Substrate-Assisted Atomic Dispersion of Cobalt for Alkaline Water Electrolysis.

Atomically dispersed single-atom catalysts have recently attracted broad research interest due to their high atom efficiency and unique catalytic performance. In this study, atomic dispersion of cobalt is achieved using a chemical bath deposition method on a highly stable alkali titanate film (Ti/KTiO). These films were characterized using a variety of techniques, with atomic dispersion confirmed via grazing incidence X-ray absorption spectroscopy and ab initio modeling of single-atom systems. This modeling indicated that the alkali ion incorporated into the film facilitates atomic dispersion. Experimentally, the Ti/KTiO-supported Co(OH)2 catalysts exhibited remarkable electrochemical performance, with an overpotential of 163 mV to achieve a current density of 10 mA cm-2 with a catalyst loading of ∼0.1 mg cm-2 and high stability. These results show the potential of Ti/KTiO/Co(OH)2 catalysts for atomically efficient hydrogen production.

Quantifying DNA damage following light sheet and confocal imaging of the mammalian embryo.

Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts. In this study, we employed DNA damage as a sensitive indicator of photodamage. We use light sheet microscopy with excitation at a wavelength of 405 nm for imaging embryo autofluorescence and compare its performance to laser scanning confocal microscopy. At an equivalent signal-to-noise ratio for images acquired with both modalities, light sheet microscopy reduced image acquisition time by ten-fold, and did not induce DNA damage when compared to non-imaged embryos. In contrast, imaging with confocal microscopy led to significantly higher levels of DNA damage within embryos and had a higher photobleaching rate. Light sheet imaging is also capable of inducing DNA damage within the embryo but requires multiple cycles of volumetric imaging. Collectively, this study confirms that light sheet microscopy is faster and safer than confocal microscopy for imaging live embryos, indicating its potential as a label-free diagnostic for embryo quality.

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.

UVA Hyperspectral Light-Sheet Microscopy for Volumetric Metabolic Imaging: Application to Preimplantation Embryo Development.

Cellular metabolism is a key regulator of energetics, cell growth, regeneration, and homeostasis. Spatially mapping the heterogeneity of cellular metabolic activity is of great importance for unraveling the overall cell and tissue health. In this regard, imaging the endogenous metabolic cofactors, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), with subcellular resolution and in a noninvasive manner would be useful to determine tissue and cell viability in a clinical environment, but practical use is limited by current imaging techniques. In this paper, we demonstrate the use of phasor-based hyperspectral light-sheet (HS-LS) microscopy using a single UVA excitation wavelength as a route to mapping metabolism in three dimensions. We show that excitation solely at a UVA wavelength of 375 nm can simultaneously excite NAD(P)H and FAD autofluorescence, while their relative contributions can be readily quantified using a hardware-based spectral phasor analysis. We demonstrate the potential of our HS-LS system by capturing dynamic changes in metabolic activity during preimplantation embryo development. To validate our approach, we delineate metabolic changes during preimplantation embryo development from volumetric maps of metabolic activity. Importantly, our approach overcomes the need for multiple excitation wavelengths, two-photon imaging, or significant postprocessing of data, paving the way toward clinical translation, such as in situ, noninvasive assessment of embryo viability.

Mechanical Exfoliation of Expanded Graphite to Graphene-Based Materials and Modification with Palladium Nanoparticles for Hydrogen Storage.

Hydrogen is a promising green fuel carrier that can replace fossil fuels; however, its storage is still a challenge. Carbon-based materials with metal catalysts have recently been the focus of research for solid-state hydrogen storage due to their efficacy and low cost. Here, we report on the exfoliation of expanded graphite (EG) through high shear mixing and probe tip sonication methods to form graphene-based nanomaterial ShEG and sEG, respectively. The exfoliation processes were optimized based on electrochemical capacitance measurements. The exfoliated EG was further functionalized with palladium nanoparticles (Pd-NP) for solid-state hydrogen storage. The prepared graphene-based nanomaterials (ShEG and sEG) and the nanocomposites (Pd-ShEG and Pd-sEG) were characterized with various traditional techniques (e.g., SEM, TEM, EDX, XPS, Raman, XRD) and the advanced high-resolution pair distribution function (HRPDF) analysis. Electrochemical hydrogen uptake and release (QH) were measured, showing that the sEG decorated with Pd-NP (Pd-sEG, 31.05 mC cm-2) and ShEG with Pd-NP (Pd-ShEG, 24.54 mC cm-2) had a notable improvement over Pd-NP (9.87 mC cm-2) and the composite of Pd-EG (14.7 mC cm-2). QH showed a strong linear relationship with an effective surface area to volume ratio, indicating nanoparticle size as a determining factor for hydrogen uptake and release. This work is a promising step toward the design of the high-performance solid-state hydrogen storage devices through mechanical exfoliation of the substrate EG to control nanoparticle size and dispersion.

Investigation of refractive index dynamics during in vitro embryo development using off-axis digital holographic microscopy.

Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. The phase of the recorded hologram was numerically retrieved, from which the refractive index of the embryo was calculated. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.

Experimentally unsupervised deconvolution for light-sheet microscopy with propagation-invariant beams.

Deconvolution is a challenging inverse problem, particularly in techniques that employ complex engineered point-spread functions, such as microscopy with propagation-invariant beams. Here, we present a deep-learning method for deconvolution that, in lieu of end-to-end training with ground truths, is trained using known physics of the imaging system. Specifically, we train a generative adversarial network with images generated with the known point-spread function of the system, and combine this with unpaired experimental data that preserve perceptual content. Our method rapidly and robustly deconvolves and super-resolves microscopy images, demonstrating a two-fold improvement in image contrast to conventional deconvolution methods. In contrast to common end-to-end networks that often require 1000-10,000s paired images, our method is experimentally unsupervised and can be trained solely on a few hundred regions of interest. We demonstrate its performance on light-sheet microscopy with propagation-invariant Airy beams in oocytes, preimplantation embryos and excised brain tissue, as well as illustrate its utility for Bessel-beam LSM. This method aims to democratise learned methods for deconvolution, as it does not require data acquisition outwith the conventional imaging protocol.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device.

PURPOSE: Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. METHODS: The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). RESULTS: Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. CONCLUSION: The Pod and Garage system minimized the volume of cryoprotectant at vitrification-by ~ 1000-fold-improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

The effect of discrete wavelengths of visible light on the developing murine embryo.

PURPOSE: A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. METHODS: Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. RESULTS: Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. CONCLUSION: Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo.

STUDY QUESTION: Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo? SUMMARY ANSWER: Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos. WHAT IS KNOWN ALREADY: Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos. STUDY DESIGN, SIZE, DURATION: The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations. MAIN RESULTS AND THE ROLE OF CHANCE: An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis. STUDY FUNDING/COMPETING INTEREST(S): K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest.

Does artificial intelligence have a role in the IVF clinic?

The success of IVF has remained stagnant for a decade. The focus of a great deal of research is to improve on the current ~30% success rate of IVF. Artificial intelligence (AI), or machines that mimic human intelligence, has been gaining traction for its potential to improve outcomes in medicine, such as cancer diagnosis from medical images. In this commentary, we discuss whether AI has the potential to improve fertility outcomes in the IVF clinic. Based on existing research, we examine the potential of adopting AI within multiple facets of an IVF cycle, including egg/sperm and embryo selection, as well as formulation of an IVF treatment regimen. We discuss both the potential benefits and concerns of the patient and clinician in adopting AI in the clinic. We outline hurdles that need to be overcome prior to implementation. We conclude that AI has an important future in improving IVF success.

Optical imaging of cleavage stage bovine embryos using hyperspectral and confocal approaches reveals metabolic differences between on-time and fast-developing embryos.

The assessment of embryo quality aims to enhance subsequent pregnancy and live birth outcomes. Metabolic analysis of embryos has immense potential in this regard. As a step towards this goal, here we assess the metabolism of bovine embryos using label-free optical imaging. We compared embryos defined as either on-time or fast-developing, as fast dividing embryos are more likely to develop to the blastocyst stage. Specifically, bovine embryos at 48 (Day 2) and 96 (Day 4) hours post fertilization were fixed and separated based on morphological assessment: on-time (Day 2: 2 cell; Day 4: 5-7 cell) or fast-developing (Day 2: 3-7 cell; Day 4: 8-16 cell). Embryos with different developmental rates on Day 2 and Day 4 were correlated with metabolic activity and DNA damage. Confocal microscopy was used to assess metabolic activity by quantification of cellular autofluorescence specific for the endogenous fluorophores NAD(P)H and FAD with a subsequent calculation of the optical redox ratio. Separately, hyperspectral microscopy was employed to assess a broader range of endogenous fluorophores. DNA damage was determined using γH2AX immunohistochemistry. Hyperspectral imaging showed significantly lower abundance of endogenous fluorophores in fast-developing compared to on-time embryos on Day 2, indicating a lower metabolic activity. On Day 4 of development there was no difference in the abundance of FAD between on-time and fast-developing embryos. There was, however, significantly higher levels of NAD(P)H in fast-developing embryos leading to a significantly lower optical redox ratio when compared to on-time embryos. Collectively, these results demonstrate that fast-developing embryos present a 'quiet' metabolic pattern on Day 2 and Day 4 of development, compared to on-time embryos. There was no difference in the level of DNA damage between on-time and fast-developing embryos on either day of development. To our knowledge, this is the first collective use of confocal and hyperspectral imaging in cleavage-stage bovine embryos in the absence of fluorescent tags.