Analysis of cellular behavior is significant for studying cell cycle and detecting anti-cancer drugs. It is a very difficult task for image processing to isolate individual cells in confocal microscopic im- ages of non-stained live cell cultures. Because these images do not have adequate textural varia- tions. Manual cell segmentation requires massive labor and is a time consuming process. This pa- per describes an automated cell segmentation method for localizing the cells of Chinese hamster ovary cell culture. Several kinds of high-dimensional feature descriptors, K-means clustering me- thod and Chan-Vese model-based level set are used to extract the cellular regions. The region ex- tracted are used to classify phases in cell cycle. The segmentation results were experimentally as- sessed. As a result, the proposed method proved to be significant for cell isolation. In the evalua- tion experiments, we constructed a database of Chinese Hamster Ovary Cell’s microscopic images which includes various photographing environments under the guidance of a biologist.
foreign protein and without previous light exposure, we also stud- ied muscle fibers by acute in vivo staining. Normal and denervated muscle fibers were acutely injected with fluorescent oligonucle- otides at different time points after denervation. Like the nucEGFP labeling, this technique provides staining of the set of nuclei envel- oped by a single sarcolemma, excluding satellite cells and stromal cells (35) (Figure 2A). By this staining method, 136 fibers from 15 different animals were studied. No differences in the number of nuclei were observed either in the fast, glycolytic musculus exten- sor digitorum longus (EDL) or the slow soleus muscle for up to 21 days after denervation (Figure 2B). During this time, the average estimated cross-sectional area of the injected fibers calculated from measurements of the fiber diameter observed through the micro- scope, was reduced by 51% and 55% in EDL and soleus, respectively (Figure 2C). This was similar to the 54% and 58% atrophy observed in other parts of these muscles when the whole muscle was studied on cryosections postmortem (Figure 2D). Thus, the acute in vivo imaging, confirming the findings obtained by time-lapse micros- copy, showed that there was no loss of nuclei after denervation. Moreover, the acute approach excluded the possibility that the constant nuclear number is related to the presence of foreign pro- tein or repeated imaging.
Analysis of time lapse photographs such as those in Figure 4(a) (i)-(iv) allowed the electrode- droplet separation to be deduced as a function of time. Potentiometric measurements were converted to corresponding pH values using a calibration curve such as that shown in Figure S2 (see Supporting Information, section S2). Figure 4(b) shows a typical resulting pH versus electrode-droplet separation profile. As the droplet interface approached within a sufficiently close distance to the probe electrode (electrode-droplet separation ≤ 100 µm), the pH increase corresponds to a local jeffamine D230 concentration increase. pH measurements were then converted into concentration of jeffamine D230, with a typical profile shown in Figure 4c. The concentration of a weak base can be calculated from the pH of the solution so long as the pKa is known, which was calculated to be ~9.4 for jeffamine D230. This is achieved using the Henderson-Hasselbalch equation:
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In spite of remarkable potential of the time lapse micro- scopy to address various questions of circadian biology, there is a very limited number of data analysis software available. Commercially available software Metamorph (Universal Imaging Corp), Imaris (Bitplane A.G.) and Dia- Track (Semasopht) incorporate modules to track objects and to measure intensity in a region of interest. However, the analysis of the reporter protein level in the described above time lapse microscopy datasets using these software requires a lot of manual interventions. Metamorph inter- rupts tracking in every valley of the circadian cycle; there- fore the user has to manually complete the trace. This is mainly due to the high variation of intensity in the repor- ter protein level from one frame to another. Approaches based on intensity threshold or on template matching are not able to perform a correct tracking. In addition, a man- ual analysis is unreasonably time-consuming and subject to errors in observer judgment.
with reconstitution potential. We further observed cells continuously with a high temporal (every 2 min) and spatial resolution using time lapse microscopy and recorded their divisional kinetics as well as fate potential by single cell tracking for at least 4 days. We cultured cells on stroma as well as in stroma free suspension culture and used self renewal as well as differentiation promoting culture conditions to be able to compare the effects of different environments on HSC behaviour and to distinguish extrinsically from intrinsically controlled HSC behaviour. In order to assess asymmetric fate potential of individual HSC we used two different readouts, generation time of daughter cells as well as loss of reconstitution potential as assessed by a live marker. And we compared the asymmetric fate potential of HSC as well as their divisional kinetics to MPP, which are closely related cells that have lost reconstitution potential. This enabled us to distinguish characteristics that are unique to true HSC from behaviour common to all immature hematopoietic progenitor cells. Therefore, the identification of HSC specific properties, especially regarding their divisional behaviour and fate choice, is a first step required to be able to expand HSC for the use in clinical transplantations.
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The microscopy images that we used as input contain a well boundary, which we remove by applying a pre calculated boundary mask (see Fig. 8.8(b) for boundary- removed image). These images also contain extracellular material and noise that could easily confuse a classifier. To remove the noise, we introduce a fully automatic approach to computing a bounding box that encloses the embryo. To this end, we first determine the largest connected component in the boundary-removed intensity image and compute the centroid of this component. Each image contains one em- bryo only, so the centroid can be computed as the point within the component with maximum shortest distance to the region boundary (see Section 3.2.1 for details). We then crop the image around this centroid to obtain an image of size 151 × 151. The result is shown in Fig. 8.8(c). The dimension of the bounding box reflects the size of the fully developed embryo and is determined by the known optical setup of the image acquisition system. After processing all the training images in this manner, we normalise the results by subtracting the mean intensity taken over the whole dataset. Cell counting is invariant to rotation. Therefore, we generate additional training instances by applying arbitrary rotations and mirroring to the original data. Par- ticularly, images are randomly rotated by an integer multiple of 90 degrees. With mirroring enabled, there are 8 possibilities for the randomly transformed output im- age. This reduces overfitting and, as shown in our experiments, improves accuracy. 126.96.36.199 Network Architecture
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Time-dose and concentration dependency of MLN-8237 to inhibit Aurora A and B was tested in LS141 by choosing a range of concentrations from 10 nM to 10 µM from 12 to 48 hours of exposure. We elected to monitor phospho histone H3 (Ser10) as this accumulates in the setting of the mitotic arrest induced by Aurora A inhibition but is in itself a substrate of Aurora B such that phospho histone H3 is inhibited in the setting of Aurora B inhibition. As shown in Figure 2A (i) induction of phospho histone H3 (ser10) after 24 hours of drug exposure occurs only at a concentration of 100 nM and not at higher or even lower doses consistent with a dose dependent Aurora A effect. Also, there was an induction of p53 and p21 in a dose dependent manner. The effect on phospho Histone H3 was further confirmed and the timing for its induction was further tested by exposing LS141 cells to 100 nM and 1000 nM of MLN-8237 over a period of 24 hours. As shown in Figure 2A (ii), with 100 nM of MLN-8237 the induction of phospho histone H3 (Ser10) occurred at approximately 12 hours, peaks at 18 hours, then starts to decline at 24 hours. It is interesting to note that at 1000 nM phospho H3S10 is completely inhibited at all time points tested. At the same time, there is induction of Aurora A protein levels at both the low and high dose conditions indicating Mitotic accumulation at both concentrations (phospho MPM2 by FACScan analysis). This clearly suggests Aurora A inhibition at 100 nM (high phospho H3S10) and Aurora B inhibition at 1000 nM dose (ablation of phospho H3S10).
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Immune targeting of CSCs presents a promising and safe approach for cancer treatment, and one of the major advantages of most immunotherapeutic strategies is low or acceptable toxicity . The previous report showed CIK cell-based therapy as an enhanced immune cell therapy in mice that can target stem-like lymphoma cells . Cancer patient-derived CIK cells killed putative CSCs of autologous metastatic melanoma , and autologous metastatic bone sarcoma and soft-tissue sarcomas , which will be still required to be confirmed by further evidence (i.e., tumor sphere formation, time-lapse imaging, in vivo experiment, etc) and in various cancers. Additionally, so far, the antitumor activity of CIK cells against CSCs of NPC is completely unexplored. Against this background, in this study, we fully investigated the effects of CIK cell treatment on stem cell-like populations in NPC as well as the underlying mechanisms by using various methods.
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Migration of T cells on the LFA-1 ligand ICAM-1 using time-lapse microscopy. When the patient ’ s T cells were allowed to migrate on ICAM-1, they showed abnormally high adhesion and slow migration when compared with control T cells. A, Images of T lymphoblasts migrating on ICAM-1 showing typical control cells with leading edges and some detached uropods (left panel) and patient cells that are more ﬁ rmly spread with less uniform leading edges (right panel). B, Migratory tracks of individual control and patient T cells (n = 10 tracks per cell type). C, Comparison of speed of migration of control and patient T cells on ICAM-1. *** 5 P , .001.
Until the publication in 2014 of a revised avian phylogeny, based on genomic data, the timing of avian diversiﬁcation has been a subject of much debate (Jarvis et al., 2014). The ﬁ rst avian divergence is con- sidered to have occurred about 100 mya when the Paleognathae (Ra- tites and Tinamous) diverged from the Neognathae (Galloanseres and Neoaves which subsequently diverged ∼ 80 mya). The Galloansere di- vergence into the Galliformes (landfowl e.g. chicken) and Anseriformes (waterfowl e.g. ducks) occurred around the time of the K-Pg extinction event (see below). The major divergences of the Neoaves into Columbea (e.g. pigeons) and Passarea (e.g. songbirds) are now dated to before the K-Pg boundary (67–69 mya). Data from the Jarvis et al. analysis and Prum et al., (2015) suggests that following the mass extinction event thought to be caused by the Chicxulub meteor strike (Schulte et al., 2010), there was a period of rapid avian speciation, with 36 lineages appearing over the relatively short period of 10–15 million years (Jarvis et al., 2014). Genomic studies have therefore, updated our under- standing of dinosaur genomics and its relationship to phenotype and diversity (Zhang et al., 2014a; Jarvis et al., 2014). The overall genomic structure (i.e. karyotype) of dinosaurs was something that had until now, been understudied and was therefore the subject of our in- vestigations.
Being asked to give a presentation about dinosaur genomes (in a conference that is fundamentally about preimplantation and prenatal diagnosis) presented somewhat of a challenge. Firstly, a title; ‘Time lapse’ - to most of the audience - conjures up time-lapse imaging of human embryos; the title that you see above was thus deliberately mischievous as the talk had nothing to do with this. Second, how, scientifically, does our work link to the rest of the conference? The point here is that techniques such as array CGH, NGS and karyomapping would not be possible for chromosome screening unless the human genome was assembled to ‘chromosome-level’; that is a genome with all the sequences assigned to their rightful place on the chromosome.
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Fig. 3. Live imaging of Flk1-myr::mCherry - and Flk1-H2B::eYFP -labeled endothelial cells revealed endothelial cell migrations at the border of the vascular and avascular space. (A,B) Depiction of entire E8.5 embryo showing four key events captured during the time lapse: yolk sac expansion (blue arrows), two regions of EC migrations, proximal to the edge of the yolk as well as distal from the edge (white arrows and white boxes), and sprouting angiogenesis/vessel anastomosis (brown box) at the edge of the yolk sac. (C,D) Select images from time-lapse sequences (4.7 h and 14.5 h) showing EC nuclei (YFP) and membrane (mCherry fluorescent protein) of cultured E8.5 embryo and yolk sac. During the culture, yolk sac and blood vessel expansion occurs around the embryos, as indicated by the blue arrows in C and the final position of the yolk sac, while the avascular spaces are maintained (selected white shapes). (E,F) Endothelial cells close to the edge of the yolk sac show directed migration trajectories as it begins to close (white arrows). E, the same image shown as in C, overlaid with the final migration trajectories to indicate the state of the tissue at 4.7 h. F similarly shows the 14.5 h image as in D, overlaid with the final trajectories. (G,H) Colored maps indicate ECs at 4.7 h (G) and 14.5 h (H) with large displacement lengths (>200 µm, green, yellow and red spheres in G,H). In the region more distal from the edge, ECs showed smaller displacement lengths (<100 µm, blue spheres in G,H). Images were acquired with a 20×, NA=1.0 objective, zoom=0.4, every 10 min for 16.5 h, z-step=2.67 µm, total z-depth=411.18 µm. Scale bar in C: 100 µm.
cells from Actin-DsRed donor mice were transferred i.v. into mesh-implanted mice and followed by intravital 2-photon laser-scanning microscopy (TPLSM) imaging (imaging for up to 3 hours). (A) Representative images of the experimental setting and schematic overview of the experimental design. (B) TPLSM images of cellular infiltrates at early stages of foreign body reaction (top row). Cells were injected 12 hours following surgery and analyzed at day 1, day 4, and day 7 (acute FBR). Imaging at day 21 (bottom row) was commenced following cell injection 1, 14, and 20 days before imaging (chronic FBR). Images were composed of 4 adjacent view fields. Mesh fibers are visible as either black or highly fluorescent coherent structures. Blue fibers show collagen fibers visualized by second harmonic generation. Scale bar: 200 μm. Statistical analysis was performed by cell counting in at least 4 view fields of surrounding mesh fibers with a penetration depth of 200 μm. (C) Flow cytometric assessment of cellular infiltration visible in B. Cells were stained for CD45, Ly6G, CD11b, F4/80, CD11c, I-A b , Ly6C, and CD16. Cells were gated as CD45 + Ly6G – DsRed + cells and for quantification of relative monocyte recruitment efficacy. (D) Flow
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Hindbrain segmentation in culture explants was followed by low-light- level video microscopy (transmitted light). The six-well culture plate was placed on the stage of a Zeiss Axiovert microscope that was surrounded by an insulated box with two enclosed warming incubators (Lyon Electric Co. Inc.; #115-020) that maintained the explant cultures at 38°C for the duration of the time-lapse filming, with only mild temperature fluctuations. For better image resolution, we modified the six-well culture plate by making a hole in the bottom of one of the wells into which a 25 mm round glass coverslip was sealed using silicone grease. The membrane of the Millipore culture insert is 12.7 µm thick and becomes transparent when moist. Images were obtained through a 10X objective (Zeiss Plan-Neofluar, Numerical Aper- ture (NA)=0.30) and recorded with a Hamamatsu 2400 silicon intensified target (SIT) camera. Each image was processed with frame averaging (16 frames) using the VidIm software package (Belford, Stolberg and Fraser, unpublished). VidIm controlled the shutters (Uniblitz, model D122) and the recording of images every 2 minutes onto either a video optical memory disk recorder (OMDR; Panasonic 3038) or digitally to magneto-optical disk (Pinnacle). Images collected to the OMDR can conveniently be played back, allowing for adjustments to the specimen or microscope. Images which were digitally recorded were played back as a movie using the image
The inversion resistivity model is shown in Fig. 5, with the resistivity differences shown in the same figure. A slight reduction in resistivity between 500 and 700 m begins on day 9 and slowly increases with time, culminating with a drop of roughly 15% in resistivity by the final day. There is also a slight ( ≈ 5%) increase in resistivity in the area between 100 and 500 m. Finally, there is also a reduction in resistiv- ity at depth; however, this is smaller than the resistivity drop in the higher zone. Notably this would be near the penetra- tion depth of the data, and would be less constrained than the other areas of the model. The entire model resistivity is between 1.25 and 16 m. When considering the commence- ment date of pumping, shown as a dashed line in Fig. 5, we see that the resistivity changes occur slightly before the com- mencement of dewatering. In the synthetic model, temporal Fig. 4 Schematic of survey extents. A schematic of the site used for
This screen is the home screen of the human machine interface (HMI) interface which was developed for this system. This screen contains basic parameters for the user to select. User can use the slider to select the time required for the lapse. An angle selection drop down is provided to enable user for selecting the degree of rotations desired. Once the user completes making these selections then the user can click on the start button to start the Time Lapse session. Figure 6 provides an over view of the main screen.
lapse experiment, where the cells were then imaged for Hoechst 33342 fluorescence. An algorithm coded in MATLAB (The MathWorks, Natick, MA, USA) was constructed to quantify the integrated nuclear fluorescence of Hoechst 33342 in individual cells. From this algorithm, a cell cycle histogram is generated from all cells imaged in a single field of view, allowing DNA content to be estimated. The ploidy of each mitotic cell was manually determined by summing the amount of DNA content observed in the respective daughter cells. For the FUCCI sensor (ThermoFisher Scientific, Waltham, MA, USA) experiments, cells were co-transduced with 80 particles per cell (PPC) Premo geminin-GFP (G2/M reagent) and 80 PPC Premo Cdt1-RFP (G1/S reagent) for 24 hours. The cells were then washed, released into complete media and imaged 12 hours later at 20 minute intervals for 18 hours. Individual cells expressing both vectors were scored for completion of mitosis, an abortive mitosis or mitotic bypass.
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software [9–12]. Since we needed a simple and readily adaptable tool for the quantitative analysis of large amounts of time-lapse microscopy data, we established the TLM-Quant pipeline for data processing and analyses based on open-source software. This pipeline was validated using a custom-built fluorescence microscopy set-up and B. subtilis strains producing GFP from promoters that direct either homogenous, heterogeneous, or bistable gene expression, as described by Botella et al. . Importantly, the TLM-Quant pipeline was then effectively implemented in a large-scale systems biological analysis on the global network reorganization during dynamic adaptations of B. subtilis metabolism to nutritional shifts between the preferred carbon sources glucose and malate . In the latter study, TLM-Quant allowed us to verify the absence of heterogeneity in the expression of genes involved in central carbon metabolism. The respective datasets can be queried at https://basysbio.ethz.ch/ openbis/index.html?viewMode = SIM-
Cells were grown and transfected in 35mm glass-bottomed culture dishes (Iwaki brand; Asahi Techno Glass Corporation, Japan) obtained from Bibby Sterilin. Transfections were performed using GeneJuice (Novagen) according to the manufacturers instructions. 12-18h after transfection the standard cell culture medium was replaced by 2ml of pre-warmed medium supplemented with 20mM HEPES. The cells were transferred to a Zeiss Axiovert 200 inverted microscope with a heated chamber enclosing the microscope stage (Solent Scientific, UK) allowing the temperature to be maintained at 37 ° C throughout imaging. Cells were imaged by fluorescence microscopy using a Zeiss Plan Apo 63X/1.4NA oil immersion lens. Time-lapse images were captured at 3-10s intervals for durations of 5-10min using Ludl shutters and a Hamamatsu Orca II ER camera. Images were obtained using 2x2 binning with exposure times of less than 350ms/frame. An excitation/emission
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Monocyte-derived dendritic cells (MDDC) stimulate CD8 ⴙ cytotoxic T lymphocytes (CTL) by presenting endogenous and exog- enous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 de- pletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse micros- copy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stoma- titis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demon- strate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the anti- viral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.
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