(Research Papers) Time-Resolved Fluorescence Measurements Using Microlens Array and Area Imaging Devices

Abstract

Time-resolved fluorescence (TRF) assay formats are frequently used technologies in high-throughput screening. In this article, we have characterised the novel Plate::Vision(2) 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments. For TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y/SA-APC at saturating concentrations. Both the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.

Computer vision-based analytical chemistry applied to determining iron in commercial pharmaceutical formulations

“…The CVAC technique has been increasingly used in research laboratories and commercial laboratory equipment for more than two decades [26,[28][29][30][31][32][33][34][35][36][37][38][39][40][41]. Lowcost commercial digital cameras and scanners have been gradually incorporated into the analytical laboratory, where they are being increasingly used for forensic [42,43], telemedicine [27,48] and, obviously, analytical purposes [26,44,46,47,49,50,[53][54][55].…”

Using digital photography to implement the McFarland method

“…Analytical imaging (CCD) methods are increasingly being used in Raman spectroscopy [11], chemiluminescence spectroscopy [12] or electron transmission microscopy [13] at research laboratories. This has promoted the development of dedicated commercial equipment where analyses are implemented on inductively coupled plasma-atomic emission spectroscopy (ICP-AES) [14], mass spectrometers [15] or fast-scan systems [16]. In biological research, CCDs have been used to capture digital images for UV-fluorescing substances with a variety of purposes including documentation and quantitative analysis in, for example, electrophoretic separations of nucleic acids and proteins-even in in vivo tests in the latter case [17].…”

Quantitative colorimetric-imaging analysis of nickel in iron meteorites

“…As regards commercial availability, chemical imaging analysis has so far been used with inductively coupled plasma atomic emission spectrometers (ICP-AES) [29], mass spectrometers [30] and high speed screening systems [31]. On the remote sensing field, digital imaging has been applied to evaluation of the photosynthetic biomass [32]; to the quantitative determination of chlorophyll quantitation [33]; and, to the analysis of vegetation [34].…”