Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

Viewing early life without labels: optical approaches for imaging the early embryo†.

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: (1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, (2) highlight the recent application of these technologies in the field of reproductive biology, and (3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

Machine learning in time-lapse imaging to differentiate embryos from young vs old mice†.

Time-lapse microscopy for embryos is a non-invasive technology used to characterize early embryo development. This study employs time-lapse microscopy and machine learning to elucidate changes in embryonic growth kinetics with maternal aging. We analyzed morphokinetic parameters of embryos from young and aged C57BL6/NJ mice via continuous imaging. Our findings show that aged embryos accelerated through cleavage stages (from 5-cells) to morula compared to younger counterparts, with no significant differences observed in later stages of blastulation. Unsupervised machine learning identified two distinct clusters comprising of embryos from aged or young donors. Moreover, in supervised learning, the extreme gradient boosting algorithm successfully predicted the age-related phenotype with 0.78 accuracy, 0.81 precision, and 0.83 recall following hyperparameter tuning. These results highlight two main scientific insights: maternal aging affects embryonic development pace, and artificial intelligence can differentiate between embryos from aged and young maternal mice by a non-invasive approach. Thus, machine learning can be used to identify morphokinetics phenotypes for further studies. This study has potential for future applications in selecting human embryos for embryo transfer, without or in complement with preimplantation genetic testing.

Label-free, high-throughput holographic imaging to evaluate mammalian gametes and embryos†.

Assisted reproduction is one of the significant tools to treat human infertility. Morphological assessment is the primary method to determine sperm and embryo viability during in vitro fertilization cycles. It has the advantage of being a quick, convenient, and inexpensive means of assessment. However, visual observation is of limited predictive value for early embryo morphology. It has led many to search for other imaging tools to assess the reproductive potential of a given embryo. The limitations of visual assessment apply to both humans and animals. One recent innovation in assisted reproduction technology imaging is interferometric phase microscopy, also known as holographic microscopy. Interferometric phase microscopy/quantitative phase imaging is the next likely progression of analytical microscopes for the assisted reproduction laboratory. The interferometric phase microscopy system analyzes waves produced by the light as it passes through the specimen observed. The microscope collects the light waves produced and uses the algorithm to create a hologram of the specimen. Recently, interferometric phase microscopy has been combined with quantitative phase imaging, which joins phase contrast microscopy with holographic microscopy. These microscopes collect light waves produced and use the algorithm to create a hologram of the specimen. Unlike other systems, interferometric phase microscopy can provide a quantitative digital image, and it can make 2D and 3D images of the samples. This review summarizes some newer and more promising quantitative phase imaging microscopy systems for evaluating gametes and embryos. Studies clearly show that quantitative phase imaging is superior to bright field microscopy-based evaluation methods when evaluating sperm and oocytes prior to IVF and embryos prior to transfer. However, further assessment of these systems for efficacy, reproducibility, cost-effectiveness, and embryo/gamete safety must take place before they are widely adopted.

High resolution dynamic ultrasound atlas of embryonic and fetal development of the common marmoset.

PURPOSE: The common marmoset (Callithrix jacchus) provides an ideal model to study early development of primates, and an in vivo platform to validate conclusions from in vitro studies of human embryos and embryo models. Currently, however, no established staging atlas of marmoset embryonic development exists. Using high-resolution, longitudinal ultrasound scans on live pregnant marmosets, we present the first dynamic in vivo imaging of entire primate gestation beginning with attachment until the last day before birth. METHODS: Our study unveils the first dynamic images of an in vivo attached mammalian embryo developing in utero, and the intricacies of the delayed development period unique to the common marmoset amongst primates, revealing a window for somatic interventions. RESULTS: Established obstetric and embryologic measurements for each scan were used comparatively with the standardized Carnegie staging of human development to highlight similarities and differences. Our study also allows for tracking the development of major organs. We focus on the ontogeny of the primate heart and brain. Finally, input ultrasound images were used to train deep neural networks to accurately determine the gestational age. All our ultrasounds and staging data recording are posted online so that the atlas can be used as a community resource toward monitoring and managing marmoset breeding colonies. CONCLUSION: The temporal and spatial resolution of ultrasound achieved in this study demonstrates the promise of noninvasive imaging in the marmoset for the in vivo study of primate-specific aspects of embryonic and fetal development.

The visible skeleton 2.0: phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT.

For decades, clearing and staining with Alcian Blue and Alizarin Red has been the gold standard to image vertebrate skeletal development. Here, we present an alternate approach to visualise bone and cartilage based on X-ray microCT imaging, which allows the collection of genuine 3D data of the entire developing skeleton at micron resolution. Our novel protocol is based on ethanol fixation and staining with Ruthenium Red, and efficiently contrasts cartilage matrix, as demonstrated in whole E16.5 mouse foetuses and limbs of E14 chicken embryos. Bone mineral is well preserved during staining, thus the entire embryonic skeleton can be imaged at high contrast. Differences in X-ray attenuation of ruthenium and calcium enable the spectral separation of cartilage matrix and bone by dual energy microCT (microDECT). Clearing of specimens is not required. The protocol is simple and reproducible. We demonstrate that cartilage contrast in E16.5 mouse foetuses is adequate for fast visual phenotyping. Morphometric skeletal parameters are easily extracted. We consider the presented workflow to be a powerful and versatile extension to the toolkit currently available for qualitative and quantitative phenotyping of vertebrate skeletal development.

Longitudinal surface measurements of human blastocysts show that the dynamics of blastocoel expansion are associated with fertilization method and ongoing pregnancy.

BACKGROUND: Despite all research efforts during this era of novel time-lapse morphokinetic parameters, a morphological grading system is still routinely being used for embryo selection at the blastocyst stage. The blastocyst expansion grade, as evaluated during morphological assessment, is associated with clinical pregnancy. However, this assessment is performed without taking the dynamics of blastocoel expansion into account. Here, we studied the dynamics of blastocoel expansion by comparing longitudinal blastocoel surface measurements using time-lapse embryo culture. Our aim was to first assess if this is impacted by fertilization method and second, to study if an association exists between these measurement and ongoing pregnancy. METHODS: This was a retrospective cohort study including 225 couples undergoing 225 cycles of in vitro fertilization (IVF) treatment with time-lapse embryo culture. The fertilization method was either conventional IVF, intracytoplasmic sperm injection (ICSI) with ejaculated sperm or ICSI with sperm derived from testicular sperm extraction (TESE-ICSI). This resulted in 289 IVF embryos, 218 ICSI embryos and 259 TESE-ICSI embryos that reached at least the full blastocyst stage. Blastocoel surface measurements were performed on time-lapse images every hour, starting from full blastocyst formation (tB). Linear mixed model analysis was performed to study the association between blastocoel expansion, the calculated expansion rate (µm2/hour) and both fertilization method and ongoing pregnancy. RESULTS: The blastocoel of both ICSI embryos and TESE-ICSI embryos was significantly smaller than the blastocoel of IVF embryos (beta -1121.6 µm2; 95% CI: -1606.1 to -637.1, beta -646.8 µm2; 95% CI: -1118.7 to 174.8, respectively). Still, the blastocoel of transferred embryos resulting in an ongoing pregnancy was significantly larger (beta 795.4 µm2; 95% CI: 15.4 to 1575.4) and expanded significantly faster (beta 100.9 µm2/hour; 95% CI: 5.7 to 196.2) than the blastocoel of transferred embryos that did not, regardless of the fertilization method. CONCLUSION: Longitudinal blastocyst surface measurements and expansion rates are promising non-invasive quantitative markers that can aid embryo selection for transfer and cryopreservation. TRIAL REGISTRATION: Our study is a retrospective observational study, therefore trial registration is not applicable.

High sensitivity X-ray phase contrast imaging by laboratory grating-based interferometry at high Talbot order geometry.

X-ray phase contrast imaging is a powerful analysis technique for materials science and biomedicine. Here, we report on laboratory grating-based X-ray interferometry employing a microfocus X-ray source and a high Talbot order (35th) asymmetric geometry to achieve high angular sensitivity and high spatial resolution X-ray phase contrast imaging in a compact system (total length <1 m). The detection of very small refractive angles (∼50 nrad) at an interferometer design energy of 19 keV was enabled by combining small period X-ray gratings (1.0, 1.5 and 3.0 µm) and a single-photon counting X-ray detector (75 µm pixel size). The performance of the X-ray interferometer was fully characterized in terms of angular sensitivity and spatial resolution. Finally, the potential of laboratory X-ray phase contrast for biomedical imaging is demonstrated by obtaining high resolution X-ray phase tomographies of a mouse embryo embedded in solid paraffin and a formalin-fixed full-thickness sample of human left ventricle in water with a spatial resolution of 21.5 µm.

Solution NMR in human embryo culture media as an option for assessment of embryo implantation potential.

NMR offers the potential to holistically screen hundreds of metabolites and has already proved to be a powerful technique able to provide a global picture of metabolic changes in a wide range of biological systems underlying complex and multifactorial matrixes. This review covers the literature until May 2020 centered on the early prediction of the viability of in vitro developed embryos using several analytical techniques, including NMR. Nowadays, the predominant non-invasive technique for selecting viable embryos is based on morphology, where variables associated with the rate of cleavage and blastocyst formation are evaluated by the embryologist following standardized criteria that are somewhat subjective. This morphological approach is therefore inadequate for the prediction of embryo quality, and several studies have focused on developing new non-invasive methods using molecular approaches based particularly on metabolomics. This review outlines the potential of NMR as one of these non-invasive in vitro methods based on the analysis of spent embryo culture media.

Evaluation of artificial intelligence using time-lapse images of IVF embryos to predict live birth.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the prediction of live births based on embryo images? DESIGN: The AI system was created by using the Attention Branch Network associated with deep learning to predict the probability of live birth from 141,444 images recorded by time-lapse imaging of 470 transferred embryos, of which 91 resulted in live birth and 379 resulted in non-live birth that included implantation failure, biochemical pregnancy and clinical miscarriage. The possibility that the calculated confidence scores of each embryo and the focused areas visualized in each embryo image can help predict subsequent live birth was examined. RESULTS: The AI system for the first time successfully visualized embryo features in focused areas that had potential to distinguish between live and non-live births. No visual feature of embryos were visualized that were associated with live or non-live births, although there were many images in which high-focused areas existed around the zona pellucida. When a cut-off level for the confidence score was set at 0.341, the live birth rate was significantly greater for embryos with a score higher than the cut-off level than for those with a score lower than the cut-off level (P < 0.001). In addition, the live birth rate of embryos with good morphological quality and confidence scores higher than 0.341 was 41.1%. CONCLUSIONS: The authors have created an AI system with a confidence score that is useful for non-invasive selection of embryos that could result in live birth. Further study is necessary to improve selection accuracy.

Three-dimensional live imaging of bovine embryos by optical coherence tomography.

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.

Day 3 time lapse selection is beneficial for the patients with no good-quality embryos.

We aimed to evaluate whether or not time lapse selection was beneficial for the cleavage-stage embryo transfers. The study included 838 infertile women with good ovarian reserve (obtaining more than 8 oocytes) from January 2018 to August 2019. Based on the transferred embryos with different grades (grade I, II and III), the patients were divided into day 3 selection with conventional morphology (CM) and day 3 selection with time lapse (TL) groups. For the grade I and II embryos, we observed that CM and TL had similar implantation, clinical pregnancy and ongoing pregnancy (p > .05) rates. For the grade III embryos, we observed that CM group showed slightly lower implantation (36.74 versus 41.03%, p = .261) and clinical pregnancy (56.82 versus 64.10%, p = .182) rates than TL group. CM group showed significantly lower ongoing pregnancy (47.35 versus 59.83%, p = .025) rate than TL group. And we observed that CM group had significantly higher blastulation (38.93 versus 26.61%, p = .019) rate than TL group. We concluded that TL selection was beneficial to the patients with no good-quality embryos in the first cleavage-stage embryo transfers.

Towards the selection of embryos with the greatest implantation potential.

Choosing the most suitable embryo remains challenging as the standard approach to select top-quality embryos for transfer rely on static morphological assessment. It is completed after fertilisation, on days 3 and 5 post oocyte retrieval and evaluates the size and number of blastomeres, presence of nucleation and percentage of fragmentation for cleavage stage embryos. Because of the limited number of observations during the morphological assessment, morphokinetic development of embryos has been implemented. It shows a broader image of embryo behaviour with precise evaluation of the timing of events. Yet, studies are inconsistent and debatable in predicting the parameters to identify chromosomal abnormalities. Pre-implantation genetic testing detects dysmorphic embryos and correlate their developmental potential to the assessed morphology. However, the clinical utility of PGT-aneuploidy remains controversial. The future relies on newly described scoring systems such as artificial intelligence and non-invasive PGT, yet their application and actual success rate still lacks supportive evidence.

A bioimage informatics platform for high-throughput embryo phenotyping.

High-throughput phenotyping is a cornerstone of numerous functional genomics projects. In recent years, imaging screens have become increasingly important in understanding gene-phenotype relationships in studies of cells, tissues and whole organisms. Three-dimensional (3D) imaging has risen to prominence in the field of developmental biology for its ability to capture whole embryo morphology and gene expression, as exemplified by the International Mouse Phenotyping Consortium (IMPC). Large volumes of image data are being acquired by multiple institutions around the world that encompass a range of modalities, proprietary software and metadata. To facilitate robust downstream analysis, images and metadata must be standardized to account for these differences. As an open scientific enterprise, making the data readily accessible is essential so that members of biomedical and clinical research communities can study the images for themselves without the need for highly specialized software or technical expertise. In this article, we present a platform of software tools that facilitate the upload, analysis and dissemination of 3D images for the IMPC. Over 750 reconstructions from 80 embryonic lethal and subviable lines have been captured to date, all of which are openly accessible at mousephenotype.org. Although designed for the IMPC, all software is available under an open-source licence for others to use and develop further. Ongoing developments aim to increase throughput and improve the analysis and dissemination of image data. Furthermore, we aim to ensure that images are searchable so that users can locate relevant images associated with genes, phenotypes or human diseases of interest.

Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia.

PURPOSE: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. METHODS: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. RESULTS: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. CONCLUSIONS: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Head-Mounted Display-Based Microscopic Imaging System with Customizable Field Size and Viewpoint.

In recent years, the use of microinjections has increased in life science and biotechnology fields; specific examples include artificial insemination and gene manipulation. Microinjections are mainly performed based on visual information; thus, the operator needs high-level skill because of the narrowness of the visual field. Additionally, microinjections are performed as the operator views a microscopic image on a display; the position of the display requires the operator to maintain an awkward posture throughout the procedure. In this study, we developed a microscopic image display apparatus for microinjections based on a view-expansive microscope. The prototype of the view-expansive microscope has problems related to the variations in brightness and focal blur that accompany changes in the optical path length and amount of reflected light. Therefore, we propose the use of a variable-focus device to expand the visual field and thus circumvent the above-mentioned problems. We evaluated the observable area of the system using this variable-focus device. We confirmed that the observable area is 261.4 and 13.9 times larger than that of a normal microscope and conventional view-expansive microscopic system, respectively. Finally, observations of mouse embryos were carried out by using the developed system. We confirmed that the microscopic images can be displayed on a head-mounted display in real time with the desired point and field sizes.

QUEST MRI assessment of fetal brain oxidative stress in utero.

PURPOSE: To achieve sufficient precision of R1 (=1/T1) maps of the fetal brain in utero to perform QUEnch-assiSTed (QUEST) MRI in which a significant anti-oxidant-induced reduction in R1 indicates oxidative stress. METHODS: C57BL/6 mouse fetuses in utero were gently and non-surgically isolated and secured using a homemade 3D printed clip. Using a commercial receive-only surface coil, brain maps of R1, an index sensitive to excessive and continuous free radical production, were collected using either a conventional Cartesian or a non-Cartesian (periodically rotated overlapping parallel lines with enhanced reconstruction) progressive saturation sequence. Data were normalized to the shortest TR time to remove bias. To assess oxidative stress, brain R1 maps were acquired on the lipopolysaccharide (LPS) model of preterm birth ±â€¯rosiglitazone (ROSI, which has anti-oxidant properties); phosphate buffered saline (PBS) controls ±â€¯ROSI were similarly studied. RESULTS: Sufficient quality R1 maps were generated by a combination of the 3D printed clip, surface coil detection, non-Cartesian sequence, and normalization scheme ensuring minimal fetal movement, good detection sensitivity, reduced motion artifacts, and minimal baseline variations, respectively. In the LPS group, the combined caudate-putamen and thalamus region R1 was reduced (p < 0.05) with ROSI treatment consistent with brain oxidative stress; no evidence for oxidative stress was found in the pons region. In the PBS control group, brain R1's did not change with ROSI treatment. CONCLUSION: The sensitivity and reproducibility of the combined approaches described herein enabled first-time demonstration of regional oxidative stress measurements of the fetal brain in utero using QUEST MRI.

Screening of angiogenesis inhibitors using a 3D vascular microfluidic chip to achieve contraception.

Angiogenesis plays a vital role in the process of embryo implantation, as it improves endometrial receptivity and guides embryo implantation, thus creating a favorable environment for subsequent development of the embryo. Hence, a theory of achieving contraception by inhibiting angiogenesis was put forward. Here, we screened the drugs inhibiting angiogenesis using cell scratch wound assay and a 3D biomimetic vascular microfluidic chip, then observed the effect of them on contraception by injecting these drugs into fertilized mice and observing if the embryos were implanted. We preliminarily verify the feasibility of contraception by inhibiting angiogenesis and gives a new direction in the development of contraceptive pills.

Insights from imaging the implanting embryo and the uterine environment in three dimensions.

Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo.