A review of different deep learning techniques for sperm fertility prediction

Sperm morphology analysis (SMA) is a significant factor in diagnosing male infertility. Therefore, healthy sperm detection is of great significance in this process. However, the traditional manual microscopic sperm detection methods have the disadvantages of a long detection cycle, low detection accuracy in large orders, and very complex fertility prediction. Therefore, it is meaningful to apply computer image analysis technology to the field of fertility prediction. Computer image analysis can give high precision and high efficiency in detecting sperm cells. In this article, first, we analyze the existing sperm detection techniques in chronological order, from traditional image processing and machine learning to deep learning methods in segmentation and classification. Then, we analyze and summarize these existing methods and introduce some potential methods, including visual transformers. Finally, the future development direction and challenges of sperm cell detection are discussed. We have summarized 44 related technical papers from 2012 to the present. This review will help researchers have a more comprehensive understanding of the development process, research status, and future trends in the field of fertility prediction and provide a reference for researchers in other fields

Approaches for assessing the presence and impact of thyroid hormone disrupting chemicals in delphinid cetaceans

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2006Cetacean blubber is a primary site for lipid storage, which the animal utilizes during periods of energetic stress. It is important to understand how the blubber responds to factors such as ontogeny, water temperature, reproductive status, and nutritional state because blubber is also the primary bioaccumulation site for persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). During periods of lipid mobilization such as lactation, PCBs from the blubber are mobilized into the circulatory system and may cause toxic effects. One particular toxic mechanism may include the induction of cytochrome P450 enzymes in the integument and liver, which could enhance the biotransformation of PCBs to hydroxylated metabolites (OH-PCBs). OH-PCBs may then interfere with thyroid hormone dependent neurodevelopment. The goals of these studies were to investigate the relationships between lipid dynamics and PCB effects and to devise a quantitative approach to assess neurodevelopment in delphinid cetaceans. Blubber morphology, cytochrome P450 1A1 (CYP1A1) expression in the skin-blubber biopsy, blubber and plasma PCBs, and plasma OH-PCBs were assessed in bottlenose dolphins (Tursiops truncatus). In addition, magnetic resonance (MR) images of the postmortem brain in situ were obtained from Atlantic white-sided dolphin (Lagenorhynchus acutus) specimens.These results showed that: 1) Factors such as ontogeny, water temperature, and reproductive status affected blubber morphology in bottlenose dolphins. In response to warmer water, the lipid content of the blubber decreased and this appeared to involve loss of lipids from adipocytes in the middle blubber layer. Similar to the effects of starvation on blubber morphology, lactation decreased adipocyte size predominantly in the deeper blubber, 2) CYP1A1 levels in the deep blubber were significantly related to the total plasma TEQ98 concentrations, adipocyte shrinkage, and plasma OH-PCB levels, 3) Through in situ MR imaging of stranded, Atlantic white-sided dolphin specimens, the size of brain structures that depend on thyroid hormones for maturation could be measured accurately. Future studies can use this technique, coupled with chemical analysis of brain regions, to determine if thyroid hormone disrupting chemicals in delphinid cetaceans are associated with changes in the size of brain structures.Funding for this research was provided by an Environmental Protection Agency STAR fellowship (U-91616101-2) awarded to Eric Montie, NOAA contract #WC1330- 02SE0257, NOAA contract #JHT04P1226, NOAA Fisheries Marine Mammal Health and Stranding Response Program, the Florida Protect Wild Dolphins License Plate Fund, the National Woman’s Farm and Garden Association Scholarship awarded to Eric Montie, Shields MRI and CT of Cape Cod, the Quebec Labrador Fund/Atlantic Center for the Environment, Woods Hole Oceanographic Institution Academic Programs Office, Office of Naval Research, and NOAA Fisheries Marine Mammal Health and Stranding Response Program

Electron microscopy and new technological approaches to investigate structural elements of the mitotic apparatus in Saccharomyces cerevisiae and Xenopus laevis

The mitotic cell cycle is a complex process which leads to the chromosome segregation from the mother cell to the two daughter cells and transmit the full genetic background necessary to grow and adapt to the permanently evolving environment. The equal partitioning of the duplicated chromosomes is finely regulated by the cytoskeleton which on purpose organises into a mitotic spindle. In eukaryotes like Xenopus laevis and Saccharomyces cerevisiae the mitotic spindle is mainly composed of three components: first the microtubules (MT) are organised in a spindle emanating from the chromosomes and focusing at the poles. Second they connect to the chromosomes via the kinetochore complexes (KT) and third focus at the microtubule organising centres (MTOC), also called centrosomes. We have focused our interest on structural analysis of the S. cerevisiae centrosome, called the Spindle Pole Body (SPB), at the molecular level, and on the MTs organisation in the meiotic spindle midzone of X. laevis. With S. cerevisiae we have used cryo-electron tomography on vitreous sections (CETOVIS) to investigate the three dimensional (3D) molecular structure of the SPB. The samples were vitrified by high pressure freezing (HPF) and sectioned in vitreous state. Acquiring instant snapshots close to native state of the SPB in vivo, we confirmed previous observations done on plunge frozen or freeze substituted material. The Spc42 central crystal protein are organised with a defined spacing of 107Å, but the plaques composing the SPB could not all be identified. Mainly, the central and the outer plaque were visible, in contrast with the plastic tomography results where the inner plaque appeared dense and compact. Unfortunately, the very low signal to noise ratio prevented us from extracting details structural information about the SPB in its native state. The project concerning the X. laevis meiotic spindle focused on the 3D organisation of the MT within the spindle. Their interactions with the MTOC and the KT have been extensively studied over the past, but a high resolution structural map is still missing and has long been awaited by scientists to put all the knowledge into a 3D context. Furthermore, information about the individual MT is missing like their average length distribution, the existence of short MTs and the end morphologies distribution. To study this complex and large structure, we have developed a novel correlative light to electron microscopy (CLEM) approach combined with electron tomography. We cryo-fixed by HPF, for the first time to our knowledge, spindle assembled in X. laevis egg extracts and prepared them for a structural electron tomographic study. We reconstructed three quarter of a meiotic spindle midzone and identified three subcategories of MT bundle organisation. Finally, we have used our CLEM method to develop a new technology that should facilitate future CLEM work. Recent advances in plastic polymer transformation and micro-injection moulding (µIM) allowed us to create a cryocaspule designed for an easy correlative microscopy and transfer for HPF

Novel approaches for image analysis of in vitro epithelial cultures with application to silver nanoparticle toxicity

A novel imaging approach was developed for the purpose of counting cells from phase contrast microscopy images of laboratory grown (in vitro} cultures of epithelial cells. Validation through comparison with standard laboratory cell counting techniques showed this approach provided consistent and comparable results, whilst overcoming limitations of these existing techniques, such as operator variability and sample destruction. The imaging approach was subsequently applied to investigate the effects of silver nanoparticles (AgNP} on H400 oral keratinocytes. Concurrent investigations into antimicrobial effects of AgNP were performed on Escherichia coli, Staphylococcus aureus and Streptococcus mutans to provide models for Gram-positive and Gram-negative infection, and to compare with the literature and oral keratinocyte toxicity. It was found that AgNP elicit size-, dose- and time-dependent growth inhibition in both human cells and bacteria, although bacterial inhibition was not achieved without significant cytotoxicity at the same concentrations

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Holography

Holography - Basic Principles and Contemporary Applications is a collection of fifteen chapters, describing the basic principles of holography and some recent innovative developments in the field. The book is divided into three sections. The first, Understanding Holography, presents the principles of hologram recording illustrated with practical examples. A comprehensive review of diffraction in volume gratings and holograms is also presented. The second section, Contemporary Holographic Applications, is concerned with advanced applications of holography including sensors, holographic gratings, white-light viewable holographic stereograms. The third section of the book Digital Holography is devoted to digital hologram coding and digital holographic microscopy

Analysis of the Barr body with super-resolution microscopy

X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment

Object Detection in medical imaging

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor in Information Management, specialization in Information and Decision SystemsArtificial Intelligence, assisted by deep learning, has emerged in various fields of our society. These systems allow the automation and the improvement of several tasks, even surpassing, in some cases, human capability. Object detection methods are used nowadays in several areas, including medical imaging analysis. However, these methods are susceptible to errors, and there is a lack of a universally accepted method that can be applied across all types of applications with the needed precision in the medical field. Additionally, the application of object detectors in medical imaging analysis has yet to be thoroughly analyzed to achieve a richer understanding of the state of the art. To tackle these shortcomings, we present three studies with distinct goals. First, a quantitative and qualitative analysis of academic research was conducted to gather a perception of which object detectors are employed, the modality of medical imaging used, and the particular body parts under investigation. Secondly, we propose an optimized version of a widely used algorithm to overcome limitations commonly addressed in medical imaging by fine-tuning several hyperparameters. Thirdly, we develop a novel stacking approach to augment the precision of detections on medical imaging analysis. The findings show that despite the late arrival of object detection in medical imaging analysis, the number of publications has increased in recent years, demonstrating the significant potential for growth. Additionally, we establish that it is possible to address some constraints on the data through an exhaustive optimization of the algorithm. Finally, our last study highlights that there is still room for improvement in these advanced techniques, using, as an example, stacking approaches. The contributions of this dissertation are several, as it puts forward a deeper overview of the state-of-the-art applications of object detection algorithms in the medical field and presents strategies for addressing typical constraints in this area.A Inteligência Artificial, auxiliada pelo deep learning, tem emergido em diversas áreas da nossa sociedade. Estes sistemas permitem a automatização e a melhoria de diversas tarefas, superando mesmo, em alguns casos, a capacidade humana. Os métodos de detecção de objetos são utilizados atualmente em diversas áreas, inclusive na análise de imagens médicas. No entanto, esses métodos são suscetíveis a erros e falta um método universalmente aceite que possa ser aplicado em todos os tipos de aplicações com a precisão necessária na área médica. Além disso, a aplicação de detectores de objetos na análise de imagens médicas ainda precisa ser analisada minuciosamente para alcançar uma compreensão mais rica do estado da arte. Para enfrentar essas limitações, apresentamos três estudos com objetivos distintos. Inicialmente, uma análise quantitativa e qualitativa da pesquisa acadêmica foi realizada para obter uma percepção de quais detectores de objetos são empregues, a modalidade de imagem médica usada e as partes específicas do corpo sob investigação. Num segundo estudo, propomos uma versão otimizada de um algoritmo amplamente utilizado para superar limitações comumente abordadas em imagens médicas por meio do ajuste fino de vários hiperparâmetros. Em terceiro lugar, desenvolvemos uma nova abordagem de stacking para aumentar a precisão das detecções na análise de imagens médicas. Os resultados demostram que, apesar da chegada tardia da detecção de objetos na análise de imagens médicas, o número de publicações aumentou nos últimos anos, evidenciando o significativo potencial de crescimento. Adicionalmente, estabelecemos que é possível resolver algumas restrições nos dados por meio de uma otimização exaustiva do algoritmo. Finalmente, o nosso último estudo destaca que ainda há espaço para melhorias nessas técnicas avançadas, usando, como exemplo, abordagens de stacking. As contribuições desta dissertação são várias, apresentando uma visão geral em maior detalhe das aplicações de ponta dos algoritmos de detecção de objetos na área médica e apresenta estratégias para lidar com restrições típicas nesta área

Unraveling nanoscale alterations in liver cell fenestrations - Morphological studies via optical super-resolution microscopy approaches

The endothelium makes up the innermost cell layer of blood vessels. It consists of a thin layer of simple squamous cells, forming an interface between circulating blood and the surrounding tissue. Endothelial cells of different vascular beds are specialized according to tissue-specific functions. For this project emphasis was placed upon high-resolution methods enabling the study of liver sinusoidal endothelial cells (LSECs) below the diffraction limit of visible light (~200 nm). LSECs have unusual morphology with as much of 20% of their surface covered with cellular fenestrations - holes through the cells of 50-300 nm diameter. These allow bi-directional flow of plasma from the sinusoids to the surrounding hepatocytes, while retaining blood cells in the sinusoidal lumen. Little is known about the function of fenestrations, their regulation, and their role in the transfer of metabolites, viruses, lipoproteins and pharmaceuticals to other cells of the liver. There are two major challenges with the study of LSEC fenestrations; i) the majority have diameters smaller than the diffraction limit of visible light and; ii) they disappear rapidly in cultured LSEC, and there are no cell line alternatives that express fenestrations. To address the first challenge, the project used classical super resolution imaging technologies such as scanning electron microscopy, and two novel super-resolution optical microscopy modalities: dSTORM (direct stochastic optical reconstruction microscopy) and SIM (structured illumination microscopy) to study the in vitro effects of xanthines, sildenafil and oxidized LDL on LSEC fenestrations. One of the xanthines, theobromine, and sildenafil increased both the frequency and diameter of fenestrations in cultured LSEC. While oxidized LDL caused major disruptions in LSEC fenestration morphology. Finally, to address the second challenge, namely the rapid loss of fenestrations in LSEC, a cryopreservation method for freshly isolated LSEC was developed such that they can be used at researchers’ convenience, rather than directly after isolation from liv

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model