Typical time intervals between acquisitions of three-dimensional (3-D) images of the same cell in live cell imaging are in the orders of minutes. In the meantime, the live cell can move in a water basin on the stage. This movement can hamper the studies of intranuclear processes. We propose a fast point-based image registration method for the suppression of the movement of a cell as a whole in the image data. First, centroids of certain intracellular objects are computed for each image in a time-lapse series. Then, a matching between the centroids, which have the maximal number of pairs, is sought between consecutive point sets by a 3-D extension of a two-dimensional fast point pattern matching method, which is invariant to rotation, translation, local distortion, and extra/missing points. The proposed 3-D extension assumes rotations only around the z axis to retain the complexity of the original method. The final step involves computing the optimal fully 3-D transformation between images from corresponding points in the least-squares manner. The robustness of the method was evaluated on generated data. The results of the simulations show that the method is very precise and its correctness can be estimated. This article also presents two practical application examples, namely the registration of images of HP1 domains and the registration of images of telomeres. More than 97% of time-consecutive images were successfully registered. The results show that the method is very well suited to live cell imaging.
- MeSH
- Algorithms MeSH
- Artifacts MeSH
- Microscopy, Fluorescence methods MeSH
- Image Interpretation, Computer-Assisted methods MeSH
- Cells, Cultured cytology MeSH
- Humans MeSH
- Cell Movement MeSH
- Reproducibility of Results MeSH
- Pattern Recognition, Automated methods MeSH
- Sensitivity and Specificity MeSH
- Subtraction Technique MeSH
- Information Storage and Retrieval methods MeSH
- Artificial Intelligence MeSH
- Microscopy, Video methods MeSH
- Image Enhancement methods MeSH
- Imaging, Three-Dimensional methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
The successful development of visualization techniques for live cell imaging leads to the development of suitable software for the acquisition and processing of multidimensional image data. This report compares several possible approaches to image acquisition and processing in confocal in vivo microscopy and suggests new alternatives to the published methods. Special attention is paid to spinning disk systems based either on a classical Nipkow disk or on the microlens principle. This study shows how to optimize image acquisition process in live cell studies using camera binning feature and how to perform object tracking using a new fast image registration method based on the graph theory.
- MeSH
- Algorithms MeSH
- Cell Physiological Phenomena * MeSH
- Microscopy, Confocal instrumentation MeSH
- Luminescent Proteins metabolism MeSH
- Image Cytometry instrumentation methods MeSH
- Image Processing, Computer-Assisted methods MeSH
- Software * MeSH
- Green Fluorescent Proteins MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- Luminescent Proteins MeSH
- Green Fluorescent Proteins MeSH
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.
- Keywords
- 3D imaging, diffraction-limited spot detection, fluorescence microscopy, parameter sensitivity,
- MeSH
- Algorithms MeSH
- Microscopy, Fluorescence methods MeSH
- Microscopy, Confocal methods MeSH
- Image Processing, Computer-Assisted methods MeSH
- Sensitivity and Specificity MeSH
- Imaging, Three-Dimensional methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Movement of labelled plasmid DNA relative to heterochromatin foci in nuclei, visualized with HP1-GFP, was studied using live-cell imaging and object tracking. In addition to Brownian motion of plasmid DNA we found a pronounced, non-random movement of plasmid DNA towards the nearest HP1 focus, while time-lapse microscopy showed that HP1 foci are relatively immobile and positionally stable. The movement of plasmid DNA was much faster than that of the HP1 foci. Contact of transgene DNA with an HP1 focus usually resulted in cessation of the directional motion. Moreover, the motion of plasmid DNA inside the heterochromatin compartment was more restricted (limited to 0.25 microm) than when the plasmid DNA was outside heterochromatin (R = 0.7 microm). Three days after transfection most of the foreign labelled DNA colocalized with centromeric heterochromatin.
- MeSH
- Biological Transport genetics physiology MeSH
- Cell Nucleus physiology MeSH
- Chromosomal Proteins, Non-Histone genetics physiology MeSH
- DNA genetics physiology MeSH
- Heterochromatin MeSH
- Chromobox Protein Homolog 5 MeSH
- Humans MeSH
- Microscopy MeSH
- Cell Line, Tumor MeSH
- Plasmids genetics physiology MeSH
- Transfection MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromosomal Proteins, Non-Histone MeSH
- DNA MeSH
- Heterochromatin MeSH
- Chromobox Protein Homolog 5 MeSH
Tracking motile cells in time-lapse series is challenging and is required in many biomedical applications. Cell tracks can be mathematically represented as acyclic oriented graphs. Their vertices describe the spatio-temporal locations of individual cells, whereas the edges represent temporal relationships between them. Such a representation maintains the knowledge of all important cellular events within a captured field of view, such as migration, division, death, and transit through the field of view. The increasing number of cell tracking algorithms calls for comparison of their performance. However, the lack of a standardized cell tracking accuracy measure makes the comparison impracticable. This paper defines and evaluates an accuracy measure for objective and systematic benchmarking of cell tracking algorithms. The measure assumes the existence of a ground-truth reference, and assesses how difficult it is to transform a computed graph into the reference one. The difficulty is measured as a weighted sum of the lowest number of graph operations, such as split, delete, and add a vertex and delete, add, and alter the semantics of an edge, needed to make the graphs identical. The measure behavior is extensively analyzed based on the tracking results provided by the participants of the first Cell Tracking Challenge hosted by the 2013 IEEE International Symposium on Biomedical Imaging. We demonstrate the robustness and stability of the measure against small changes in the choice of weights for diverse cell tracking algorithms and fluorescence microscopy datasets. As the measure penalizes all possible errors in the tracking results and is easy to compute, it may especially help developers and analysts to tune their algorithms according to their needs.
- MeSH
- Algorithms MeSH
- Cell Line MeSH
- Cell Tracking methods MeSH
- Time-Lapse Imaging methods MeSH
- Microscopy, Fluorescence MeSH
- Humans MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.
- Keywords
- endothelial differentiation, induced pluripotent stem cells, peripheral blood mononuclear cells,
- MeSH
- Biomarkers metabolism MeSH
- Cell Differentiation physiology MeSH
- Human Umbilical Vein Endothelial Cells cytology metabolism MeSH
- Endothelial Cells cytology metabolism MeSH
- Fibroblasts cytology metabolism MeSH
- Neovascularization, Physiologic physiology MeSH
- Induced Pluripotent Stem Cells cytology metabolism MeSH
- Cells, Cultured MeSH
- Leukocytes, Mononuclear cytology metabolism MeSH
- Humans MeSH
- Regenerative Medicine methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Biomarkers MeSH
Apoptosis is a natural form of cell death involved in many physiological changes in the cell. Defects in the process of apoptosis can lead to serious diseases. During some apoptotic pathways, proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin degradation together with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interacting partners like flap endonuclease 1, DNase I, and exonuclease III were already suggested, but also other interacting partners were proposed. We conducted a living-cell confocal fluorescence microscopy followed by an image analysis of fluorescence resonance energy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins during apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin degradation. The possible existence of a degradation complex consisting of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investigated.
- MeSH
- Antigens, Neoplasm chemistry genetics metabolism MeSH
- Apoptosis * MeSH
- Cell Nucleus enzymology pathology MeSH
- Time Factors MeSH
- DNA-Binding Proteins chemistry genetics metabolism MeSH
- DNA Topoisomerases, Type II chemistry genetics metabolism MeSH
- Endodeoxyribonucleases chemistry genetics metabolism MeSH
- HeLa Cells MeSH
- Histones chemistry genetics metabolism MeSH
- Protein Interaction Domains and Motifs MeSH
- Microscopy, Confocal MeSH
- Protein Conformation MeSH
- Humans MeSH
- Protein Interaction Mapping MeSH
- Models, Molecular MeSH
- Recombinant Fusion Proteins metabolism MeSH
- Chromatin Assembly and Disassembly * MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Transfection MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Antigens, Neoplasm MeSH
- DNA-Binding Proteins MeSH
- DNA Topoisomerases, Type II MeSH
- Endodeoxyribonucleases MeSH
- endonuclease G MeSH Browser
- Histones MeSH
- Recombinant Fusion Proteins MeSH
We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.
- MeSH
- Tumor Suppressor p53-Binding Protein 1 MeSH
- Leukemia, Promyelocytic, Acute metabolism pathology MeSH
- Time-Lapse Imaging MeSH
- DNA chemistry metabolism MeSH
- Intracellular Signaling Peptides and Proteins metabolism MeSH
- Intranuclear Inclusion Bodies metabolism ultrastructure MeSH
- Microscopy, Confocal instrumentation methods MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- DNA Damage MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Tumor Suppressor p53-Binding Protein 1 MeSH
- DNA MeSH
- Intracellular Signaling Peptides and Proteins MeSH
- TP53BP1 protein, human MeSH Browser
In this work, we have studied the structural and functional linkage between lamin A/C, nuclear actin, and organization of chromosome territories (CTs) in mammary carcinoma MCF-7 cells. Selective down-regulation of lamin A/C expression led to disruption of the lamin A/C perinuclear layer and disorganization of lamin-bound emerin complexes at the inner nuclear membrane. The silencing of lamin A/C expression resulted in a decrease in the volume and surface area of chromosome territories, especially in chromosomes with high heterochromatin content. Inhibition of actin polymerization led to relaxation of the structure of chromosome territories, and an increase in the volumes and surface areas of the chromosome territories of human chromosomes 1, 2 and 13. The results show an important role of polymeric actin in the organization of the nuclei and the chromosome territories.
- MeSH
- Actins metabolism MeSH
- Cell Nucleus metabolism MeSH
- Down-Regulation MeSH
- Genome, Human genetics MeSH
- Microscopy, Confocal MeSH
- Lamin Type A metabolism MeSH
- Humans MeSH
- Chromosomes, Human metabolism MeSH
- Cell Line, Tumor MeSH
- Cell Shape MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Actins MeSH
- lamin C MeSH Browser
- Lamin Type A MeSH
OBJECTIVE: The study aimed to provide a description of a new and a hopeful possibility in the treatment of severe vulvodynia, which does not respond to treatments used so far. MATERIALS AND METHODS: The use of radiofrequency therapy in vulvodynia treatment is described for the first time. This method was suggested by a neurosurgeon after applying all available possibilities. RESULT: In this article, we are reporting on the successful use of the pulsed radiofrequency treatment in a patient with intractable chronic vulvodynia. CONCLUSIONS: To our knowledge, this is the first report of a successful use of pulsed radiofrequency in the treatment of chronic vulvodynia. If efficacy of pulsed radiofrequency is confirmed by more studies, it would be a welcome addition to the treatment modalities used to treat this sometimes truly intractable condition.
