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Lloyd W.R.,University of Michigan | Wilson R.H.,University of Michigan | Chang C.-W.,University of Michigan | Gillispie G.D.,Fluorescence Innovations, Inc. | Mycek M.-A.,University of Michigan
Biomedical Optics Express | Year: 2010

A fiber-optic system was developed to rapidly acquire tissue fluorescence wavelength-time matrices (WTMs) with high signal-to-noise ratio (SNR). The essential system components (473 nm microchip laser operating at 3 kHz repetition frequency, fiber-probe assemblies, emission monochromator, photomultiplier tube, and digitizer) were assembled into a compact and clinically-compatible unit. Data were acquired from fluorescence standards and tissue-simulating phantoms to test system performance. Fluorescence decay waveforms with SNR > 100 at the decay curve peak were obtained in less than 30 ms. With optimized data transfer and monochromator stepping functions, it should be feasible to acquire a full WTM at 5 nm emission wavelength intervals over a 200 nm range in under 2 seconds. © 2010 Optical Society of America.


Muretta J.M.,University of Minnesota | Kyrychenko A.,University of Kansas Medical Center | Ladokhin A.S.,University of Kansas Medical Center | Kast D.J.,University of Minnesota | And 2 more authors.
Review of Scientific Instruments | Year: 2010

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 105 times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats. © 2010 American Institute of Physics.


Gauer J.W.,University of Minnesota | Sisk R.,University of Minnesota | Murphy J.R.,University of Minnesota | Jacobson H.,University of Minnesota | And 3 more authors.
Biophysical Journal | Year: 2012

The C2A domain is one of two calcium ion (Ca2+)- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca 2+ sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca2+ and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca2+- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca2+- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I. © 2012 by the Biophysical Society.


Peng Y.,Montana State University | Veneziano S.E.,Montana State University | Gillispie G.D.,Fluorescence Innovations, Inc. | Broderick J.B.,Montana State University
Journal of Biological Chemistry | Year: 2010

Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 Å from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the Tm of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed ↔ open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Gruber S.J.,University of Minnesota | Cornea R.L.,University of Minnesota | Li J.,University of Minnesota | Peterson K.C.,Fluorescence Innovations, Inc. | And 7 more authors.
Journal of Biomolecular Screening | Year: 2014

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 to many other targets. © 2013 Society for Laboratory Automation and Screening.


Cornea R.L.,University of Minnesota | Gruber S.J.,University of Minnesota | Lockamy E.L.,University of Minnesota | Muretta J.M.,University of Minnesota | And 8 more authors.
Journal of Biomolecular Screening | Year: 2013

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS. © 2013 Society for Laboratory Automation and Screening.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase II | Award Amount: 1.50M | Year: 2015

DESCRIPTION provided by applicant We seek to develop and commercialize a powerful technology platform to increase dramatically the effectiveness of drug discovery using high throughput screening HTS based on fluorescence lifetime FLT readouts of FRET in live cells This breakthrough is enabled by a combination of two complementary and synergistic technologies fluorescent biosensor engineering University of Minnesota UMN and fluorescence instrumentation engineering Fluorescence Innovations FI In Phase I we achieved our aims producing the first truly high throughput and high precision applications of FLT in living cells applied to a specific biosensor SERCA We also surpassed our aims by developing new instrumentation for high throughput spectral recording carrying out a SERCA screen on a compund library and developing FRET biosensors based on several new targets In addition several pharmaceutical companies expressed interest in this technology platform so UMN and FI started a new drug discovery company Photonic Pharma for the ultimate commercialization of these combined technologies In Phase II we will further develop this technology platform and apply it to a diverse array of targets thus clearly demonstrating the high potential for commercialization In In Aim we will seek improvements in hardware and software that will enhance future commercialization potential including a new instrument that combines FLT and spectral recording increased digitizer speed higher density plates connectivity with robotics and software improvements In Aim in order to demonstrate that our technology platform is widely applicable we will expand our list of biosensors focusing on targets of high commercial potential for which world leading experts are in close proximity at UMN These disease targets include heart failure drug abuse and addiction inflammation and cancer and muscle dystonia Biosensors will be engineered and expressed in live cells and screened against standard test and validation libraries to optimize the screening assay We will then carry out several large scale compound screens using optimized HTS assays Hit compounds will be retested as a function of compound concentration dose response and then subjected to functional assays performed by the above world class experts at UMN Promising hits may become leads for future medicinal chemistry development but the primary goal is to validate the discovery platform We are confident that the research conducted under these aims will set us up nicely for the commercialization phase by validating our business plan to combine UMN expertise in biosensor engineering and cell culture with FI expertise in instrumentation led by Photonic Pharma an emerging early phase drug discovery start up company Thus our business plan is not to sell instruments but to leverage our unique combination of biosensor and instrumentation expertise to develop and apply a technology platform that accelerates the early phase of drug discovery PUBLIC HEALTH RELEVANCE Fluorescence Innovations Inc in collaboration with the University of Minnesota proposes to greatly improve the technology for drug discovery during the crucial early stages of the process This technology has great commercial potential because it will make the process of drug discovery much more effective and efficient and is applicable to a wide range of health issues including heart failure diabetes cancer and drug addiction


PubMed | University of Minnesota and Fluorescence Innovations, Inc.
Type: | Journal: Journal of biomolecular screening | Year: 2016

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCAs distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCAs ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.


PubMed | University of Minnesota and Fluorescence Innovations, Inc.
Type: | Journal: Journal of biomolecular screening | Year: 2016

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 253.93K | Year: 2013

DESCRIPTION (provided by applicant): This project will establish proof-of-concept for a powerful and versatile implementation of live-cell assays in a true high-throughput screening (HTS) format for small-molecule drug discovery. The technological basis isfluorescence lifetime (FLT) readout of FRET between fluorescent fusion proteins. Lifetime measurement is needed in HTS to overcome the low precision of conventional fluorescence intensity measurements, which is particularly severe in live-cell assays. However, conventional lifetime technology, i.e., time-correlated single-photon counting (TCSPC), takes at least 10 seconds per sample to obtain adequate precision for HTS. Thus, whether carried out in a microplate reader or in fluorescence lifetime imaging microscopy (FLIM), TCSPC is much too slow for practical HTS. Our team has taken an entirely fresh and creative approach, which critically relies on the revolutionary NovaFluor PR fluorescence lifetime microplate reader developed by Fluorescence Innovations.NovaFluor employs Direct Waveform Recording (DWR), an exceptionally fast and precise fluorescence lifetime method recently developed in collaboration between FI and the Thomas research group at the University of Minnesota. DWR provides precision and resolution equivalent to TCSPC while dramatically increasing the speed of data acquisition. Of all the existing fluorescence lifetime methods, only DWR offers both the speed and precision needed for effective HTS. Our other breakthrough innovation is Cells-and-Wells (CNW). We simultaneously measure the response of hundreds of cells in a microplate well, after excitation with a pulsed laser, and the lifetime readout provides HTS data as fast as any intensity-based assay employing purified protein targets, but withan order of magnitude better precision and resolution. Aim 1 is to demonstrate the CNW method on two well-defined test systems, cleavage of a labeled peptide by caspase-3 and ubiquitination of -synuclein, in order to optimize procedures in cell handling,data acquisition, instrument configuration, and data reduction. Aim 2 is to develop a high-performance assay for an important protein target, SERCA, the sarco(endo)plasmic reticulum Ca-ATPase, which is key to calcium regulation in all mammalian cells, andof particular interest in heart failure therapies. The Thomas group leads the world in developing spectroscopic probes of SERCA. Aim 3 is to conduct a first-pass screening with the LOPAC library. This work will set the stage for a more comprehensive exploration of chemical space in Phase II, leading to successful commercialization of FLT technology for drug discovery. The significance of this project stems from the clear potential of FLT in live cells to revolutionize HTS, resulting in a vastly improved input into the drug discovery process. We envision our approach will enable successful drug discovery campaigns for a wide range of targets and systems that currently can only be screened by fluorescence intensity. The high potential significance of fluorescence lifetime in HTS will make this a high-impact project, even in Phase I. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Fluorescence Innovations, Inc., in collaboration with the University of Minnesota proposes to greatly improve the technology for drug discovery. This technology has great commercial potential, because it will make the process of drug discovery much more effective and efficient, particularly for the discovery of drugs for the treatment of heart failure.

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