scholarly journals Single molecule diffusion coefficient estimation by image analysis of simulated CCD images to aid high-throughput screening

2009 ◽  
Author(s):  
Pengfei Song ◽  
L.M. Davis ◽  
G.R. Bashford
Keyword(s):  
Image Analysis ◽  
Diffusion Coefficient ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Screening ◽  
Coefficient Estimation

scholarly journals ZF-AutoML: An Easy Machine-Learning-Based Method to Detect Anomalies in Fluorescent-Labelled Zebrafish

Inventions ◽  
2019 ◽  
Vol 4 (4) ◽  
pp. 72
Author(s):  
Ryota Sawaki ◽  
Daisuke Sato ◽  
Hiroko Nakayama ◽  
Yuki Nakagawa ◽  
Yasuhito Shimada
Keyword(s):  
Machine Learning ◽  
Image Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Learning Program ◽  
High Throughput Analysis ◽  
Easy Method ◽  
High Fecundity

Background: Zebrafish are efficient animal models for conducting whole organism drug testing and toxicological evaluation of chemicals. They are frequently used for high-throughput screening owing to their high fecundity. Peripheral experimental equipment and analytical software are required for zebrafish screening, which need to be further developed. Machine learning has emerged as a powerful tool for large-scale image analysis and has been applied in zebrafish research as well. However, its use by individual researchers is restricted due to the cost and the procedure of machine learning for specific research purposes. Methods: We developed a simple and easy method for zebrafish image analysis, particularly fluorescent labelled ones, using the free machine learning program Google AutoML. We performed machine learning using vascular- and macrophage-Enhanced Green Fluorescent Protein (EGFP) fishes under normal and abnormal conditions (treated with anti-angiogenesis drugs or by wounding the caudal fin). Then, we tested the system using a new set of zebrafish images. Results: While machine learning can detect abnormalities in the fish in both strains with more than 95% accuracy, the learning procedure needs image pre-processing for the images of the macrophage-EGFP fishes. In addition, we developed a batch uploading software, ZF-ImageR, for Windows (.exe) and MacOS (.app) to enable high-throughput analysis using AutoML. Conclusions: We established a protocol to utilize conventional machine learning platforms for analyzing zebrafish phenotypes, which enables fluorescence-based, phenotype-driven zebrafish screening.

scholarly journals Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening

2001 ◽  
Vol 6 (1) ◽  
pp. 11-18
Author(s):  
Andreas A. Scheel ◽  
Bettina Funsch ◽  
Michael Busch ◽  
Gabriele Gradl ◽  
Johannes Pschorr ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
High Throughput Screening ◽  
Free Ligand ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Model Systems ◽  
Fluorescence Signal ◽  
Distribution Analysis

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

2003 ◽  
Vol 8 (1) ◽  
pp. 19-33 ◽  
Author(s):  
Ulrich Haupts ◽  
Martin Rüdiger ◽  
Stephen Ashman ◽  
Sandra Turconi ◽  
Ryan Bingham ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Fluorescence Intensity ◽  
Intensity Distribution ◽  
High Throughput Screening ◽  
Biological Data ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Fluorescence Intensity Distribution Analysis ◽  
Detection Technologies

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. ( Journal of Biomolecular Screening 2003:19-33)

scholarly journals 48-spot single-molecule FRET setup with periodic acceptor excitation

2017 ◽  
Author(s):  
Antonino Ingargiola ◽  
Maya Segal ◽  
Angelo Gulinatti ◽  
Ivan Rech ◽  
Ivan Labanca ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Acquisition Time ◽  
Enzymatic Reactions ◽  
Single Molecule Fret ◽  
Array Detectors ◽  
Donor And Acceptor

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time.Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Image Analysis and Classification for High-Throughput Screening of Embryonic Stem Cells

2015 ◽  
pp. 17-31
Author(s):  
Laura Casalino ◽  
Pasqua D’Ambra ◽  
Mario R. Guarracino ◽  
Antonio Irpino ◽  
Lucia Maddalena ◽  
...  
Keyword(s):  
Stem Cells ◽  
Image Analysis ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem

Quantitative digital image analysis of chromogenic assays for high throughput screening of α-amylase mutant libraries

2009 ◽  
Vol 31 (8) ◽  
pp. 1197-1201 ◽  
Author(s):  
Manoharan Shankar ◽  
Ramachandran Priyadharshini ◽  
Paramasamy Gunasekaran
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Digital Image ◽  
High Throughput Screening ◽  
Digital Image Analysis

scholarly journals Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers

2014 ◽  
Vol 20 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Marios Stylianou ◽  
Hanna Uvell ◽  
José Pedro Lopes ◽  
Per-Anders Enquist ◽  
Mikael Elofsson ◽  
...  
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Antifungal Drugs ◽  
Width Ratio ◽  
High Morbidity ◽  
Candida Spp ◽  
New Approach ◽  
Length Width

Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus, morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably, Z′ factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal. Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.

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