Computer vision for sequential non-invasive microscopy imaging cytometry with applications in embryology

Many in vitro cytometric methods requires the sample to be destroyed in the process. Using image analysis of non-invasive microscopy techniques it is possible to monitor samples undisturbed in their natural environment, providing new insights into cell development, morphology and health. As the effect on the sample is minimized, imaging can be sustained for long un-interrupted periods of time, making it possible to study temporal events as well as individual cells over time. These methods are applicable in a number of fields, and are of particular importance in embryological studies, where no sample interference is acceptable. Using long term image capture and digital image cytometry of growing embryos it is possible to perform morphokinetic screening, automated analysis and annotation using proper software tools. By literature reference, one such framework is suggested and the required methods are developed and evaluated. Results are shown in tracking embryos, embryo cell segmentation, analysis of internal cell structures and profiling of cell growth and activity. Two related extensions of the framework into three dimensional embryo analysis and adherent cell monitoring are described

A Modular and Open-Source Framework for Virtual Reality Visualisation and Interaction in Bioimaging

Life science today involves computational analysis of a large amount and variety of data, such as volumetric data acquired by state-of-the-art microscopes, or mesh data from analysis of such data or simulations. The advent of new imaging technologies, such as lightsheet microscopy, has resulted in the users being confronted with an ever-growing amount of data, with even terabytes of imaging data created within a day. With the possibility of gentler and more high-performance imaging, the spatiotemporal complexity of the model systems or processes of interest is increasing as well. Visualisation is often the first step in making sense of this data, and a crucial part of building and debugging analysis pipelines. It is therefore important that visualisations can be quickly prototyped, as well as developed or embedded into full applications. In order to better judge spatiotemporal relationships, immersive hardware, such as Virtual or Augmented Reality (VR/AR) headsets and associated controllers are becoming invaluable tools. In this work we present scenery, a modular and extensible visualisation framework for the Java VM that can handle mesh and large volumetric data, containing multiple views, timepoints, and color channels. scenery is free and open-source software, works on all major platforms, and uses the Vulkan or OpenGL rendering APIs. We introduce scenery's main features, and discuss its use with VR/AR hardware and in distributed rendering. In addition to the visualisation framework, we present a series of case studies, where scenery can provide tangible benefit in developmental and systems biology: With Bionic Tracking, we demonstrate a new technique for tracking cells in 4D volumetric datasets via tracking eye gaze in a virtual reality headset, with the potential to speed up manual tracking tasks by an order of magnitude. We further introduce ideas to move towards virtual reality-based laser ablation and perform a user study in order to gain insight into performance, acceptance and issues when performing ablation tasks with virtual reality hardware in fast developing specimen. To tame the amount of data originating from state-of-the-art volumetric microscopes, we present ideas how to render the highly-efficient Adaptive Particle Representation, and finally, we present sciview, an ImageJ2/Fiji plugin making the features of scenery available to a wider audience.:Abstract Foreword and Acknowledgements Overview and Contributions Part 1 - Introduction 1 Fluorescence Microscopy 2 Introduction to Visual Processing 3 A Short Introduction to Cross Reality 4 Eye Tracking and Gaze-based Interaction Part 2 - VR and AR for System Biology 5 scenery — VR/AR for Systems Biology 6 Rendering 7 Input Handling and Integration of External Hardware 8 Distributed Rendering 9 Miscellaneous Subsystems 10 Future Development Directions Part III - Case Studies C A S E S T U D I E S 11 Bionic Tracking: Using Eye Tracking for Cell Tracking 12 Towards Interactive Virtual Reality Laser Ablation 13 Rendering the Adaptive Particle Representation 14 sciview — Integrating scenery into ImageJ2 & Fiji Part IV - Conclusion 15 Conclusions and Outlook Backmatter & Appendices A Questionnaire for VR Ablation User Study B Full Correlations in VR Ablation Questionnaire C Questionnaire for Bionic Tracking User Study List of Tables List of Figures Bibliography Selbstständigkeitserklärun

Dynamic hypoxic pre-conditioning of cells seeded in tissue-engineered scaffold to improve neovascularisation

Introduction: Tissue engineering (TE) is the potential solution to the global shortage of tissue and organs. However, the lack of adequate angiogenesis to TE scaffolds during the initial stages of implantation has hindered its success in vivo. Mesenchymal stem cells (MSC) have the most established track record for translational regenerative therapy and have been widely used in combination with TE scaffolds. Hypoxia is one of the main potentiators for upregulating angiogenic factors in MSC. However, fine-tuning their cellular function and behaviour is still not fully understood. This study aims to help increase the understanding of this process by determining the effects of in vitro hypoxic conditioning on enhancement of angiogenesis of MSC for the purpose of pre-clinical translational for TE application. Methods: The angiogenic potential of 3 different tissue sources (bone marrow, umbilical cord and adipose) MSC were initially determined for downstream pre-clinical application. We established the appropriate regime for in vitro dynamic hypoxia conditions in 2D and 3D hydrogel to enhance MSC angiogenic pathway using real-time continuous oxygen sensors and angiogenic cytokine profiling. Cell metabolism and proliferation effects were also evaluated using intravital Realtime-glo, D-luciferin (on transduced MSC) and microscopic Live-Dead stain techniques. We optimised seeding of cells on the tissue engineered dermal (INTEGRA®) for in vivo translational purpose and used targeted in vitro and ex vivo angiogenesis assays, which helped to determine aspects of the MSC conditioned media on endothelial migration, proliferation, morphogenesis and matrix degradation. Finally, the functional reproducibility of the in vitro angiogenic response was assessed using in vivo angiogenesis CAM assay and murine diabetic wound healing models. Results: Adipose derived MSC (adMSC) were found to have the most angiogenic potential in response to hypoxic conditioning. Dynamic hypoxia (DH) regime of changing oxygen levels from 21% to 1% when transitioning from T-flask subculture to multiwell plate seeding was most effective at eliciting pro-angiogenic response from adMSC for both in vitro 2D and 3D models compared to controls using static normoxia (21% oxygen) and static hypoxia (1%). Low seeding density of adMSC was found to be the most appropriate to ensure optimised cell adherence and survival post-seeding on TE dermal scaffold (INTEGRA®). It also minimised on localised hypoxic gradient induced oxidative stress by the seeded cells when compared to high seeding density techniques found on non-invasive oxygen monitoring. Conditioned media from DH seeded adMSC was shown to have enhanced angiogenic proteomic profile compared to the controls. In vitro angiogenesis assays showed better human endothelial cell migration and morphogenesis in scratch assay and tubular formation assay compared to controls. Preliminary ex vivo organ assay results using novel human umbilical arterial rings showed better endothelial out-sprouting and migration through embedded matrix compared to controls. Results from in vivo transplantation of adMSC seeded INTEGRA® scaffold showed a mixed response in the CAM assay, highlighting an unaccounted scaffold effect from INTEGRA® from the host. Histological sections showed increased vascular and host tissue infiltration into the scaffold. When evaluating the functional angiogenesis in murine wound healing models, although DH adMSC seeded scaffolds showed non-statistically significant increased rate of wound closure, there was significantly greater vessel density within the scaffold on histological evaluation in this group compared to controls. Conclusion: The results provide a better comprehension of how cells behave in 2D and 3D environments when cultured in dynamically changing oxygen environments. The study addresses important issues, such as the effects of chronic hypoxia on MSC, and how dynamic hypoxia can enhance angiogenic signalling. It also offers a crucial understanding of the in vitro oxygen culture environments for future research applications. Further insight into cell-scaffold interaction during in vivo transplantation was also established. The importance of having an appropriate in vivo model to determine if such in vitro angiogenic enhancement would translate to functionally improving neoangiogenesis and subsequent tissue regeneration in vivo was also highlighted in this study. Improving and advancing research into optimising and evaluating the in vitro environment for clinical application will undoubtedly have a huge impact on the future of cell therapy for regenerative medicine purposes

Molecular, morphological, and kinetic diagnosis of human preimplantation embryo viability

There have been phenomenal advances in the field of reproductive medicine and success rates following in vitro fertilisation have improved dramatically in recent years. The aim of this project was to improve our understanding of human preimplantation embryo development by identifying potential markers of viability that may aid us in selecting the best embryo for uterine transfer in the clinical embryology laboratory. Investigations into the distribution of cytoskeletal F-actin in human embryos demonstrated that a highly organised actin cortex is important for embryo cleavage and continued development to the blastocyst stage. Whilst they are polarised in the mouse from the oocyte to the blastocyst, the regulatory proteins leptin and STAT3 are co-localised only at the oocyte stage in humans, and are distributed within different cytoplasmic domains in human cleavage stage embryos and blastocysts. Whether polarity in humans is predetermined in the oocyte remains elusive, but none of the evidence generated in this thesis supports this idea. Leptin transiently activates STAT3 via the long form of the leptin receptor, and most significantly in the ICM of human day 6 blastocysts. Morphological features of blastocysts that can be visualised microscopically, such as a double ICM and cytoplasmic projections connecting the ICM to the TE, provide clues to their viability and may help us to choose the most suitable embryo from a cohort when deciding which to transfer. Nuclear volumes may in future contribute to this selection. Using time lapse technology to study cleavage patterns is now a routine occurrence in the clinical embryology laboratory. The results in this thesis show that distinctive patterns of divisions and the site at which blastocysts hatch can provide us with more information than a snap-shot morphological evaluation. Finally, contributing to the development of modelling software and predictive algorithms for the study of human embryos, particularly in time lapse imaging, means that our understanding of this fascinating area of medicine will continue to progress

Cell markers for air chamber development in Marchantia polymorpha

Marchantia polymorpha, thanks to its small genome and its simple cellular architecture, is an excellent model for the study of developmental processes. Marchantia can propagate asexually producing small flat disc-like propagules, called gemmae, that develop into adult plants. Gemmae have an open mode of development, which facilitates the live observation of developmental processes. The fundamental processes underlying morphogenesis in plants are poorly understood and epidermal patterning represents one of the key events during plant development. Marchantia produces characteristic epidermal structures called air chambers that represent an example of a self-organising, three-dimensional morphological unit that has a modular and repetitive arrangement, similar to the one observed in stomata meristemoid cells in vascular plants. To date, the cellular scale and the genetic description of air chamber development are poorly resolved. Therefore, in this dissertation I applied modern genetic and microscopy tools to better map the process of air chamber morphogenesis at a cellular scale and to generate genetic markers to understand the steps involved in this developmental process. Similar tools have been used in Arabidopsis thaliana, and they have shown that patterning and cell differentiation can be dissected into discrete steps that provide insights into the mechanisms controlling the process and can also represent valuable targets for engineering the entire process. In my thesis, I developed high resolution imaging techniques to produce a systematic description of air chamber development. I generated genetic resources to identify potential candidate genes involved in air chamber development by comparing the transcriptome of wild type plants and the nopperabo1 (nop1) mutants that are unable to produce air chambers. Candidate genes were used to build a collection of cellular markers to be screened for their potential involvement in air chamber patterning. The screening led to the identification of two potential transcription factors, MpbHLH24 and MpWRKY10

X-ray computed tomography analysis of mouse embryonic heart

Rentgenová počítačová tomografie je univerzální technika umožňující nedestruktivní trojrozměrné (3D) zobrazování. Je široce používána v průmyslu pro metrologii a charakterizaci materiálů. V poslední době našla svůj význam také v biologii a vývojové vědě, kde 3D rekonstruovaná data poskytují komplexnější informace o vzorku než konvenční 2D zobrazovací metody. Kromě toho může vytvářet vysoce kvalitní snímky různých biologických vzorků. Překážkou při zobrazování biologických vzorků může být velmi podobný koeficient útlumu měkkých tkání, a proto je nutné použít barvení chemickou látkou. Tato práce si klade za cíl pomocí mikropočítačové tomografie vizualizovat a kvalitativně vyhodnotit srdce embryonálních myší E17.5 a představit nejlepší barvící protokol. Dále byla zavedena metodika pro zvýraznění rozdílů mezi myší divokého typu a mutantem. Rigaku nano 3DX byl použit pro experimenty s mezi-komorovou přepážkou myšího srdce a po nastavení specifických parametrů zařízení byl použit algoritmus pro zvýraznění fáze. Nakonec byla provedena validace $\mu$ CT dat se snímkami z konfokálního mikroskopu.X-ray computed tomography is a versatile technique allowing non-destructive three-dimensional (3D) imaging. It is widely used in industry for metrology and material characterisation. Lately, it has found its importance also in biology and developmental science, where the 3D reconstructed data provide more comprehensive information about the sample than the conventional 2D imaging methods. Moreover, it can produce high-quality images of various biological samples. The obstacle in imaging biological samples might be a very similar attenuation coefficient of soft tissue, and therefore staining with chemical substance must be used. This thesis aims to use microcomputed tomography to visualise and qualitatively evaluate embryonic mouse hearts E17.5 and introduce the best staining protocol. Furthermore, the methodology for highlighting differences between wild type mice and mutant were introduced. Rigaku nano 3DX was used for the experiments with mouse heart interventricular septum, and after considering the specific setting of the device, the phase retrieval algorithm was applied. Finally, the validation of the $\mu$CT data was performed by comparing pictures from the confocal microscope.

Cardiac multi-scale investigation of the right and left ventricle ex vivo: a review

The heart is a complex multi-scale system composed of components integrated at the subcellular, cellular, tissue and organ levels. The myocytes, the contractile elements of the heart, form a complex three-dimensional (3D) network which enables propagation of the electrical signal that triggers the contraction to efficiently pump blood towards the whole body. Cardiovascular diseases (CVDs), a major cause of mortality in developed countries, often lead to cardiovascular remodeling affecting cardiac structure and function at all scales, from myocytes and their surrounding collagen matrix to the 3D organization of the whole heart. As yet, there is no consensus as to how the myocytes are arranged and packed within their connective tissue matrix, nor how best to image them at multiple scales. Cardiovascular imaging is routinely used to investigate cardiac structure and function as well as for the evaluation of cardiac remodeling in CVDs. For a complete understanding of the relationship between structural remodeling and cardiac dysfunction in CVDs, multi-scale imaging approaches are necessary to achieve a detailed description of ventricular architecture along with cardiac function. In this context, ventricular architecture has been extensively studied using a wide variety of imaging techniques: ultrasound (US), optical coherence tomography (OCT), microscopy (confocal, episcopic, light sheet, polarized light), magnetic resonance imaging (MRI), micro-computed tomography (micro-CT) and, more recently, synchrotron X-ray phase contrast imaging (SR X-PCI). Each of these techniques have their own set of strengths and weaknesses, relating to sample size, preparation, resolution, 2D/3D capabilities, use of contrast agents and possibility of performing together with in vivo studies. Therefore, the combination of different imaging techniques to investigate the same sample, thus taking advantage of the strengths of each method, could help us to extract the maximum information about ventricular architecture and function. In this review, we provide an overview of available and emerging cardiovascular imaging techniques for assessing myocardial architecture ex vivo and discuss their utility in being able to quantify cardiac remodeling, in CVDs, from myocyte to whole organ