The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform

David D. Thomas - One of the best experts on this subject based on the ideXlab platform.

  • discovery of enzyme modulators via high Throughput Time resolved fret in living cells
    Journal of Biomolecular Screening, 2014
    Co-Authors: Simon J Gruber, Kurt C. Peterson, Gregory D. Gillispie, Ji Li, Razvan L. Cornea, Tory M Schaaf, Russell Dahl, Krisztina M Zsebo, Seth L Robia, David D. Thomas
    Abstract:

    We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-Throughput fluorescence lifeTime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing Throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable...

  • High Throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-Throughput Time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifeTimes across 384 wells with short (< 5 min.) read Times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. Time) yield lifeTimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifeTime is better than 2%. These features will enable high-Throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells
    Biophysical Journal, 2014
    Co-Authors: Simon J Gruber, Kurt C. Peterson, Gregory D. Gillispie, Razvan L. Cornea, Seth L Robia, David D. Thomas
    Abstract:

    We have used a “2-color” SERCA (sarco/endo-plasmic reticulum calcium ATPase) biosensor and a high-Throughput fluorescence lifeTime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. We used a construct derived from canine cardiac SERCA, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop. This 2-color SERCA was stably expressed in HEK-GnTI- cells, a strain that can be grown in monolayers or in suspension, as needed for automated transfer to multiwell plates. Fluorescence resonance energy transfer (FRET) was measured from GFP to RFP using the FLT-PR, which increases precision by a factor of 30 over a conventional intensity-based plate-reader, without sacrificing Throughput. FRET was highly sensitive to both known activators and inhibitors of SERCA. We screened a small (1280-compound) chemical library and identified nine compounds that significantly affect 2-color SERCA FLT. Three of these compounds affected FRET in a dose-dependent manner, and all three were found to inhibit SERCA function. Two of the hits were known SERCA inhibitors and the third was novel (Gruber et al., J. Biol. Screen, in press). This assay is being extended to several human isoforms of SERCA, for therapeutic applications to heart failure, muscular dystrophy, diabetes, and cancer. This assay is ready for a large-scale HTS campaign, and is adaptable to numerous protein targets. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220), to SJG (AHA 13PRE13230005), and to SLR (R01 HL106189).

  • Screening for SERCA Activators using a High-Throughput Time-Resolved FRET Assay
    Biophysical Journal, 2013
    Co-Authors: Ji Li, Joseph M. Muretta, Kurt C. Peterson, Gregory D. Gillispie, Holly R. Langer, Razvan L. Cornea, David D. Thomas
    Abstract:

    We used a prototype Time-resolved fluorescence lifeTime microplate reader to carry out a high-Throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifeTime plate reader was made possible by our recent development of fast Time-resolved fluorescence by direct waveform recording, which achieves 105 higher Throughput than the conventional single-photon counting technology. using this plate reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the Time-resolved microplate reader has at least 10x higher precision than a conventional intensity-based microplate reader, raising the quality index (z') of our assay from marginal, in the intensity reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the Time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

Kevin K Tsia - One of the best experts on this subject based on the ideXlab platform.

  • high Throughput Time stretch imaging flow cytometry for multi class classification of phytoplankton
    Optics Express, 2016
    Co-Authors: Anson H L Tang, Kenneth K Y Wong, Hayden Kwokhay So, Kevin K Tsia
    Abstract:

    Time-stretch imaging has been regarded as an attractive technique for high-Throughput imaging flow cytometry primarily owing to its real-Time, continuous ultrafast operation. Nevertheless, two key challenges remain: (1) sufficiently high Time-stretch image resolution and contrast is needed for visualizing sub-cellular complexity of single cells, and (2) the ability to unravel the heterogeneity and complexity of the highly diverse population of cells – a central problem of single-cell analysis in life sciences – is required. We here demonstrate an optofluidic Time-stretch imaging flow cytometer that enables these two features, in the context of high-Throughput multi-class (up to 14 classes) phytoplantkton screening and classification. Based on the comprehensive feature extraction and selection procedures, we show that the intracellular texture/morphology, which is revealed by high-resolution Time-stretch imaging, plays a critical role of improving the accuracy of phytoplankton classification, as high as 94.7%, based on multi-class support vector machine (SVM). We also demonstrate that high-resolution Time-stretch images, which allows exploitation of various feature domains, e.g. Fourier space, enables further sub-population identification – paving the way toward deeper learning and classification based on large-scale single-cell images. Not only applicable to biomedical diagnostic, this work is anticipated to find immediate applications in marine and biofuel research.

  • High-Throughput Time-stretch imaging cellular assay based on a high-speed spinning platform
    2016 IEEE Photonics Conference (IPC), 2016
    Co-Authors: Anson H L Tang, Kenneth K Y Wong, Antony C. S. Chan, P. Yeung, Barbara P. Chan, Kevin K Tsia
    Abstract:

    We propose and demonstrate a new strategy for high-Throughput, biochemical-specific, single-cell imaging cytometry, based on integration of a high-speed spinning planar platform (900-3600 rpm) and ultrafast optical Time-stretch imaging (at a line-scan rate of 11 MHz).

Yang Yi - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of workflow resources allocation with cost constraint
    Computer Supported Cooperative Work in Design III, 2007
    Co-Authors: Zhijiao Xiao, Huiyou Chang, Yang Yi
    Abstract:

    A resource allocation method is proposed to determine the proper number of resources added to each resource class with cost constraint in order to optimize workflow Time performance. The average Throughput Time of work- flow instances is used to measure the workflow Time performance. An approach which calculates the average Throughput Time of workflow instances is pro- posed. An improved genetic algorithm is presented to realize the allocation method. Experimental results show that the algorithm has good evolution per- formance and is superior to other allocation methods.

  • CSCWD (Selected Papers) - Optimization of workflow resources allocation with cost constraint
    Computer Supported Cooperative Work in Design III, 2006
    Co-Authors: Zhijiao Xiao, Huiyou Chang, Yang Yi
    Abstract:

    A resource allocation method is proposed to determine the proper number of resources added to each resource class with cost constraint in order to optimize workflow Time performance. The average Throughput Time of workflow instances is used to measure the workflow Time performance. An approach which calculates the average Throughput Time of workflow instances is proposed. An improved genetic algorithm is presented to realize the allocation method. Experimental results show that the algorithm has good evolution performance and is superior to other allocation methods.

Anson H L Tang - One of the best experts on this subject based on the ideXlab platform.

  • high Throughput Time stretch imaging flow cytometry for multi class classification of phytoplankton
    Optics Express, 2016
    Co-Authors: Anson H L Tang, Kenneth K Y Wong, Hayden Kwokhay So, Kevin K Tsia
    Abstract:

    Time-stretch imaging has been regarded as an attractive technique for high-Throughput imaging flow cytometry primarily owing to its real-Time, continuous ultrafast operation. Nevertheless, two key challenges remain: (1) sufficiently high Time-stretch image resolution and contrast is needed for visualizing sub-cellular complexity of single cells, and (2) the ability to unravel the heterogeneity and complexity of the highly diverse population of cells – a central problem of single-cell analysis in life sciences – is required. We here demonstrate an optofluidic Time-stretch imaging flow cytometer that enables these two features, in the context of high-Throughput multi-class (up to 14 classes) phytoplantkton screening and classification. Based on the comprehensive feature extraction and selection procedures, we show that the intracellular texture/morphology, which is revealed by high-resolution Time-stretch imaging, plays a critical role of improving the accuracy of phytoplankton classification, as high as 94.7%, based on multi-class support vector machine (SVM). We also demonstrate that high-resolution Time-stretch images, which allows exploitation of various feature domains, e.g. Fourier space, enables further sub-population identification – paving the way toward deeper learning and classification based on large-scale single-cell images. Not only applicable to biomedical diagnostic, this work is anticipated to find immediate applications in marine and biofuel research.

  • High-Throughput Time-stretch imaging cellular assay based on a high-speed spinning platform
    2016 IEEE Photonics Conference (IPC), 2016
    Co-Authors: Anson H L Tang, Kenneth K Y Wong, Antony C. S. Chan, P. Yeung, Barbara P. Chan, Kevin K Tsia
    Abstract:

    We propose and demonstrate a new strategy for high-Throughput, biochemical-specific, single-cell imaging cytometry, based on integration of a high-speed spinning planar platform (900-3600 rpm) and ultrafast optical Time-stretch imaging (at a line-scan rate of 11 MHz).

Robert K. Henderson - One of the best experts on this subject based on the ideXlab platform.

  • A High-Throughput Time-Resolved Mini-Silicon Photomultiplier With Embedded Fluorescence LifeTime Estimation in 0.13 $\mu$m CMOS
    IEEE Transactions on Biomedical Circuits and Systems, 2012
    Co-Authors: David Tyndall, David Day-uei Li, Jochen Arlt, Abigail Johnston, Justin A. Richardson, Robert K. Henderson
    Abstract:

    We describe a miniaturized, high-Throughput, Time-resolved fluorescence lifeTime sensor implemented in a 0.13 m CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of fluorescence lifeTime estimations on a single device. Detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with 400 ps output pulses and a 10% fill-factor. An array of Time-to-digital converters (TDCs) with ≈50 ps resolution records up to 8 photon events during each excitation period. Data from the TDC array is then processed using a centre-of-mass method (CMM) pre-calculation to produce fluorescence lifeTime estimations in real-Time. The sensor is believed to be the first reported implementation of embedded fluorescence lifeTime estimation. The system is demonstrated in a practical laboratory environment with measurements of a variety of fluorescent dyes with different single exponential lifeTimes, successfully showing the sensor's ability to overcome the classic pile-up limitation of Time-correlated single photon counting (TCSPC) by over an order of magnitude.