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Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening *

  • Alessandro Esposito
    Correspondence
    To whom correspondence should be addressed: Laser Analytics Group, Dept. of Chemical Engineering, University of Cambridge, Pembroke St., Cambridge CB2 3RA, UK. Tel.: 44-1223-334193; Fax: 49-551-39-123-46;
    Affiliations
    Cell Biophysics Group, European Neuroscience Institute-Göttingen, Waldweg 33, 37073 Göttingen, Germany

    Deutsche Forschungsgemeinschaft (DFG) Center for Molecular Physiology of the Brain (CMPB), 37073 Göttingen, Germany
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  • Christoph P. Dohm
    Footnotes
    Affiliations
    Deutsche Forschungsgemeinschaft (DFG) Center for Molecular Physiology of the Brain (CMPB), 37073 Göttingen, Germany

    Department of Neurology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany
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  • Matthias Bähr
    Affiliations
    Deutsche Forschungsgemeinschaft (DFG) Center for Molecular Physiology of the Brain (CMPB), 37073 Göttingen, Germany

    Department of Neurology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany
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  • Fred S. Wouters
    Affiliations
    Cell Biophysics Group, European Neuroscience Institute-Göttingen, Waldweg 33, 37073 Göttingen, Germany

    Deutsche Forschungsgemeinschaft (DFG) Center for Molecular Physiology of the Brain (CMPB), 37073 Göttingen, Germany
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  • Author Footnotes
    * This work was supported in part by the DFG Research Center for Molecular Physiology of the Brain and the Network of European Neuroscience Institutes (ENI-NET) consortium. The European Neuroscience Institute-Göttingen is jointly funded by the Göttingen University Medical School, the Max Planck Society, and Schering AG. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
    ** Supported by the NeuroNE network of excellence within the 6th framework program of the European Union.
Open AccessPublished:May 16, 2007DOI:https://doi.org/10.1074/mcp.T700006-MCP200
      Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of α-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant α-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.
      Above and beyond the isolation and identification of proteins, the field of proteomics faces the challenges of detecting protein cellular localization and quantifying molecular states such as protein conformations, protein-protein interactions, and post-translational modifications. In the past decade, Förster resonance energy transfer (FRET)
      The abbreviations used are: FRET, Förster resonance energy transfer; CCD, charge-coupled device; CV, coefficient of variation; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; FLIM, fluorescence lifetime imaging microscopy; (u)HTS, (ultra)high throughput screening; ICAS, image cytometry for analysis and sorting; ICCD, intensified charge-coupled device; REACh, resonance energy-accepting chromoprotein; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CHO, Chinese hamster ovary; R6G, rhodamine 6G.
      1The abbreviations used are: FRET, Förster resonance energy transfer; CCD, charge-coupled device; CV, coefficient of variation; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; FLIM, fluorescence lifetime imaging microscopy; (u)HTS, (ultra)high throughput screening; ICAS, image cytometry for analysis and sorting; ICCD, intensified charge-coupled device; REACh, resonance energy-accepting chromoprotein; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CHO, Chinese hamster ovary; R6G, rhodamine 6G.
      and fluorescence lifetime imaging microscopy (FLIM) have proven to be instrumental for the quantitative imaging of these biochemical states in single cells (
      • Wouters F.S.
      The physics and biology of fluorescence microscopy in the life sciences.
      ). Similarly the analysis of different cellular populations (cellomes) will also benefit from these imaging methods. Quantitative multiparametric microscopy is a very young field in which advances in liquid/sample handling robotics and information technology are gradually being integrated into automated microscopes (
      • Schubert W.
      • Bonnekoh B.
      • Pommer A.J.
      • Philipsen L.
      • Bockelmann R.
      • Malykh Y.
      • Gollnick H.
      • Friedenberger M.
      • Bode M.
      • Dress A.W.
      Analyzing proteome topology and function by automated multidimensional fluorescence microscopy.
      ,
      • Eggeling C.
      • Brand L.
      • Ullmann D.
      • Jager S.
      Highly sensitive fluorescence detection technology currently available for HTS.
      ). These automated imaging systems merge the high content image information with the high throughput volumes provided by their automation and unsupervised operation.
      Screening techniques have now reached (ultra)high throughput levels, i.e. they are capable of performing more than 105 assays/day in microliter volumes. Such a high throughput is necessary for applications where (bio)chemical libraries are tested, e.g. for drug discovery and interactomics research (
      • Croston G.E.
      Functional cell-based uHTS in chemical genomic drug discovery.
      ). Although the advance in throughput scale is necessary, it is often accompanied by low information content. Evidently multiparametric detection at high numbers would present a powerful tool. Moreover the screening reproducibility and estimators need to respect comparatively high quality standards, e.g. coefficient of variations (CVs) and z-scores should not exceed 5% and should be higher than 0.5, respectively.
      High content applications typically involve the quantitative and multiparametric analysis of the effect of analytes or other perturbing conditions on cellular behavior (
      • Giuliano K.A.
      • Cheung W.S.
      • Curran D.P.
      • Day B.W.
      • Kassick A.J.
      • Lazo J.S.
      • Nelson S.G.
      • Shin Y.
      • Taylor D.L.
      Systems cell biology knowledge created from high content screening.
      ). The understanding of molecular mechanisms underlying disease, for instance, requires high resolution information because the screens target the cellular and/or subcellular level. Such applications aim at the monitoring of molecular pathways: the localization and interactions of biomolecules and their altered behavior in response to drugs or pathogens. An automated fluorescence lifetime imaging microscope capable of unsupervised operation was developed to provide the basis for a scalable screening platform that combines high throughput levels and high content information gained from quantitative multiparametric imaging.
      Fluorescent protein engineering offers a wide variety of genetically expressible fluorescent biosensors, e.g. for the detection of ion concentration, pH, molecular oxygen, proteolytic and chaperone activity, and ubiquitination, many of which can be quantitatively detected by fluorescence lifetime sensing (
      • Bunt G.
      • Wouters F.S.
      Visualization of molecular activities inside living cells with fluorescent labels.
      ). Their exquisite selectivity is derived from the fact that these biosensors can be targeted to specific proteins of interest, organelles, and other subcompartments of the cell. In addition, a wide variety of site-specific orthogonal protein labeling strategies using synthetic dyes is available nowadays, e.g. FlAsH (fluorescein arsenical hairpin binder), ReAsH (resorufin arsenical hairpin binder), SnapTag, HaloTag (Promega), and CoA binding (
      • Bunt G.
      • Wouters F.S.
      Visualization of molecular activities inside living cells with fluorescent labels.
      ). The availability of commercial systems for automated fluorescence imaging is constantly growing (
      • Eggeling C.
      • Brand L.
      • Ullmann D.
      • Jager S.
      Highly sensitive fluorescence detection technology currently available for HTS.
      ,
      • Ramm P.
      Image-based screening: a technology in transition.
      ,
      • Mitchison T.J.
      Small-molecule screening and profiling by using automated microscopy.
      ). Moreover recent works demonstrate the usefulness of time-resolved fluorescence assays in screening (
      • Fowler A.
      • Swift D.
      • Longman E.
      • Acornley A.
      • Hemsley P.
      • Murray D.
      • Unitt J.
      • Dale I.
      • Sullivan E.
      • Coldwell M.
      An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases.
      ,
      • Turconi S.
      • Bingham R.P.
      • Haupts U.
      • Pope A.J.
      Developments in fluorescence lifetime-based analysis for ultra-HTS.
      ).
      In this work, an automated FLIM that is based on state-of-the-art technology, i.e. intensified charge-coupled devices (ICCDs), is described. We recently introduced new all-solid-state technologies (
      • Esposito A.
      • Oggier T.
      • Gerritsen H.C.
      • Lustenberger F.
      • Wouters F.S.
      All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing.
      ,
      • Esposito A.
      • Gerritsen H.C.
      • Lustenberger F.
      • Oggier T.
      • Wouters F.S.
      Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics.
      ) that will enable the construction of cost-effective and turnkey systems that do not require specialized knowledge for their maintenance and operation.
      In light of the presented results and novel technologies, we envisage comparatively inexpensive and simple high throughput and high content quantitative screening platforms to become available in the near future. These systems would provide a substantial impulse to the recent and actively expanding fields of drug discovery, interactomics, cellomics, and proteomics.

      DISCUSSION

      FRET operates at intermolecular distances on the scale of protein dimensions (<10 nm) and exhibits sensitivity to changes in the Ångstrom range. FLIM provides a non-invasive, fast, and quantitative FRET measurement, thus giving access to molecular information like protein-protein interactions and conformational changes. Furthermore lifetime sensing was used for the quantification of oxygen content, ion concentration, and pH and can be used to map biochemical events in living cells (
      • Bunt G.
      • Wouters F.S.
      Visualization of molecular activities inside living cells with fluorescent labels.
      ), proving its value for molecular proteomics studies. The diversity of available synthetic dyes with sensing capabilities for different small molecules and conditions can be exploited by FLIM to create new sensitive and reproducible assays for a variety of cellular functions. This holds particularly true for those dyes that respond with otherwise difficult to calibrate quantum yield changes and that are now avoided in favor of ratiometric dyes.
      Such detailed and quantitative information is equally important for the life sciences and the screening industry. It was shown (see Fig. 1) that an automated FLIM, capable of unsupervised operation, provides very high throughput with good reproducibility (CV < 5%) and sensitivity (high z-score). An assay is considered robust when its statistical z-score exceeds 0.5 (
      • Golla R.
      • Seethala R.
      A sensitive, robust high-throughput electrochemiluminescence assay for rat insulin.
      ). With the coefficient of variation in our studies, this stringent statistical requirement can be fulfilled with 20% lifetime difference detected in a single well. High sensitivity and reliability are of crucial importance for the FRET-based detection of protein-protein interactions and protein conformational changes. Furthermore assays can be performed in a variable environment, e.g. in cells and in “homogeneous” assay formats that do not require washing steps, by the virtue of the independence of the fluorescence lifetime from fluorophore concentration. FLIM screening platforms could be used for the validation of protein-protein interaction found by other (u)HTS approaches. One such application example is shown for the screening of ubiquitination levels of α-synuclein and its familial mutations that are causative for Parkinson disease. With its high throughput, automated FLIM systems could be directly used for the screening of fluorescently labeled genomics banks or drug libraries.
      Our experiments also exemplify that the scalability of an automated microscope allows the analysis of samples that do not respect a standardized format: we showed the unsupervised imaging of microtiter plates (Fig. 1), bacterial plates (Fig. 2), and microscope slides (Fig. 3, Fig. 4, Fig. 5). Other samples like tissue slices, electrophoresis gels, DNA or protein arrays, and nanotiter plates could also be easily accommodated.
      Fig. 2 shows the screening of bacterial colonies. Besides screening for optimization of fluorescent proteins and fluorescent biosensors by random mutagenesis, fluorescence lifetime-based assays could be performed in bacteria as a biological model system that carries the advantage of the simplicity of sample handling, biochemistry, and retrieval of genetic/proteomic compositions.
      The microscope stores the relative position of each imaged object. The sample can therefore be revisited iteratively for real time data analysis. In addition to the “inventory” use of the platform in cell screening, the platform can therefore also be used to “hunt” for rare events with the aim of sample retrieval. Single colonies, cells, or cellular subpopulations could be isolated, for instance, by photogelation procedures (
      • Fulwyler M.
      • Hanley Q.S.
      • Schnetter C.
      • Young I.T.
      • Jares-Erijman E.A.
      • Arndt-Jovin D.
      • Jovin T.M.
      Selective photoreactions in a programmable array microscope (PAM): photoinitiated polymerization, photodecaging, and photochromic conversion.
      ) or laser microdissection and pressure catapulting (
      • Burgemeister R.
      New aspects of laser microdissection in research and routine.
      ) techniques. The protein or genetic content of the objects with specific lifetime properties can then be analyzed by the relevant techniques.
      These two modes of operation are generally known as image cytometry for analysis and sorting (ICAS) (
      • Fulwyler M.
      • Hanley Q.S.
      • Schnetter C.
      • Young I.T.
      • Jares-Erijman E.A.
      • Arndt-Jovin D.
      • Jovin T.M.
      Selective photoreactions in a programmable array microscope (PAM): photoinitiated polymerization, photodecaging, and photochromic conversion.
      ). ICAS is suitable for adherent cells and tissues where flow cytometric techniques cannot be used. Our work shows that the highly informative and sensitive fluorescence lifetime parameter can be used for the selection of cells for ICAS.
      Fig. 3 demonstrates the unsupervised cellular imaging and data analysis of extended surfaces. Data acquisition with six phase images was performed here to analyze the lifetime heterogeneity (
      • Esposito A.
      • Gerritsen H.C.
      • Wouters F.S.
      Fluorescence lifetime heterogeneity resolution in the frequency-domain by Lifetime Moments Analysis (LiMA).
      ) and to compensate for photobleaching (
      • van Munster E.B.
      • Gadella Jr., T.W.
      Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.
      ). In the case of FRET imaging, the quantification of lifetime heterogeneity by lifetime moment analysis can provide a measure of the molecular fraction that undergoes FRET, e.g. the relative concentration of interacting proteins and their average intermolecular distance. When photobleaching and lifetime heterogeneity of the fluorophores can be neglected, the rapid lifetime determination algorithm that requires only two phase-dependent images can be used. Under these conditions, the screening of an entire 4-well Labtek chamber would take a third of the current time, i.e. 30 min. The maximal cell density and transfection efficiency that allow single cells to be distinguished amount to ∼100,000 cells in this format. Therefore, a maximum of 200,000 cells/h can be screened with a 20× objective. The screening can be repeated over time by imaging extended surfaces or a user-defined group of cells (data not shown). This enables the measurement of temporal responses over a high number of cells.
      Figs. 3 and 4 (see also Supplemental Figs. 2–7) exemplify how cellular subpopulations can be analyzed by imaging single cells. The differences between the two co-transfection conditions used would be impossible to resolve when only the averages over these large numbers of cells were considered. The analysis of cell populations is important for the understanding of the regulation and molecular mechanisms of biological events as biological models are usually heterogeneous. The capability of screening and segmenting diverse cellular populations combined with the possibility to detect protein-protein interactions can offer a significant advantage for the fields of cellular proteomics and interactomics.
      Quantitative multiparametric microscopy and automated unsupervised microscopy are comparatively young techniques that attract a growing number of industrial and academic research groups. This work represents an advance in the combination of these technologies and demonstrates that current technologies can be used for the construction of an unsupervised FLIM system for high throughput and high content screening. Several commercial automated systems could be adapted for lifetime sensing, immediately offering a powerful tool for the screening community. The experiments presented in this work represent well defined benchmarks for the characterization of the quality of the data that are generated and for the application of software solutions for the detailed statistical analyses that can be performed. FRET assays enjoy an increasing popularity in the life sciences and represent the major application of our platform. The feasibility of sensitive FRET assays on our platform is demonstrated by its high quality and sensitivity. A lifetime difference of 300 ps can be clearly separated. Furthermore taking into account the CV, 95% of the cells could be successfully classified. This difference corresponds to a FRET efficiency of ∼12% with fully separated distributions. In the same experiment, three populations differing by only ∼6% were still reliably separated. The ubiquitination assay (Fig. 5) shows that differences of ∼4% can be distinguished (wild type versus A30P mutant) with high statistical significance (p < 0.01, Student's t test) by the use of the same fluorescence assays used in conventional microscopy.
      This remarkable resolution in the biochemical event of protein ubiquitination is only achieved by the automation of the lifetime microscope, combining high throughput with high content information; large cell numbers in the sample were subjected to the uniquely quantitative determination of FRET by lifetime microscopy. The cell-based statistics identify differences in the ubiquitination of disease-related mutant forms of the α-synuclein protein. These forms are ubiquitinated less than the wild type α-synuclein. All three α-synuclein proteins seem to share two basal states of ubiquitination, but the wild type protein possesses an additional high ubiquitination state that drives the average significantly upward. The cellular response to the presence of α-synuclein ubiquitination substrates is thus intrinsically heterogeneous, a fact that would be lost if this modification is measured by biochemical means with imaging approaches that do not resolve the individual responses (e.g. plate readers) or by manual acquisition of only a few cells. Because ubiquitination of unwanted proteins, leading up to their proteasomal digestion, is an integral part of the detoxification machinery of the cell, the observed altered behavior of the familial disease-causing α-synuclein mutants is important for the understanding of the pathophysiology of Parkinson disease. These novel findings are in agreement with the slower proteolytic degradation of A53T α-synuclein observed in pulse-chase biochemical experiments (
      • Bennett M.C.
      • Bishop J.F.
      • Leng Y.
      • Chock P.B.
      • Chase T.N.
      • Mouradian M.M.
      Degradation of α-synuclein by proteasome.
      ). Furthermore the clinical hallmark of this disease is the presence of intracellular inclusion bodies, called Lewy bodies, which contain ubiquitinated α-synuclein (
      • Tofaris G.K.
      • Razzaq A.
      • Ghetti B.
      • Lilley K.S.
      • Spillantini M.G.
      Ubiquitination of α-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function.
      ), and ubiquitin-proteasomal dysfunction is generally considered to be an important feature of Parkinson disease (
      • McNaught K.S.P.
      • Olanow C.W.
      • Halliwell B.
      • Isacson O.
      • Jenner P.
      Failure of the ubiquitin-proteasome system in Parkinson's disease.
      ). However, it is not known in what way these observations are causally connected. The high sensitivity afforded by our screen is crucial because, given the progression of Parkinson disease over decades, even minor impediments of α-synuclein degradation could favor the formation of aggregates. The mutations have been suggested to inhibit proteasomal activity (27), but differences in their ubiquitination levels were never reported by conventional biochemical methods. For the first time, it was shown that the ubiquitination level of α-synuclein can be quantitatively imaged in cells and that the mutants are significantly less ubiquitinated. As the mutants exhibit an increased tendency toward self-aggregation, giving rise to neurotoxicity, their decreased ubiquitination might indicate that their altered proteolytic processing contributes to aggregation. On the other hand, ubiquitination might represent a mechanism that protects α-synuclein from aggregation. These issues warrant further research.

      Acknowledgments

      We thank Prof. Gerhard Braus and Dr. Lars Fichtner for access to liquid handling robotics and Dirk Lange for valuable assistance.

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