Nuclear instance segmentation and tracking for preimplantation mouse embryos.

For investigations into fate specification and morphogenesis in time-lapse images of preimplantation embryos, automated 3D instance segmentation and tracking of nuclei are invaluable. Low signal-to-noise ratio, high voxel anisotropy, high nuclear density, and variable nuclear shapes can limit the performance of segmentation methods, while tracking is complicated by cell divisions, low frame rates, and sample movements. Supervised machine learning approaches can radically improve segmentation accuracy and enable easier tracking, but they often require large amounts of annotated 3D data. Here we first report a novel mouse line expressing near-infrared nuclear reporter H2B-miRFP720. We then generate a dataset (termed BlastoSPIM) of 3D images of H2B-miRFP720-expressing embryos with ground truth for nuclear instances. Using BlastoSPIM, we benchmark seven convolutional neural networks and identify Stardist-3D as the most accurate instance segmentation method. With our BlastoSPIM-trained Stardist-3D models, we construct a complete pipeline for nuclear instance segmentation and lineage tracking from the 8-cell stage to the end of preimplantation development (>100 nuclei). Finally, we demonstrate BlastoSPIM's usefulness as pre-train data for related problems, both for a different imaging modality and for different model systems.

A novel ground truth dataset enables robust 3D nuclear instance segmentation in early mouse embryos.

For investigations into fate specification and cell rearrangements in live images of preimplantation embryos, automated and accurate 3D instance segmentation of nuclei is invaluable; however, the performance of segmentation methods is limited by the images' low signal-to-noise ratio and high voxel anisotropy and the nuclei's dense packing and variable shapes. Supervised machine learning approaches have the potential to radically improve segmentation accuracy but are hampered by a lack of fully annotated 3D data. In this work, we first establish a novel mouse line expressing near-infrared nuclear reporter H2B-miRFP720. H2B-miRFP720 is the longest wavelength nuclear reporter in mice and can be imaged simultaneously with other reporters with minimal overlap. We then generate a dataset, which we call BlastoSPIM, of 3D microscopy images of H2B-miRFP720-expressing embryos with ground truth for nuclear instance segmentation. Using BlastoSPIM, we benchmark the performance of five convolutional neural networks and identify Stardist-3D as the most accurate instance segmentation method across preimplantation development. Stardist-3D, trained on BlastoSPIM, performs robustly up to the end of preimplantation development (> 100 nuclei) and enables studies of fate patterning in the late blastocyst. We, then, demonstrate BlastoSPIM's usefulness as pre-train data for related problems. BlastoSPIM and its corresponding Stardist-3D models are available at: blastospim.flatironinstitute.org.

Extracellular Microenvironmental Control for Organoid Assembly.

Organoids, which are multicellular clusters with similar physiological functions to living organs, have gained increasing attention in bioengineering. As organoids become more advanced, methods to form complex structures continue to develop. There is evidence that the extracellular microenvironment can regulate organoid quality. The extracellular microenvironment consists of soluble bioactive molecules, extracellular matrix, and biofluid flow. However, few efforts have been made to discuss the microenvironment optimal to engineer specific organoids. Therefore, this review article examines the extent to which engineered extracellular microenvironments regulate organoid quality. First, we summarize the natural tissue and organ's unique chemical and mechanical properties, guiding researchers to design an extracellular microenvironment used for organoid engineering. Then, we summarize how the microenvironments contribute to the formation and growth of the brain, lung, intestine, liver, retinal, and kidney organoids. The approaches to forming and evaluating the resulting organoids are also discussed in detail. Impact statement Organoids, which are multicellular clusters with similar physiological function to living organs, have been gaining increasing attention in bioengineering. As organoids become more advanced, methods to form complex structures continue to develop. This review article focuses on recent efforts to engineer the extracellular microenvironment in organoid research. We summarized the natural organ's microenvironment, which informs researchers of key factors that can influence organoid formation. Then, we summarize how these microenvironmental controls significantly contribute to the formation and growth of the corresponding brain, lung, intestine, liver, retinal, and kidney organoids. The approaches to forming and evaluating the resulting organoids are discussed in detail, including extracellular matrix choice and properties, culture methods, and the evaluation of the morphology and functionality through imaging and biochemical analysis.

Stretchable, anti-bacterial hydrogel activated by large mechanical deformation.

Hydrogels have been used extensively to deliver functional molecular cargos in response to external mechanical force. However, the intrinsic brittleness of gels restricts the applicable range of strain to 0.1, thus limiting the range of molecular release rate that may be controlled. Also, uncontrollable molecular diffusion, which is especially prominent in small molecules, reduces the role of mechanical stimulus on the release rate. As such, we hypothesized that these challenges would be resolved by combining cyclodextrin, which may form guest-host complexes with small molecular cargos, with a stretchable hydrogel system. We examined this hypothesis by synthesizing cyclodextrin acrylate and incorporating it into a polyacrylamide gel that can be stretched by 100% of its original length. In the absence of external stretching, hydrogels containing cyclodextrin acrylate with a degree of acryloyl group substitution (DSA) of 2.3 presented a lower molecular release rate than hydrogels without cyclodextrin acrylate. More interestingly, the polyacrylamide-cyclodextrin hydrogel system displayed an increased molecular release rate corresponding to the degree of stretching, particularly in the gels containing cyclodextrin acrylate with a DSA of 2.3. As such, this stretchable gel loaded with quinine was used to inhibit the growth of E. coli in lysogeny broth only when the gel was stretched. We believe the results of this study would be valuable for improving the quality of controlled molecular delivery and subsequent efficacy of molecular cargos.