Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

10.3791/62828 ◽  
2021 ◽  
Author(s):  
Koen M. O. Galenkamp ◽  
Cheska Marie Galapate ◽  
Yijuan Zhang ◽  
Cosimo Commisso
Keyword(s):  
Automated Imaging

scholarly journals Live-Cell Imaging of Caspase Activation for High-Content Screening

2009 ◽  
Vol 14 (8) ◽  
pp. 956-969 ◽  
Author(s):  
Christophe Antczak ◽  
Toshimitsu Takagi ◽  
Christina N. Ramirez ◽  
Constantin Radu ◽  
Hakim Djaballah
Keyword(s):  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Exposure Period ◽  
Live Cells ◽  
Assay Method ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Automated Imaging ◽  
First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

scholarly journals Development of a Kinetic Assay for Late Endosome Movement

2014 ◽  
Vol 19 (7) ◽  
pp. 1070-1078 ◽  
Author(s):  
Milan Esner ◽  
Felix Meyenhofer ◽  
Michael Kuhn ◽  
Melissa Thomas ◽  
Yannis Kalaidzidis ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Membrane Integrity ◽  
Living Cells ◽  
Mechanisms Of Action ◽  
Added Value ◽  
Kinetic Assay ◽  
Late Endosomes ◽  
Automated Imaging ◽  
Phenotypic Clustering ◽  
Large Compound

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

Measuring the Confluence of iPSCs using an Automated Imaging System.

10.3791/61225 ◽  
2020 ◽  
Author(s):  
Valentina Magliocca ◽  
Maria Vinci ◽  
Tiziana Persichini ◽  
Franco Locatelli ◽  
Marco Tartaglia ◽  
...  
Keyword(s):  
Imaging System ◽  
Automated Imaging

scholarly journals Development and validation of the automated imaging differentiation in parkinsonism (AID-P): a multicentre machine learning study

2019 ◽  
Vol 1 (5) ◽  
pp. e222-e231 ◽  
Author(s):  
Derek B Archer ◽  
Justin T Bricker ◽  
Winston T Chu ◽  
Roxana G Burciu ◽  
Johanna L McCracken ◽  
...  
Keyword(s):  
Machine Learning ◽  
Learning Study ◽  
Automated Imaging ◽  
Development And Validation

A Peltier-cooled CCD Camera

1991 ◽  
Vol 9 (1) ◽  
pp. 158-159 ◽  
Author(s):  
B. D. Carter ◽  
M. C. B. Ashley
Keyword(s):  
Dark Current ◽  
Cooling System ◽  
Ccd Camera ◽  
Radiative Heating ◽  
Peltier Effect ◽  
Charge Coupled Device ◽  
Automated Imaging ◽  
Cooled Ccd ◽  
Ccd Detectors ◽  
Careful Design

AbstractWe describe the application of Peltier effect cooling to charge coupled device (CCD) detectors. We are developing this technique to produce a CCD camera which requires low maintenance, yet has sufficiently small dark-current for long exposure imaging. This camera will be used in an automated imaging telescope at Siding Spring Observatory. The design principles used to maximise cooling of the detector, and hence minimise dark-current, are discussed. A small dark-current can be obtained only if great care is taken to reduce or eliminate convective, conductive and radiative heating of the chip. In addition, a path of high thermal conductivity must be provided for the heat removed from the CCD. A recent laboratory test of our cooling system demonstrates that careful design can lead to sufficiently low CCD dark-current for many astronomical applications.

Automated imaging of circulating fluorocytes for the diagnosis of erythropoietic protoporphyria: a pilot study for population screening

2008 ◽  
Vol 15 (4) ◽  
pp. 199-203 ◽  
Author(s):  
Kin-Chong Lau ◽  
Ching-Wan Lam
Keyword(s):  
Pilot Study ◽  
High Throughput ◽  
Imaging System ◽  
Blood Film ◽  
Population Screening ◽  
Fresh Blood ◽  
Erythropoietic Protoporphyria ◽  
High Throughput Analysis ◽  
Automated Imaging ◽  
Film Method

Objectives To improve the traditional fresh blood film method to a high-throughput analysis of the presence of circulating fluorescent red cells (fluorocytes) in erythropoietic protoporphyria (EPP) using an automated imaging system. Methods Based on the autofluorescence of protoporphyrin, we used an automatic image acquisition platform for examining fluorocytes in peripheral blood with minimal sample preparation. The image acquisition is easy-to-use under automated operations of excitation, focusing, detection and data analysis. Quality image and semi-quantitative fluorescence measurement of fluorocytes can be generated in a single step. For high-throughput analysis, the platform can image more than 200 96-well micro-plates, i.e. 19200 samples, in approximately 10 hours. Importantly, the reagent cost of analysis is negligible. Results In this pilot study, three EPP patients were diagnosed and 4000 normal individuals were screened for EPP by this method. Our results showed that the method can distinguish the overt case and asymptomatic carriers. It gives reliable evidence for rapid EPP screening. Conclusion This automated imaging system provides multiple advantages that improve the traditional fresh blood film method as a more effective diagnostic tool and facilitates population screening for EPP. As fluorocytes are present in the umbilical cord blood of EPP patients, this high-throughput method can be potentially used for newborn screening of EPP.

scholarly journals Automated imaging cytometry reveals dysplastic indices of colonic serrated adenomas

2020 ◽  
pp. FSO459
Author(s):  
Nicholas S Samel ◽  
Qin Huang ◽  
Hiroshi Mashimo
Keyword(s):  
Dna Content ◽  
Chromosome Instability ◽  
Neoplastic Progression ◽  
Hyperplastic Polyps ◽  
Dna Index ◽  
Tissue Sections ◽  
Automated Imaging ◽  
Imaging Cytometry ◽  
The Mean ◽  
Serrated Adenomas

Aim: Left-sided colonic serrated adenomas (L-SAs) were evaluated for aneuploidy using automated imaging cytometry to quantify DNA content and compared with normal colonic tissues (NCT), tubular adenomas (TA), left-sided hyperplastic polyps (L-HP) and adenocarcinomas. Materials & methods: We used standard paraffin-embedded Feulgen-stained tissue sections. Results: The mean DNA index (DI) of NCT was 0.95, L-HP was 1.08, TA was 1.22, L-SA was 1.11 and adenocarcinomas was 1.46. DI of L-SA was statistically higher than that of NCT, but not statistically different from L-HP. Conclusion: This study demonstrates that DIs correlate with the described neoplastic progression of L-SA, TA and L-SA compared with NCT and suggests that L-SA may be involved in a chromosome instability pathway of neoplastic progression.

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