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  • 1
    Keywords: MICROSCOPY ; IMAGES ; PAIRS ; LIVING CELLS ; COMPUTER-PROGRAM ; RESONANCE ENERGY-TRANSFER ; transfer efficiency ; fluorescent proteins ; STATE ; BY-CELL BASIS
    Abstract: Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. (c) 2013 International Society for Advancement of Cytometry.
    Type of Publication: Journal article published
    PubMed ID: 23843167
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  • 2
    Keywords: MOLECULE ; CHROMATIN ; CRYSTAL-STRUCTURE ; FLUORESCENCE ; ANGSTROM RESOLUTION ; COMPACTION ; RESONANCE ENERGY-TRANSFER ; CORE PARTICLE ; ACCESSIBILITY ; CONFORMATIONAL DYNAMICS
    Abstract: Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule Forster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA. (c) 2013 International Society for Advancement of Cytometry.
    Type of Publication: Journal article published
    PubMed ID: 23843180
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  • 3
    Keywords: segmentation ; RESOLUTION ; IN-SITU HYBRIDIZATION ; STEM-CELLS ; RECONSTRUCTION ; CHROMOSOMES ; FLUORESCENCE MICROSCOPY ; automation ; IMAGE-ANALYSIS ; INTERPHASE CELL-NUCLEI
    Abstract: Reliable 3D detection of diffraction-limited spots in fluorescence microscopy images is an important task in subcellular observation. Generally, fluorescence microscopy images are heavily degraded by noise and non-specifically stained background, making reliable detection a challenging task. In this work, we have studied the performance and parameter sensitivity of eight recent methods for 3D spot detection. The study is based on both 3D synthetic image data and 3D real confocal microscopy images. The synthetic images were generated using a simulator modeling the complete imaging setup, including the optical path as well as the image acquisition process. We studied the detection performance and parameter sensitivity under different noise levels and under the influence of uneven background signal. To evaluate the parameter sensitivity, we propose a novel measure based on the gradient magnitude of the F1 score. We measured the success rate of the individual methods for different types of the image data and found that the type of image degradation is an important factor. Using the F1 score and the newly proposed sensitivity measure, we found that the parameter sensitivity is not necessarily proportional to the success rate of a method. This also provided an explanation why the best performing method for synthetic data was outperformed by other methods when applied to the real microscopy images. On the basis of the results obtained, we conclude with the recommendation of the HDome method for data with relatively low variations in quality, or the Sorokin method for image sets in which the quality varies more. We also provide alternative recommendations for high-quality images, and for situations in which detailed parameter tuning might be deemed expensive. (c) 2015 International Society for Advancement of Cytometry.
    Type of Publication: Journal article published
    PubMed ID: 26033916
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  • 4
    Keywords: ALGORITHMS ; SUPPORT ; GENES ; FLUORESCENCE MICROSCOPY ; IMAGE-ANALYSIS ; BIOIMAGE INFORMATICS
    Abstract: Computational approaches for automatic analysis of image-based high-throughput and high-content screens are gaining increased importance to cope with the large amounts of data generated by automated microscopy systems. Typically, automatic image analysis is used to extract phenotypic information once all images of a screen have been acquired. However, also in earlier stages of large-scale experiments image analysis is important, in particular, to support and accelerate the tedious and time-consuming optimization of the experimental conditions and technical settings. We here present a novel approach for automatic, large-scale analysis and experimental optimization with application to a screen on neuroblastoma cell lines. Our approach consists of cell segmentation, tracking, feature extraction, classification, and model-based error correction. The approach can be used for experimental optimization by extracting quantitative information which allows experimentalists to optimally choose and to verify the experimental parameters. This involves systematically studying the global cell movement and proliferation behavior. Moreover, we performed a comprehensive phenotypic analysis of a large-scale neuroblastoma screen including the detection of rare division events such as multi-polar divisions. Major challenges of the analyzed high-throughput data are the relatively low spatio-temporal resolution in conjunction with densely growing cells as well as the high variability of the data. To account for the data variability we optimized feature extraction and classification, and introduced a gray value normalization technique as well as a novel approach for automatic model-based correction of classification errors. In total, we analyzed 4,400 real image sequences, covering observation periods of around 120 h each. We performed an extensive quantitative evaluation, which showed that our approach yields high accuracies of 92.2% for segmentation, 98.2% for tracking, and 86.5% for classification. (c) 2015 International Society for Advancement of Cytometry
    Type of Publication: Journal article published
    PubMed ID: 25630981
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  • 5
    Abstract: Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability.
    Type of Publication: Journal article published
    PubMed ID: 25297339
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  • 6
    Abstract: The advent of novel strategies to generate leukemia-associated-antigen (LAA)-specific T cells for adoptive immunotherapies creates a demand for standardized good laboratory practice (GLP)-compliant enumeration assays to provide a secure clinical environment-whether it is to identify potential donors, define therapeutic doses for transplantation, or monitor clinical success. Here, we introduce a no-wash assay based on single-platform cell enumeration and Streptamer staining to determine the Wilms' tumor antigen 1 (WT1)-specific T cell immunity in clinical samples. We analyzed the performance of the WT1-specific MHC Streptamers in direct comparison to CMV- and EBV-specific MHC Streptamer staining by spiking antigen-specific T cells in PBMCs. The accuracy of the assay was high for all performed experiments with a mean recovery of 94% and a linear regression of 0.988. Differences were apparent regarding the limit of detection/quantification (LOD/LOQ). While results obtained for WT1 yielded an LOD/LOQ of 0.08 +/- 0.04% and 0.11 +/- 0.06% (1.33 +/- 0.32 cells/microl and 1.9 +/- 0.14 cells/microl), the overall LOD/LOQ was notably lower and accounted to 0.02 +/- 0.02% and 0.05 +/- 0.03% (0.60 +/- 0.03 cells/microl and 1.27 +/- 0.58 cells/microl). Subsequent screening of 22 healthy individuals revealed significantly higher values for WT1 (0.04 +/- 0.02% and 1.5 +/- 0.9 cells/microl) than for the irrelevant HIV pol (0.016 +/- 0.01% and 0.5 +/- 0.4 cells/microl). In contrast, no increased frequencies were observed for WT1-specific T cells compared to HIV-specific T cells using a classical wash-protocol. These findings strongly suggest the use of no-wash single-platform assays in combination with MHC Streptamer staining for the detection of low affinity LAA-specific T cells due to its high accuracy and sensitivity. (c) 2017 International Society for Advancement of Cytometry.
    Type of Publication: Journal article published
    PubMed ID: 28544366
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  • 7
    Keywords: CANCER ; MICROSCOPY ; INFORMATION ; QUANTIFICATION ; POPULATION ; NUCLEI ; ACQUISITION ; NUMBER ; IN-SITU HYBRIDIZATION ; FISH ; LENGTH ; CHILDREN ; FLUORESCENCE ; INTERPHASE NUCLEI ; flow-FISH ; SOCIETY ; senescence ; telomere ; automatic ; HUMAN-CELLS ; ALT
    Abstract: To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations. (c) 2005 international Society for Analytical Cytology
    Type of Publication: Journal article published
    PubMed ID: 16228977
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  • 8
    Keywords: CELLS ; CELL ; MICROSCOPY ; MODEL ; MODELS ; imaging ; PROTEIN ; PROTEINS ; TRANSDUCTION ; COMPLEX ; MARKER ; DYNAMICS ; BIOLOGY ; RECOGNITION ; antibodies ; DIFFERENCE ; MEMBRANE ; SIGNAL-TRANSDUCTION ; REGION ; REGIONS ; PLASMA-MEMBRANE ; cholesterol ; T-LYMPHOCYTES ; LIPID RAFTS ; sensitivity ; LIVING CELLS ; DOMAINS ; HETEROGENEITY ; CORRELATION SPECTROSCOPY ; ENERGY-TRANSFER ; analysis ; methods ; technique ; USA ; correlation ; MODEL MEMBRANES ; FCS ; confocal polarization imaging ; GPI-MICRODOMAINS ; live lymphoid cells ; membrane rafts ; raft modulation
    Abstract: Lipid rafts are cholesterol- and glycosphingolipid-rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy-phosphatidylcholine was imaged with differential polarization CLSM (DP-CLSM). Mobility of the dil probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI-linked protein markers of rafts and a new anti -cholesterol antibody (AC8) in various immune cells. On the same cells., albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP-CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property-in accordance with dil mobility assessed by FCS-was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes-G(M1) and a new anti-cholesterol antibody-equivocally label the membrane rafts on a variety of cell types, while some raft-associated proteins (MHC-II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP-CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts. (c) 2007 International Society for Analytical Cytology
    Type of Publication: Journal article published
    PubMed ID: 18163467
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  • 9
    Keywords: CELLS ; CELL ; Germany ; MICROSCOPY ; MODEL ; ALGORITHMS ; CLASSIFICATION ; IMAGES ; imaging ; QUANTIFICATION ; screening ; segmentation ; SYSTEM ; GENE ; GENES ; LINES ; INFECTION ; image analysis ; BIOLOGY ; SIGNAL ; NUCLEI ; ENTRY ; virus ; IDENTIFICATION ; RATES ; LOCALIZATION ; NUCLEUS ; REPLICATION ; IV ; hepatitis C ; ASSAYS ; HIGH-THROUGHPUT ; SCREEN ; USA ; HEPATITIS-C ; WELL ; 33 ; Determination ; ROBUST ; Immunofluorescence microscopy ; AUTOMATED MICROSCOPY ; cell nucleus segmentation ; cell-based arrays ; image quality control ; quantification of viral infection ; siRNA screening
    Abstract: The identification of eukaryotic genes involved in virus entry and replication is important for understanding viral infection. Our goal is to develop a siRNA-based screening system using cell arrays and high-throughput (HT) fluorescence microscopy. A central issue is efficient, robust, and automated single-cell-based analysis of massive image datasets. We have developed an image analysis approach that comprises (i) a novel, gradient-based thresholding scheme for cell nuclei segmentation which does not require subsequent postprocessing steps for separation of clustered nuclei, (ii) quantification of the virus signal in the neighborhood of cell nuclei, (iii) localization of regions with transfected cells by combining model-based circle fitting and grid fitting, (iv) cell classification as infected or noninfected, and (v) image quality control (e.g., identification of out-of-focus images). We compared the results of our nucleus segmentation approach with a previously developed scheme of adaptive thresholding with subsequent separation of nuclear clusters. Our approach, which does not require a postprocessing step for the separation of nuclear clusters, correctly segmented 97.1% of the nuclei, whereas the previous scheme achieved 95.8%. Using our algorithm for the detection of out-of-focus images, we obtained a high discrimination power of 99.4%. Our overall approach has been applied to more than 55,000 images of cells infected by either hepatitis C or dengue virus. Reduced infection rates were correctly detected in positive siRNA controls, as well as for siRNAs targeting, for example, cellular genes involved in viral infection. Our image analysis approach allows for the automatic and accurate determination of changes in viral infection based on high-throughput single-cell-based siRNA cell array imaging experiments. (C) 2008 International Society for Advancement of cytometry
    Type of Publication: Journal article published
    PubMed ID: 19006066
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  • 10
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