human skin fibroblasts from an individual with a mutation in the lamin gene associated with dilated cardiomyopathy, healthy control fibroblasts, induced pluripotent stem cells (iPSCs), and human tumor cells. Taken together, these experiments demonstrate the ability of the microfluidic device and automated image analysis platform to provide robust, high throughput measurements of nuclear mechanical properties, including time-dependent elastic and viscous behavior, in a broad range of applications.

An automated image analysis platform for the study of weakly -adhered cells

Biofouling ◽  
2021 ◽  
pp. 1-10
Author(s):  
Zhijing Wan ◽  
Ben T. MacVicar ◽  
Shea Wyatt ◽  
Diana E. Varela ◽  
Rajkumar Padmawar ◽  
...  
Keyword(s):  
Image Analysis ◽  
Automated Image Analysis ◽  
Analysis Platform

scholarly journals Measuring Quantitative Virulence in the Wheat Pathogen Zymoseptoria tritici Using High-Throughput Automated Image Analysis

2014 ◽  
Vol 104 (9) ◽  
pp. 985-992 ◽  
Author(s):  
Ethan L. Stewart ◽  
Bruce A. McDonald
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Digital Image ◽  
Digital Image Analysis ◽  
Continuous Distribution ◽  
Automated Image Analysis ◽  
Host Specialization ◽  
Septoria Tritici Blotch ◽  
Septoria Tritici ◽  
Zymoseptoria Tritici

Zymoseptoria tritici, causal agent of Septoria tritici blotch on wheat, produces pycnidia in chlorotic and necrotic lesions on infected leaves. A high-throughput phenotyping method was developed based on automated digital image analysis that accurately measures the percentage of leaf area covered by lesions (PLACL) as well as pycnidia size and number. A seedling inoculation assay was conducted using 361 Z. tritici isolates originating from a controlled cross and two different winter wheat cultivars. Pycnidia size and density were found to be quantitative traits that showed a continuous distribution in the progeny. There was a weak correlation between pycnidia density and size (r = −0.27) and between pycnidia density and PLACL (r = 0.37). There were significant differences in PLACL and pycnidia density on resistant and susceptible cultivars. In all, >20% of the offspring exhibited significantly different pycnidia density on the two cultivars, consistent with host specialization. Automated image analysis provided greater accuracy and precision compared with traditional visual estimates of virulence. These results show that digital image analysis provides a powerful tool for measuring differences in quantitative virulence among strains of Z. tritici.

scholarly journals ScreenSeed as a novel high throughput seed germination phenotyping method

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nicolas Merieux ◽  
Pierre Cordier ◽  
Marie-Hélène Wagner ◽  
Sylvie Ducournau ◽  
Sophie Aligon ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Image Analysis ◽  
Seed Germination ◽  
High Throughput ◽  
Automated Image Analysis ◽  
Experimental Protocol ◽  
Visual Scoring ◽  
Radicle Protrusion ◽  
High Throughput Phenotyping ◽  
Fast Procedure

AbstractA high throughput phenotyping tool for seed germination, the ScreenSeed technology, was developed with the aim of screening genotype responsiveness and chemical drugs. This technology was presently used with Arabidopsis thaliana seeds to allow characterizing seed samples germination behavior by incubating seeds in 96-well microplates under defined conditions and detecting radicle protrusion through the seed coat by automated image analysis. This study shows that this technology provides a fast procedure allowing to handle thousands of seeds without compromising repeatability or accuracy of the germination measurements. Potential biases of the experimental protocol were assessed through statistical analyses of germination kinetics. Comparison of the ScreenSeed procedure with commonly used germination tests based upon visual scoring displayed very similar germination kinetics.

scholarly journals Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

2017 ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Rnai Screen ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Automated Image Analysis ◽  
C Elegans ◽  
Larval Stages ◽  
Lethal Mutations

ABSTRACTBackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the user’s requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

scholarly journals Fast-FISH Detection and Semi-Automated Image Analysis of Numerical Chromosome Aberrations in Hematological Malignancies

1998 ◽  
Vol 16 (4) ◽  
pp. 211-222 ◽  
Author(s):  
Arif Esa ◽  
Luba Trakhtenbrot ◽  
Michael Hausmann ◽  
Joachim Rauch ◽  
Frida Brok-Simoni ◽  
...  
Keyword(s):  
Image Analysis ◽  
Chromosome Aberrations ◽  
Clinical Diagnostics ◽  
Chromosome 8 ◽  
Myelogenous Leukemia ◽  
Automated Image Analysis ◽  
Cell Nuclei ◽  
Interphase Cell ◽  
Fish Technique ◽  
Numerical Chromosome

A new fluorescence in situ hybridization (FISH) technique calledFast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available α-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of theFast-FISH protocol, the microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with astandardFISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling andFast-FISH was found. Automated spot counting afterFast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility ofFast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since theFast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

scholarly journals Quantification of Lipid Droplets and Associated Proteins in Cellular Models of Obesity via High-Content/High-Throughput Microscopy and Automated Image Analysis

2009 ◽  
Vol 7 (5) ◽  
pp. 440-460 ◽  
Author(s):  
Patrick M. McDonough ◽  
Ramses M. Agustin ◽  
Randall S. Ingermanson ◽  
Patricia A. Loy ◽  
Benjamin M. Buehrer ◽  
...  
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Lipid Droplets ◽  
Automated Image Analysis ◽  
Cellular Models ◽  
Associated Proteins
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