Use Of Time-lapse Microscopy Research Articles

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. Cerevisiae.

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Dependability of the Sperm Chromatin Dispersion (SCD) Assay for Recurrent Sperm DNA Damage Evaluation of Sperm DNA Fragmentation (SDF) in Men with Presentation of Infertility-A Short Communication

How the male attributes in the generation of a healthy embryo, there exists a need for a sperm that is of good quality with minimum genome injury whose estimationis usually are done by the determination of sperm Deoxyribonucleic Acid (DNA) breakdown or the Sperm DNA Fragmentation (SDF). SDF usually guides with regards to the quality of sperm chromatin in case of men with a presentation of infertility. The causes attributed are the capacity of side actions of escalation of SDF amounts on natural fertility, embryo generation along with implantation, risk of miscarriage along with health of the newborn [1]. Earlier we detailed the role of SDF in Intracytoplasmic Sperm Injection (ICSI) with use of Time Lapse Microscopy (TLM) simultaneously [2]. SDF represents a marker of chromatin injury, takes place at the time of spermiogenesis, halt in the epididymis along withpost ejaculation inview of various factors that are not unique like varicocoele, insufficient life style like toxic exposure secondary to occupational along with experimental factors, aging as well as infections [3]. For the birth of a healthy newborn, genomically healthy sperms are necessary, while enhancement of sperms with injured chromatin in the human ejaculate might result in non stabilization of the genome at the time of fertilization as well as embryogenesis [4]. Thus it has been pointed that the evaluation of sperm chromatin at a molecularlevel might aid in its provision of the paternal factor for infertility, besides point to decide what treatment actions might be feasi ble for enhanced sperm chromatin quality [5].

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient's breast cancer brain metastatic growth within the brain microenvironment.

Optimization of a single-embryo transfer by using time-lapse microscopy in IVF and ICSI programs

Introduction. Due to refinements of assisted reproductive technology, the number of multiple pregnancies has increased substantially. Therefore, transfer of a single embryo, as opposed to multiple embryos, is a top-priority task in ART-based infertility treatment. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. The aim of the study was to assess the outcomes of single-embryo transfer following embryo incubation in a TLM-equipped incubator in patients undergoing IVF and ICSI. Methods . The study was carried out in 743 infertile women. Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 282 patients, who formed the main group; the control group consisted of 461 patients undergoing singleembryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and live birth rates. Results. The groups did not differ in the proportion of IVF and ICSI cycles, terms of age and infertility factors. In the study group, there was a higher proportion of excellent quality embryos for transfer (p = 0.001), a higher proportion of cycles with elective embryo transfer (p = 0.001) and a higher proportion of cycles with cryopreservation of embryos (p <0.001). In the subgroup of non-elective embryo transfer with using of TLM, the clinical pregnancy rate was 10% higher than in the control group (p = 0.03). The live birth rates did not differ between the TLM group and the conventional culture group depending on the type of embryo transfer. Conclusion. Our studies show that the use of time-lapse microscopy can increase the effectiveness of IVF and ICSI programs. Continuous monitoring with short intervals provides more information about the development of embryos than the standard daily assessment.

A Cost Effective and Adaptable Scratch Migration Assay.

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

Оптимизация переноса одного эмбриона у пациенток с хорошим овариальным резервом

Due to refinements of assisted reproductive technology, the number of multiple pregnancies has increased substantially. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. This study aimed to assess the outcomes of single-embryo transfer of autologous oocytes performed on day 5 of embryo incubation in a TLM-equipped system in patients with good ovarian reserve. The study was carried out in 208 infertile women with good ovarian reserve (over 8 oocytes retrieved). Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 95 patients, who formed the main group; the control group consisted of 113 patients undergoing single-embryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and pregnancy loss. Two subgroups were identified in each group of the participants: the 5SET subgroup (nonelective single-embryo transfer), which included 45 patients from the main group and 67 controls, and the 5eSET subgroup (elective single-embryo transfer), which consisted of 50 main group patients and 46 controls. The groups did not differ in terms of age, infertility factors and infertility duration. The quality of transferred embryos was excellent or good in all main group patients (100%); in the control group, the quality of transferred embryos was excellent or good in 93.8% of cases (p = 0.037). Clinical pregnancies were achieved in 64.2% of women in the main group and in 60.2% of controls (p = 0.65). Delivery rates were 54% and 51.1% in the 5eSET and 5SET subgroups of the main group, respectively (p = 0.940). For the control group, delivery rates were 54.4% and 34.3% in the 5eSET and 5SET subgroups, respectively (p = 0.052, Fisher exact test). Elective single-embryo transfer (5eSET) and the use of TLM increased the chance of pregnancy 2.17-fold (p = 0.01).

Assessment of human embryo development using morphological criteria in an era of time-lapse, algorithms and 'OMICS': is looking good still important?

With the worldwide move towards single embryo transfer there has been a renewed focus on the requirement for reliable means of assessing embryo viability. In an era of 'OMICS' technologies, and algorithms created through the use of time-lapse microscopy, the actual appearance of the human embryo as it progresses through each successive developmental stage to the blastocyst appears to have been somewhat neglected in recent years. Here we review the key features of the human preimplantation embryo and consider the relationship between morphological characteristics and developmental potential. Further, the impact of the culture environment on morphological traits, how key morphological qualities reflect aspects of embryo physiology, and how computer-assisted analysis of embryo morphology may facilitate a more quantitative approach to selection are discussed. The clinical introduction of time-lapse systems has reopened our eyes and given us a new vantage point from which to view the beauty of the initial stages of human life. Rather than a future in which the morphology of the embryo is deemed irrelevant, we propose that key features, such as multinucleation, cell size and blastocyst differentiation should be included in future iterations of selection/deselection algorithms.

The role of geophytes in stone-age hunter-–gatherer diets

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined > 680 colonies from 3 different preparations of cells over 5 days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response.

Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations.

Does use of time-lapse microscopy in the selection of embryos for transfer improve pregnancy rates? A randomized controlled trial

Does use of time-lapse microscopy in the selection of embryos for transfer improve pregnancy rates? A randomized controlled trial

Labour-Efficient In Vitro Lymphocyte Population Tracking and Fate Prediction Using Automation and Manual Review

Interest in cell heterogeneity and differentiation has recently led to increased use of time-lapse microscopy. Previous studies have shown that cell fate may be determined well in advance of the event. We used a mixture of automation and manual review of time-lapse live cell imaging to track the positions, contours, divisions, deaths and lineage of 44 B-lymphocyte founders and their 631 progeny in vitro over a period of 108 hours. Using this data to train a Support Vector Machine classifier, we were retrospectively able to predict the fates of individual lymphocytes with more than 90% accuracy, using only time-lapse imaging captured prior to mitosis or death of 90% of all cells. The motivation for this paper is to explore the impact of labour-efficient assistive software tools that allow larger and more ambitious live-cell time-lapse microscopy studies. After training on this data, we show that machine learning methods can be used for realtime prediction of individual cell fates. These techniques could lead to realtime cell culture segregation for purposes such as phenotype screening. We were able to produce a large volume of data with less effort than previously reported, due to the image processing, computer vision, tracking and human-computer interaction tools used. We describe the workflow of the software-assisted experiments and the graphical interfaces that were needed. To validate our results we used our methods to reproduce a variety of published data about lymphocyte populations and behaviour. We also make all our data publicly available, including a large quantity of lymphocyte spatio-temporal dynamics and related lineage information.

Spatial and temporal evaluation of cell attachment to printed polycaprolactone microfibres

Surface topography plays a crucial role in influencing cellular responses and has therefore been utilized in the development of numerous implantable devices. Whilst numerous studies have either investigated cell attachment or migration post-attachment, few have looked at the early-stages of this process temporally. The aim of this study was to evaluate the use of time-lapse microscopy to study the behaviour of fibroblasts cultured with polycaprolactone microfibres and to assess spatially and temporally the cell–structure interaction over a 24h period. Ordered polymeric structures were printed (predetermined) onto glass substrates using an electrohydrodynamic direct write process to produce fine (3–5μm wide) structures. Fibroblast attachment and migration were characterized as a function of distance perpendicular from structures (∼17.3, 34.6 and 51.9μm). The use of time-lapse microscopy revealed a gradual decrease in cell attachment as the distance from the microfibres was increased. The technique also revealed that some cells were attaching and detaching from the microfibre multiple times. Our findings demonstrate that time-lapse microscopy is a useful technique for evaluating early-stage cell–biomaterial interaction that is capable of recording important events that might otherwise be overlooked.

Characterisation of Hofbauer Cells in First and Second Trimester Placenta: Incidence, Phenotype, Survival in vitro and Motility

Macrophages, known as Hofbauer cells, are most abundant in placental villous stroma in the first and second trimesters. Their functions are not well defined. We have used a combination of in situ and in vitro methods to characterise these cells. Lectin histochemistry and immunohistochemistry were used to identify macrophages in situ. The lectin from Maclura pomifera (MPA) was found to mark cells bearing the CD68 antigen with optimal specificity and selectivity. MPA staining was used to show that they increase in number from mid first to mid second trimester, becoming much less abundant at term. The cells are absent from mesenchymal villi, being associated primarily with villous stroma containing the prominent interstitial channels characteristic of immature intermediate villi. A mixed stromal cell isolate was studied in monolayer culture, including the use of time-lapse microscopy. Cells from first or second trimester tissue contained a subpopulation of about 14–17% of cells that exhibited a macrophage-like morphology and expressed CD68 as well as MPA-binding glycans. These cells were short-lived in monoculture, but could persist in vitro in association with a fibroblast layer for several weeks. They could switch rapidly from a macrophage-like to a fibroblastic morphology, were highly motile and associated in clusters that rapidly formed and dissipated over periods of a few hours. These data suggest that Hofbauer cells play a role in the maturation of mesenchymal into immature intermediate-type stroma. They may be important in the excavation of stromal channels. Their prolonged viability in mixed cultures suggests a paracrine relationship with resident fibroblasts. Their location and migratory behaviour predict an ability to move rapidly around the villous stroma, perhaps within the channel system, and to make transient contacts both with other macrophages and stromal cells.

The use of time-lapse microscopy to investigate the impact of an oxygen gradient on embryo development

OBJECTIVE: There is growing clinical data that human embryonic development is improved at a reduced oxygen concentration (5%). In other mammalian species, the beneficial effects of 5% oxygen concentration on embryo viability have been established. It is not known whether reduced oxygen levels are required at all stages of development.

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of approximately 20 microm h(-1), which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.

Bet-hedging and epigenetic inheritance in bacterial cell development

Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell's ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational "memory" caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.

Structural properties and neuronal toxicity of amyotrophic lateral sclerosis–associated Cu/Zn superoxide dismutase 1 aggregates

The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis

The spindle midzone, a conspicuous network of antiparallel interdigitating nonkinetochore microtubules between separating chromosomes, plays a crucial role in regulating the initiation and completion of cytokinesis. In this study, we report the use of time-lapse microscopy and a human kinesin endoribonucleases RNase III-prepared short interfering RNA (esiRNA) library to identify Kif4 as a motor protein that translocates PRC1, a spindle midzone-associated cyclin-dependent kinase substrate protein, to the plus ends of interdigitating spindle microtubules during the metaphase-to-anaphase transition. We show that Kif4 binds to PRC1 through its "stalk plus tail" domains and Kif4 and PRC1 colocalize on the spindle midzone/midbody during anaphase and cytokinesis. Suppression of Kif4 expression by Kif4 esiRNA results in the inhibition of PRC1 translocation, a block of the midzone formation, and a failure of cytokinesis. PRC1 translocation and midzone formation can be restored, and the cytokinetic defects can be rescued in Kif4 esiRNA-treated cells by coexpression of Kif4 but not its motor dead mutant Kif4md. Furthermore, we show that cyclin-dependent kinase phosphorylation of PRC1 controls the timing of PRC1 translocation by Kif4. These results, in light of the crucial role of PRC1 in midzone formation, indicate that cell cycle-dependent translocation of PRC1 by Kif4 is essential for midzone formation and cytokinesis.

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.