Cell viability and cytotoxicity assays are highly important for drug screening and cytotoxicity tests of antineoplastic or other therapeutic drugs. Even though biochemical-based tests are very helpful to obtain preliminary preview, their results should be confirmed by methods based on direct cell death assessment. In this study, time-dependent changes in quantitative phase-based parameters during cell death were determined and methodology useable for rapid and label-free assessment of direct cell death was introduced. The goal of our study was distinction between apoptosis and primary lytic cell death based on morphologic features. We have distinguished the lytic and non-lytic type of cell death according to their end-point features (Dance of Death typical for apoptosis versus swelling and membrane rupture typical for all kinds of necrosis common for necroptosis, pyroptosis, ferroptosis and accidental cell death). Our method utilizes Quantitative Phase Imaging (QPI) which enables the time-lapse observation of subtle changes in cell mass distribution. According to our results, morphological and dynamical features extracted from QPI micrographs are suitable for cell death detection (76% accuracy in comparison with manual annotation). Furthermore, based on QPI data alone and machine learning, we were able to classify typical dynamical changes of cell morphology during both caspase 3,7-dependent and -independent cell death subroutines. The main parameters used for label-free detection of these cell death modalities were cell density (pg/pixel) and average intensity change of cell pixels further designated as Cell Dynamic Score (CDS). To the best of our knowledge, this is the first study introducing CDS and cell density as a parameter typical for individual cell death subroutines with prediction accuracy 75.4% for caspase 3,7-dependent and -independent cell death.

• MeSH
• Algorithms MeSH
• Apoptosis * drug effects   MeSH
• Cell Death * drug effects   MeSH
• Cells ultrastructure   MeSH
• Time-Lapse Imaging methods   MeSH
• Time Factors MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Optical Imaging methods   MeSH
• Cell Count MeSH
• Models, Statistical MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Double-stranded DNA breaks activate a DNA damage checkpoint in G2 phase to trigger a cell cycle arrest, which can be reversed to allow for recovery. However, damaged G2 cells can also permanently exit the cell cycle, going into senescence or apoptosis, raising the question how an individual cell decides whether to recover or withdraw from the cell cycle. Here we find that the decision to withdraw from the cell cycle in G2 is critically dependent on the progression of DNA repair. We show that delayed processing of double strand breaks through HR-mediated repair results in high levels of resected DNA and enhanced ATR-dependent signalling, allowing p21 to rise to levels at which it drives cell cycle exit. These data imply that cells have the capacity to discriminate breaks that can be repaired from breaks that are difficult to repair at a time when repair is still ongoing.

• MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• Cell Line MeSH
• Time-Lapse Imaging methods   MeSH
• Cyclin B1 genetics   metabolism   MeSH
• Microscopy, Fluorescence MeSH
• HEK293 Cells MeSH
• Cyclin-Dependent Kinase Inhibitor p21 genetics   metabolism   MeSH
• G2 Phase Cell Cycle Checkpoints genetics   MeSH
• Humans MeSH
• DNA Repair genetics   MeSH
• DNA Damage * MeSH
• Signal Transduction genetics   MeSH
• Cellular Senescence genetics   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

• MeSH
• Single-Cell Analysis methods   MeSH
• Apoptosis physiology   MeSH
• Cell Nucleus physiology   ultrastructure   MeSH
• Hep G2 Cells MeSH
• Time-Lapse Imaging methods   MeSH
• Chromatin chemistry   metabolism   ultrastructure   MeSH
• Kinetics MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• DNA Packaging MeSH
• Models, Theoretical * MeSH
• Organelle Size physiology   MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy.

• MeSH
• Aneuploidy * MeSH
• Time-Lapse Imaging methods   MeSH
• Kinetochores metabolism   MeSH
• Microscopy, Confocal MeSH
• Meiosis genetics   MeSH
• Microtubules metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Nondisjunction, Genetic * MeSH
• Oocytes metabolism   MeSH
• Chromosome Segregation genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

• MeSH
• Arabidopsis genetics   growth & development   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Time-Lapse Imaging methods   MeSH
• Exocytosis * MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Phosphatidylinositols metabolism   MeSH
• Phylogeny MeSH
• Plants, Genetically Modified MeSH
• Microscopy, Confocal MeSH
• Mutation MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Arabidopsis Proteins classification   genetics   metabolism   MeSH
• Pollen genetics   growth & development   metabolism   MeSH
• Pollen Tube genetics   growth & development   metabolism   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Homology, Nucleic Acid MeSH
• Molecular Dynamics Simulation MeSH
• Gene Expression Profiling methods   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Vesicular Transport Proteins classification   genetics   metabolism   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH

The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ∼ 3-5 minutes into the cell cycle. Furthermore, unlike what has been observed in mammalian cells, the relative spatial positioning of chromatin domains remained largely unchanged during prometaphase in the early C. elegans embryo. Live imaging of photoconverted chromatin also allowed us to detect a reproducible 180° rotation of the nuclei during cytokinesis of the one-cell embryo. Imaging of fluorescently-labeled P granules and polar bodies showed that the entire embryo rotates during the first cell division. To our knowledge, we report here the first live observation of the initial separation and subsequent mixing of parental chromatin domains during embryogenesis.

• MeSH
• Blastomeres cytology   metabolism   MeSH
• Cell Cycle MeSH
• Caenorhabditis elegans embryology   genetics   metabolism   MeSH
• Time-Lapse Imaging methods   MeSH
• Time Factors MeSH
• Chromatin genetics   metabolism   MeSH
• Embryo, Nonmammalian cytology   embryology   metabolism   MeSH
• Fertilization MeSH
• Histones genetics   metabolism   MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Mitosis MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural 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

Cíl studie: Za pomoci kontinuálního monitoringu (time--lapse) popsat časovou variabilitu raného embryonálního dělení. Posoudit vliv věku pacientek na embryonální růst. Porovnat pregnancy rate time-lapse selektovaných embryí s pregnancy rate embryí ze standardní kultivace. Typ studie: Case-control studie. Název a sídlo pracoviště: Centrum asistované reprodukce Sanus Pardubice, První privátní chirurgické centrum s.r.o. Hradec Králové Metodika: Vývoj 213 embryí od 44 pacientek byl monitorován systémem PrimoVision s frekvencí záznamu1 obrázek za 12 minut. Data byla rozdělena na záznamy pocházející od pacientek ≥ 35 let (skupina ≥ 35) a porovnána s kontrolní skupinou pacientek ≤ 32 let (skupiny ≤ 32). Z natočeného materiálu byl určen čas prvního (t2) a druhého (t3) buněčného dělení a délka cyklu mezi t2 a t3 (cc2). Kritérii pro transfer bylo souměrné dělení buněk na sudý počet buněk dceřiných, časné dělení a dosažení stadia blastocysty. Výsledky: Byly zjištěny průměrné časy sledovaných parametrů u skupiny ≥ 35: t2 = 27,0 h, t3 = 38,7 h, cc2 = 11,7 h. U skupiny ≤ 32: t2 = 27,1 h, t3 = 39,0 h, cc2 = 11,9 h bez signifikantního rozdílu mezi skupinami. Byla popsána průměrná variabilita v načasování dělení u embryí jedné pacientky jako průměrný časový rozdíl mezi začátkem dělení nejrychlejšího a nejpomalejšího embrya pro skupinu ≥ 35: t2 = 4,5 h, t3 = 5,7 h, pro skupinu ≤ 32: t2 = 4,5 h, t3 = 5,1 h bez rozdílu mezi skupinami. Mezi věkem pacientek a t2, t3 a cc2 jednotlivých embryí se nenalezla žádná závislost (p = 0,60, p = 0,81, p = 0,57). Embrya s časným dělením statisticky signifikantně setrvávala v cc2 kratší dobu než embrya pomalejší (p = 0,0001). Dosažená pregnancy rate u time-lapse selektovaných embryí byla 55 %, u embryí z běžné kultivace 47 %. Závěr: Vliv věku pacientky na načasování prvních embryonálních dělení nebyl prokázán. Nalezla se závislost mezi časem prvního buněčného dělení a dobou setrvání embrya ve fázi cc2. Hodnotu cc2 doporučujeme jako vhodnou k selekci embrya pro embryotransfer v centrech asistované reprodukce. Studie prokázala, že výběr embryí za pomoci time-lapse zvyšuje úspěšnost léčby u pacientek s věkovým faktorem.

Objective: To monitor time variability of early embryo cleavage by continual monitoring system (time-lapse). To evaluate the impact of patient age for the embryonic growth. To compare pregnancy rate of the time-lapse selected embryos with embryos after ordinary/standard cultivation. Design: Case-control study. Setting: Centre of assisted reproduction Sanus, Pardubice; PPCHC s.r.o. Hradec Kralove. Methods: Development of 213 embryos from 44 females was monitored by PrimoVision (time-lapse) system with time frequency of recording 1 image in 12 minutes. The data were evaluated in two groups: infertile patients ≥ 35 years (group ≥ 35) and control ≤ 32 years (group ≤ 32). From the collected recordings, time of the first (t2) and of the second (t3) cell cleavage and the time interval between t2 and t3 (cc2) were determined. Symmetrical cellular division to even number of daughter cells, early cleavage and the attainment of blastocyst stage have become the major selection criteria for the embryotransfer. Results: The following average values of studied parameters were found: Group ≥ 35: t2 = 27.0 hours, t3 = 38.7 hours, cc2 = 11.7 hours Group ≤ 32: t2 = 27.1 hours, t3 = 39.0 hours, cc2 = 11.9 hours, with no significant differences between both groups. Likewise, no significant difference was observed in mean variability of embryo cleavage timing in an individual patient and control: Group ≥ 35: t2 = 4.5 hours, t3 = 5.7 hours, Group ≤ 32: t2 = 4.5 hours, t3 = 5.1 hours. No relation was observed between the patient age and t2, t3 and cc2 times when evaluated by regression curve (p = 0.60, p = 0.81, p = 0.57). However, embryos of early cleavage have remained in cc2 period significantly shorter period of time compared to embryos of slower cleavage(p = 0.0001). Pregnancy rate at time-lapse selected embryos reached 55.0% while embryos from standard cultivation only 47%. Conclusion: The impact of patient age to the cleavage dynamic has not been proved. Relation between the first cell cleavage time and embryo persistence in this stage (cc2) was observed. We may hence recommend cc2 time as convenient parameter at embryo selection for embryotransfer in the centres of assisted reproduction. Our study has shown that embryo selection with time-lapse system (PrimoVision) enhances the success rate of treatment of aging patients.

• Keywords
• lidské embryo, věkový faktor neplodnosti, načasování buněčného dělení,
• MeSH
• Cell Division MeSH
• Time-Lapse Imaging * methods   statistics & numerical data   MeSH
• Time Factors MeSH
• Adult MeSH
• Embryo, Mammalian MeSH
• Embryonic Development * MeSH
• Fertilization in Vitro methods   MeSH
• Embryo Implantation MeSH
• Embryo Culture Techniques MeSH
• Humans MeSH
• Embryo Disposition MeSH
• Embryo Transfer MeSH
• Reproducibility of Results MeSH
• Cleavage Stage, Ovum cytology   MeSH
• Statistics as Topic MeSH
• Case-Control Studies MeSH
• Age Factors * MeSH
• Embryo Research MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH

Chronology of three consecutive mitotic events in human pre-implantation embryos was examined by time-lapse imaging. In zygotes producing well-formed and pregnancy-yielding expanded blastocysts, uniform time-patterning of cleavage clusters (c) and interphases (i) was revealed: i2=11+/-1, i3=15+/-1, i4=23+/-1 h / c2=15+/-5, c3=40+/-10, c4=55+/-15 min. Oppositely, shortened or prolonged durations of one or more cell cycles were strongly predictive of poor implantation and development. Furthermore, trichotomic mitosis was discovered in 17 % of cases - zygotes cleaved into 3 blastomeres and 2-cell embryos into 5-6 cells (instead of normal 2 and 4). During conventional clinical assessment, such embryos are indistinguishable from normal, often considered just-in-course of the next cell cycle. Only detailed time-lapse monitoring paced at 10-minute intervals had proven all these embryos to be absolutely unviable, even in rare cases when they reduced their hypercellularity to normal cell counts via cell-cell fusion. Overall, we demonstrate that time-lapse embryo cleavage rating (ECR) as a standalone diagnostic procedure allows for effective identification of viable early embryos with 90 % specificity, while elimination of good-looking but unviable embryos can be assumed with a specificity of 100 %. Thus, making this non-invasive and contactless approach worth of addition to routine embryo screening in clinical IVF programs.

• MeSH
• Blastocyst cytology   MeSH
• Time-Lapse Imaging methods   MeSH
• Image Interpretation, Computer-Assisted methods   MeSH
• Humans MeSH
• Preimplantation Diagnosis methods   MeSH
• Cleavage Stage, Ovum cytology   MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

• MeSH
• Anaphase MeSH
• Aneuploidy MeSH
• Time-Lapse Imaging methods   MeSH
• Histones genetics   metabolism   MeSH
• Kinetochores metabolism   MeSH
• Ubiquitin-Protein Ligase Complexes genetics   metabolism   MeSH
• Microscopy, Confocal methods   MeSH
• M Phase Cell Cycle Checkpoints MeSH
• Metaphase MeSH
• Microinjections MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oocytes cytology   metabolism   MeSH
• Chromosome Pairing * MeSH
• Proteolysis MeSH
• Chromosomes, Mammalian genetics   metabolism   MeSH
• Mammals MeSH
• Chromosome Segregation * MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Tubulin genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH