Time-resolved imaging-based CRISPRi screening

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays

Bovine oocyte exposure to perfluorohexane sulfonate (PFHxS) induces phenotypic, transcriptomic, and DNA methylation changes in resulting embryos in vitro

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage

3D surface reconstruction for lower limb prosthetic model using modified radon transform

Computer vision has received increased attention for the research and innovation on three-dimensional surface reconstruction with aim to obtain accurate results. Although many researchers have come up with various novel solutions and feasibility of the findings, most require the use of sophisticated devices which is computationally expensive. Thus, a proper countermeasure is needed to resolve the reconstruction constraints and create an algorithm that is able to do considerably fast reconstruction by giving attention to devices equipped with appropriate specification, performance and practical affordability. This thesis describes the idea to realize three-dimensional surface of the residual limb models by adopting the technique of tomographic imaging coupled with the strategy based on multiple-views from a digital camera and a turntable. The surface of an object is reconstructed from uncalibrated two-dimensional image sequences of thirty-six different projections with the aid of Radon transform algorithm and shape-from-silhouette. The results show that the main objective to reconstruct three-dimensional surface of lower limb model has been successfully achieved with reasonable accuracy as the starting point to reconstruct three-dimensional surface and extract digital reading of an amputated lower limb model where the maximum percent error obtained from the computation is approximately 3.3 % for the height whilst 7.4%, 7.9% and 8.1% for the diameters at three specific heights of the objects. It can be concluded that the reconstruction of three-dimensional surface for the developed method is particularly dependent to the effects the silhouette generated where high contrast two-dimensional images contribute to higher accuracy of the silhouette extraction. The advantage of the concept presented in this thesis is that it can be done with simple experimental setup and the reconstruction of three-dimensional model neither involves expensive equipment nor require any service by an expert to handle sophisticated mechanical scanning system