| 1. |
- Adler, Jeremy, et al.
(author)
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Quantifying Colocalization by Correlation : The Pearson Correlation Coefficient is Superior to the Mander's Overlap Coefficient
- 2010
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In: CYTOMETRY PART A. - : Wiley. - 1552-4922 .- 1552-4930. ; 77A:8, s. 733-742
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Journal article (peer-reviewed)abstract
- The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence.
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| 2. |
- Allalou, Amin, et al.
(author)
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Robust signal detection in 3D fluorescence microscopy
- 2010
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In: Cytometry. Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 77A:1, s. 86-96
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Journal article (peer-reviewed)abstract
- Robust detection and localization of biomolecules inside cells is of great importance to better understand the functions related to them. Fluorescence microscopy and specific staining methods make biomolecules appear as point-like signals on image data, often acquired in 3D. Visual detection of such point-like signals can be time consuming and problematic if the 3D images are large, containing many, sometimes overlapping, signals. This sets a demand for robust automated methods for accurate detection of signals in 3D fluorescence microscopy. We propose a new 3D point-source signal detection method that is based on Fourier series. The method consists of two parts, a detector, which is a cosine filter to enhance the point-like signals, and a verifier, which is a sine filter to validate the result from the detector. Compared to conventional methods, our method shows better robustness to noise and good ability to resolve signals that are spatially close. Tests on image data show that the method has equivalent accuracy in signal detection in comparison to Visual detection by experts. The proposed method can be used as an efficient point-like signal detection tool for various types of biological 3D image data.
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| 3. |
- Cieslar-Pobuda, Artur, et al.
(author)
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Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
- 2014
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In: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 85A:7, s. 628-635
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Journal article (peer-reviewed)abstract
- Fluorescent compounds capable of staining cells selectively without affecting their viability are gaining importance in biology and medicine. Recently, a new family of optical dyes, denoted luminescent conjugated oligothiophenes (LCOs), has emerged as an interesting class of highly emissive molecules for studying various biological phenomena. Properly functionalized LCOs have been utilized for selective identification of disease-associated protein aggregates and for selective detection of distinct cells. Herein, we present data on differential staining of various cell types, including cancer cells. The differential staining observed with newly developed pentameric LCOs is attributed to distinct side chain functionalities along the thiophene backbone. Employing flow cytometry and fluorescence microscopy we examined a library of LCOs for stainability of a variety of cell lines. Among tested dyes we found promising candidates that showed strong or moderate capability to stain cells to different extent, depending on target cells. Hence, LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells, whereas LCOs with other amino acid side chains along the backbone showed a complete lack of staining for the cells included in the study. Furthermore, for p-HTMI,a LCO functionalized with methylated imidazole moieties, the staining was dependent on the p53 status of the cells, indicating that the molecular target for the dye is a cellular component regulated by p53. We foresee that functionalized LCOs will serve as a new class of optical ligands for fluorescent classification of cells and expand the toolbox of reagents for fluorescent live imaging of different cells.
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| 4. |
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| 5. |
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| 6. |
- Gréen,, Anna, 1973-, et al.
(author)
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Translocation of Histone H1 Subtypes Between Chromatin and Cytoplasm During Mitosis in Normal Human Fibroblasts
- 2010
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In: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 77A:5, s. 478-484
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Journal article (peer-reviewed)abstract
- Histone H1 is an important constituent of chromatin which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3 and H1.5 during mitosis. H1.2 was found in chromatin during prophase, and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase it appeared in both chromatin and cytoplasm, and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but some H1.5 remained situated in chromatin during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or their phosphorylated variants, may be signalling molecules in mitosis or that they leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.
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| 7. |
- Grenvall, Carl, et al.
(author)
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Label-free somatic cell cytometry in raw milk using acoustophoresis.
- 2012
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In: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 81A:12, s. 1076-1083
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Journal article (peer-reviewed)abstract
- A microfluidic system for cell enumeration in raw milk was developed. The new method, preconditions the milk sample using acoustophoresis that removes lipid particles which are larger than a few micrometers. The acoustophoretic preprocessing eliminates the need for conventional sample preparation techniques, which include chemical solvents, cell labeling and centrifugation, and facilitates rapid cell enumeration using microscopy or coulter counter measurements. By introducing an acoustic standing wave with three pressure nodes in a microchannel at the same time as the milk sample is laminated to the channel center, lipids are acoustically driven to the closest pressure antinode at each side of the channel center and the cells in the milk sample are focused in the central pressure node. The extracted center fraction with cells becomes sufficiently clean from lipid vesicles to enable enumeration of somatic cells without any labeling step either by direct light microscopy or by coulter counting. Obtained lipid free milk fractions clearly revealed the cell fraction when analyzed by Coulter Counting. Cell counting as measured by a Coulter Counter after acoustophoretic lipid depletion aligned with the corresponding data obtained by reference measurements based on fluorescence staining and subsequent flow cytometer analysis. © 2012 International Society for Advancement of Cytometry.
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| 8. |
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| 9. |
- Koch, Christin, et al.
(author)
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CHIC - An automated approach for the detection of dynamic variations in complex microbial communities
- 2013
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In: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 83A:6, s. 561-567
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Journal article (peer-reviewed)abstract
- Altering environmental conditions change structures of microbial communities. These effects have an impact on the single-cell level and can be sensitively detected using community flow cytometry. However, although highly accurate, microbial monitoring campaigns are still rarely performed applying this technique. One reason is the limited access to pattern analysis approaches for the evaluation of microbial cytometric data. In this article, a new analyzing tool, Cytometric Histogram Image Comparison (CHIC), is presented, which realizes trend interpretation of variations in microbial community structures (i) without any previous definition of gates, by working (ii) person independent, and (iii) with low computational demand. Various factors influencing a sensitive determination of changes in community structures were tested. The sensitivity of this technique was found to discriminate down to 0.5% internal variation. The final protocol was exemplarily applied to a complex microbial community dataset, and correlations to experimental variation were successfully shown.
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| 10. |
- Kostova-Koleva, Nora N., et al.
(author)
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Histone H5chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation
- 2013
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In: Cytometry Part A. - : Wiley-Blackwell. - 1552-4922 .- 1552-4930. ; 83A:3, s. 273-279
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Journal article (peer-reviewed)abstract
- We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
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| 11. |
- Lenshof, Andreas, et al.
(author)
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Efficient Purification of CD4+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis
- 2014
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In: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 85A:11, s. 933-941
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Journal article (peer-reviewed)abstract
- Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology
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| 12. |
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| 13. |
- Rundberg Nilsson, Alexandra, et al.
(author)
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Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective.
- 2013
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In: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 83A:8, s. 721-727
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Research review (peer-reviewed)abstract
- Flow cytometry allows for identification of cellular subsets based on cell intrinsic properties, most often by the use of fluorochrome-conjugated antibodies recognizing distinct cell-surface epitopes that define the cells of interest. Advances in technical instrumentation and the availability of an ever-increasing number of fluorophores, today enables identification of multicolor defined cellular populations to a previously unreachable resolution. However, these possibilities put an increasing demand on preparation, acquisition, and subsequent analysis of the investigated samples. Identification of very rare cellular subsets, such as the bone marrow-residing hematopoietic stem cells (HSCs), causes further complexity to such analysis. Here, we discuss considerations and aspects in multicolor flow cytometry as exemplified by analysis of human and mouse HSCs. We illustrate advantages and drawbacks of polychromatic flow cytometry and propose strategies, such as the use of internal reference populations, for sample analysis. © 2013 International Society for Advancement of Cytometry.
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| 14. |
- Rönnlund, Daniel, et al.
(author)
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Spatial organization of proteins in metastasizing cells
- 2013
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In: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 83:9, s. 855-865
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Journal article (peer-reviewed)abstract
- The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell-matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and intermediate filaments.
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| 15. |
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| 16. |
- Gavrilovic, Milan, et al.
(author)
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Automated Classification of Multicolored Rolling Circle Products in Dual-Channel Wide-Field Fluorescence Microscopy
- 2011
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In: Cytometry Part A. - : Wiley. - 1552-4922. ; 79A:7, s. 518-527
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Journal article (peer-reviewed)abstract
- Specific single-molecule detection opens new possibilities in genomics and proteomics, and automated image analysis is needed for accurate quantification. This work presents image analysis methods for the detection and classification of single molecules and single-molecule interactions detected using padlock probes or proximity ligation. We use simple, widespread, and cost-efficient wide-field microscopy and increase detection multiplexity by labeling detection events with combinations of fluorescence dyes. The mathematical model presented herein can classify the resulting point-like signals in dual-channel images by spectral angles without discriminating between low and high intensity. We evaluate the methods on experiments with known signal classes and compare to classical classification algorithms based on intensity thresholding. We also demonstrate how the methods can be used as tools to evaluate biochemical protocols by measuring detection probe quality and accuracy. Finally, the method is used to evaluate single-molecule detection events in situ.
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| 17. |
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