Image cytometry still faces the problem of the quality of cell image analysis results. Degradations caused by cell preparation, optics, and electronics considerably affect most 2D and 3D cell image data acquired using optical microscopy. That is why image processing algorithms applied to these data typically offer imprecise and unreliable results. As the ground truth for given image data is not available in most experiments, the outputs of different image analysis methods can be neither verified nor compared to each other. Some papers solve this problem partially with estimates of ground truth by experts in the field (biologists or physicians). However, in many cases, such a ground truth estimate is very subjective and strongly varies between different experts. To overcome these difficulties, we have created a toolbox that can generate 3D digital phantoms of specific cellular components along with their corresponding images degraded by specific optics and electronics. The user can then apply image analysis methods to such simulated image data. The analysis results (such as segmentation or measurement results) can be compared with ground truth derived from input object digital phantoms (or measurements on them). In this way, image analysis methods can be compared with each other and their quality (based on the difference from ground truth) can be computed. We have also evaluated the plausibility of the synthetic images, measured by their similarity to real image data. We have tested several similarity criteria such as visual comparison, intensity histograms, central moments, frequency analysis, entropy, and 3D Haralick features. The results indicate a high degree of similarity between real and simulated image data. ' International Society for Advancement of CytometryKey terms digital phantom; synthetic image; procedural texture; point spread function; fluorescence optical microscope; 3D image cytometry CURRENT biomedical research relies strongly on computer-based evaluation, as the vast majority of commonly used acquisition techniques produce large numbers of numerical or visual data. Each individual technique (optical microscopy, PET, MRI, CT, ultrasound, etc. (1)) has its advantages that make it suitable for use in selected fields of research. However, each technique also has its own drawbacks (blur, noise, various aberrations) (2-4). In this sense, one should keep in mind that biomedical data supplied by scientific or diagnostic instruments always suffer from some imperfection and therefore cannot be directly used for further analysis, evaluation or measurement. To guarantee more accurate results, some preprocessing is required.Let us focus on the area of image reconstruction. Here, the search for ground truth approximation is called an image restoration (5-7), and it constitutes the best possible method for image recovery. It is done in time-reverse order; that is, the image defect caused by the phenomenon that appeared in the whole acquisition process as the first one must be eliminated as the last...

BackgroundHuman induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs).MethodsWe measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases.ResultsWe demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages.ConclusionsOur observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0522-5) contains supplementary material, which is available to authorized users.

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