A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies

Abstract

The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Due to the discovery of new molecules and new emerging bioanalytical problems, there is an ongoing demand for a technology platform that provides a broad range of assays with a user-friendly flexibility and rapid adaptability to new applications. Here we describe a highly versatile microscopy platform, VideoScan, for the rapid and simultaneous analysis of various assay formats based on fluorescence microscopic detection. The technological design is equally suitable for assays in solution, microbead-based assays and cell pattern recognition. The multiplex real-time capability for tracking of changes under dynamic heating conditions makes it a useful tool for PCR applications and nucleic acid hybridization, enabling kinetic data acquisition impossible to obtain by other technologies using endpoint detection. The paper discusses the technological principle of the platform regarding data acquisition and processing. Microbead-based and solution applications for the detection of diverse biomolecules, including antigens, antibodies, peptides, oligonucleotides and amplicons in small reaction volumes, are presented together with a high-content detection of autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility gives VideoScan a competitive edge over other bioanalytical technologies.

Adhesion of Human and Animal Escherichia coli Strains in Association with Their Virulence-Associated Genes and Phylogenetic Origins

“…Microbeads consisting of polymethylmethacrylate (PMMA) were thermotolerant, carboxylated, and coded by size (diameters, between 10 and 20 m) and by two fluorescence dyes (PolyAn GmbH, Germany). 5=-Amino-modified DNA capture probes with poly(dT) 10 spacers were cross-linked to the microbead surface using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (27). Microbeads with bound capture probes were fixed to the bottom of poly-Llysine (Sigma-Aldrich, Germany)-coated 96-well NucleoLink plates (Nunc, Germany).…”

“…The VideoScan technology implemented in the Aklides system (GA Generic Assays GmbH, Dahlewitz, Germany) is a versatile fluorescence microscope imaging technology for the analysis of fluorescent objects (27,(29)(30)(31). The VideoScan instrument was automatically guided to each well of a 96-well plate, where it focused on microbeads or cell monolayer surfaces and took images, which were saved as .bmp files.…”

“…The ultimate parameter, the referenced mean fluorescence intensity (refMFI), was referenced to the intensity of the dyes encoding the microbeads and correlated with the amount of hybridized PCR products and, therefore, with the quantity of a PCR product (27,32).…”

Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

“…Diagnostic applications include microRNA profiling, gene expression analysis, pathogen quantification, and cancer diagnostics (2,3 ). There is an ongoing development of novel devices on the basis of capillary systems, microfluidics, point-of-care testing, microbeads, and related technologies (6,(7)(8)(9)(10)(11)(12).…”

RKWard: A Comprehensive Graphical User Interface and Integrated Development Environment for Statistical Analysis withR

“…In contrast, RKWard limits itself to generate R code from GUI settings. Yang, Liu, Liu, Qian, Zhang, and Hu 2011;Rödiger et al 2011Rödiger et al , 2012. Dialogs for statistical procedures in RKWard do not necessarily show a one-to-one correspondence to the underlying steps in R, but are rather oriented at statistical tasks.…”