AutoMitoNetwork: Software for analyzing mitochondrial networks in autofluorescence images to enable label-free cell classification.

High-resolution mitochondria imaging in combination with image analysis tools have significantly advanced our understanding of cellular function in health and disease. However, most image analysis tools for mitochondrial studies have been designed to work with fluorescently labeled images only. Additionally, efforts to integrate features describing mitochondrial networks with machine learning techniques for the differentiation of cell types have been limited. Herein, we present AutoMitoNetwork software for image-based assessment of mitochondrial networks in label-free autofluorescence images using a range of interpretable morphological, intensity, and textural features. To demonstrate its utility, we characterized unstained mitochondrial networks in healthy retinal cells and in retinal cells exposed to two types of treatments: rotenone, which directly inhibited mitochondrial respiration and ATP production, and iodoacetic acid, which had a milder impact on mitochondrial networks via the inhibition of anaerobic glycolysis. For both cases, our multi-dimensional feature analysis combined with a support vector machine classifier distinguished between healthy cells and those treated with rotenone or iodoacetic acid. Subtle changes in morphological features were measured including increased fragmentation in the treated retinal cells, pointing to an association with metabolic mechanisms. AutoMitoNetwork opens new options for image-based machine learning in label-free imaging, diagnostics, and mitochondrial disease drug development.

Ageing and Polypharmacy in Mesenchymal Stromal Cells: Metabolic Impact Assessed by Hyperspectral Imaging of Autofluorescence.

The impact of age on mesenchymal stromal cell (MSC) characteristics has been well researched. However, increased age is concomitant with increased prevalence of polypharmacy. This adjustable factor may have further implications for the functionality of MSCs and the effectiveness of autologous MSC procedures. We applied hyperspectral microscopy of cell autofluorescence-a non-invasive imaging technique used to characterise cytometabolic heterogeneity-to identify changes in the autofluorescence signals of MSCs from (1) young mice, (2) old mice, (3) young mice randomised to receive polypharmacy (9-10 weeks of oral therapeutic doses of simvastatin, metoprolol, oxycodone, oxybutynin and citalopram), and (4) old mice randomised to receive polypharmacy. Principal Component Analysis and Logistic Regression Analysis were used to assess alterations in spectral and associated metabolic characteristics. Modelling demonstrated that cells from young mice receiving polypharmacy had less NAD(P)H and increased porphyrin relative to cells from old control mice, allowing for effective separation of the two groups (AUC of ROC curve > 0.94). Similarly, cells from old polypharmacy mice were accurately separated from those from young controls due to lower levels of NAD(P)H (p < 0.001) and higher porphyrin (p < 0.001), allowing for an extremely accurate logistic regression (AUC of ROC curve = 0.99). This polypharmacy regimen may have a more profound impact on MSCs than ageing, and can simultaneously reduce optical redox ratio (ORR) and increase porphyrin levels. This has implications for the use of autologous MSCs for older patients with chronic disease.

Diagnostic and prognostic biomarkers for tubulointerstitial fibrosis.

Renal fibrosis is the final common pathophysiological pathway in chronic kidney disease (CKD) regardless of the underlying cause of kidney injury. Tubulointerstitial fibrosis (TIF) is considered to be the key pathological predictor of CKD progression. Currently, the gold-standard tool to identify TIF is kidney biopsy, an invasive method that carries risks. Non-invasive diagnostics rely on an estimation of glomerular filtration rate and albuminuria to assess kidney function, but these fail to diagnose early CKD accurately or to predict progressive decline in kidney function. In this review, we summarize the current and emerging molecular biomarkers that have been studied in various clinical settings and in animal models of kidney disease and that are correlated with the degree of TIF. We examine the potential of these biomarkers to diagnose TIF non-invasively and to predict disease progression. We also examine the potential of new technologies and non-invasive diagnostic approaches in assessing TIF. Limitations of current and potential biomarkers are discussed and knowledge gaps identified.

Terahertz imaging: a diagnostic technology for prevention of grass seed infestation.

One of the most significant problems the Australian sheep and lamb industry faces today is grass seed infestation (GSI), which occurs when seeds accumulate in the sheep's fleece and penetrate the skin, causing infection. Meat & Livestock Australia estimates that the yearly losses caused due to GSI are around AUD$47.5 M (in Australia alone). Here, we demonstrate that terahertz spectroscopy and imaging can be utilized for early detection of GSI. This is possible because terahertz waves can penetrate through sheep wool and have the appropriate wavelength for identifying the seed. Moreover, terahertz waves have non-invasive and non-ionizing properties and are ideal for non-contact and standoff detection. This work demonstrates that terahertz waves can be utilized for the early detection of seeds in the animal fleece or on the pelt as a precursor tool for the prevention of GSI.

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Multispectral autofluorescence characteristics of reproductive aging in old and young mouse oocytes.

Increasing age has a major detrimental impact on female fertility, which, with an ageing population, has major sociological implications. This impact is primarily mediated through deteriorating quality of the oocyte. Deteriorating oocyte quality with biological age is the greatest rate-limiting factor to female fertility. Here we have used label-free, non-invasive multi-spectral imaging to identify unique autofluorescence profiles of oocytes from young and aged animals. Discriminant analysis demonstrated that young oocytes have a distinct autofluorescent profile which accurately distinguishes them from aged oocytes. We recently showed that treatment with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) restored oocyte quality and fertility in aged animals, and when our analysis was applied to oocytes from aged animals treated with NMN, 85% of these oocytes were classified as having the autofluorescent signature of young animals. Spectral unmixing using the Robust Dependent Component Analysis (RoDECA) algorithm demonstrated that NMN treatment altered the metabolic profile of oocytes, increasing free NAD(P)H, protein bound NAD(P)H, redox ratio and the ratio of bound to free NAD(P)H. The frequency of oocytes with simultaneously high NAD(P)H and flavin content was also significantly increased in mice treated with NMN. Young and Aged + NMN oocytes had a smoother spectral distribution, with the distribution of NAD(P)H in young oocytes specifically differing from that of aged oocytes. Identifying the multispectral profile of oocyte autofluorescence during aging could have utility as a non-invasive and sensitive measure of oocyte quality.

Exfoliated Kidney Cells from Urine for Early Diagnosis and Prognostication of CKD: The Way of the Future?

Chronic kidney disease (CKD) is a global health issue, affecting more than 10% of the worldwide population. The current approach for formal diagnosis and prognostication of CKD typically relies on non-invasive serum and urine biomarkers such as serum creatinine and albuminuria. However, histological evidence of tubulointerstitial fibrosis is the 'gold standard' marker of the likelihood of disease progression. The development of novel biomedical technologies to evaluate exfoliated kidney cells from urine for non-invasive diagnosis and prognostication of CKD presents opportunities to avoid kidney biopsy for the purpose of prognostication. Efforts to apply these technologies more widely in clinical practice are encouraged, given their potential as a cost-effective approach, and no risk of post-biopsy complications such as bleeding, pain and hospitalization. The identification of biomarkers in exfoliated kidney cells from urine via western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence techniques, measurement of cell and protein-specific messenger ribonucleic acid (mRNA)/micro-RNA and other techniques have been reported. Recent innovations such as multispectral autofluorescence imaging and single-cell RNA sequencing (scRNA-seq) have brought additional dimensions to the clinical application of exfoliated kidney cells from urine. In this review, we discuss the current evidence regarding the utility of exfoliated proximal tubule cells (PTC), podocytes, mesangial cells, extracellular vesicles and stem/progenitor cells as surrogate markers for the early diagnosis and prognostication of CKD. Future directions for development within this research area are also identified.

Autofluorescent imprint of chronic constriction nerve injury identified by deep learning.

Our understanding of chronic pain and the underlying molecular mechanisms remains limited due to a lack of tools to identify the complex phenomena responsible for exaggerated pain behaviours. Furthermore, currently there is no objective measure of pain with current assessment relying on patient self-scoring. Here, we applied a fully biologically unsupervised technique of hyperspectral autofluorescence imaging to identify a complex signature associated with chronic constriction nerve injury known to cause allodynia. The analysis was carried out using deep learning/artificial intelligence methods. The central element was a deep learning autoencoder we developed to condense the hyperspectral channel images into a four- colour image, such that spinal cord tissue based on nerve injury status could be differentiated from control tissue. This study provides the first validation of hyperspectral imaging as a tool to differentiate tissues from nerve injured vs non-injured mice. The auto-fluorescent signals associated with nerve injury were not diffuse throughout the tissue but formed specific microscopic size regions. Furthermore, we identified a unique fluorescent signal that could differentiate spinal cord tissue isolated from nerve injured male and female animals. The identification of a specific global autofluorescence fingerprint associated with nerve injury and resultant neuropathic pain opens up the exciting opportunity to develop a diagnostic tool for identifying novel contributors to pain in individuals.

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.

Radiodynamic Therapy Using TAT Peptide-Targeted Verteporfin-Encapsulated PLGA Nanoparticles.

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.

In vivo intrathecal IL-1ß quantification in rats: Monitoring the molecular signals of neuropathic pain.

BACKGROUND: Neuropathic pain, or pain after nerve injury, is a disorder with a significant reliance on the signalling of cytokines such as IL-1ß. However, quantifying the cytokine release repeatedly over time in vivo is technically challenging. AIM: To evaluate if changes in IL-1ß are correlated with the presentation of mechanical allodynia over time, by repeatedly quantifying intrathecal IL-1ß concentrations following chronic constriction injury of the sciatic nerve in rats. Also, to establish any possible correlation between biochemical spinal marker expression and the in vivo quantification of IL-1ß. Finally, to assess the expression of the mature IL-1ß in lumbar spinal cord samples. METHOD: The Chronic Constriction Injury model (CCI) was used to initiate nerve injury in male Sprague Dawley rats and the generation of behavioural mechanical allodynia was quantified. Using an indwelling intrathecal catheter, a stainless steel (SS) wire biosensing device was repeatedly introduced to quantify intrathecal IL-1ß concentrations at three timepoints of 0, 7, and 14 days post CCI. Fixed spinal cord samples (L4-L5), collected on day 14, were imaged for the expression of glial fibrillary acidic protein (GFAP, astrocytes) and ionized calcium binding adaptor molecule 1 (IBA1, microglia). Snap frozen spinal cord tissues (L4-L5) were also processed for western blot analysis. RESULTS: Using the novel SS based biosensing device we established that CCI caused a significant increase in intrathecal IL-1ß concentrations from day 0 to day 7 (p = 0.001) and to day 14 (p < 0.0001), while the sham group did not show any significant increase. We also further showed that the degree of mechanical allodynia correlated positively with the increase in the intrathecal concentration of IL-1ß in the active CCI animals (p = 0.0007). While there was a significant increase in the ipsilateral GFAP expression in injured animals compared to sham animals (p = 0.03), we did not find any significant correlation between in vivo IL-1ß concentration on days 7 and 14 and the area of dorsal horn GFAP or IBA1 positive structures on day 14. The result of western blot analysis of whole lumbar spinal cord revealed that there was no significant change (p = 0.7579) in IL-1ß expression on day 14 in the CCI group compared to the sham group. CONCLUSION: For the first time we have established that the SS based immunosensing platform technology can repeatedly sample the intrathecal space for bioactive peptides, such as IL-1ß. Using this novel approach, we have been able to establish the correlation of the intrathecal concentration of IL-1ß with the extent of mechanical allodynia, providing a molecular biomarker of the degree of the exaggerated pain state.

Acute stress induces the rapid and transient induction of caspase-1, gasdermin D and release of constitutive IL-1ß protein in dorsal hippocampus.

The proinflammatory cytokine interleukin (IL)-1ß plays a pivotal role in the behavioral manifestations (i.e., sickness) of the stress response. Indeed, exposure to acute and chronic stressors induces the expression of IL-1ß in stress-sensitive brain regions. Thus, it is typically presumed that exposure to stressors induces the extra-cellular release of IL-1ß in the brain parenchyma. However, this stress-evoked neuroimmune phenomenon has not been directly demonstrated nor has the cellular process of IL-1ß release into the extracellular milieu been characterized in brain. This cellular process involves a form of inflammatory cell death, termed pyroptosis, which involves: 1) activation of caspase-1, 2) caspase-1 maturation of IL-1ß, 3) caspase-1 cleavage of gasdermin D (GSDMD), and 4) GSDMD-induced permeability of the cell membrane through which IL-1ß is released into the extracellular space. Thus, the present study examined whether stress induces the extra-cellular release of IL-1ß and engages the above cellular process in mediating IL-1ß release in the brain. Male Sprague-Dawley rats were exposed to inescapable tailshock (IS). IL-1ß extra-cellular release, caspase-1 activity and cleavage of GSDMD were measured in dorsal hippocampus. We found that exposure to IS induced a transient increase in the release of IL-1ß into the extracellular space immediately after termination of the stressor. IS also induced a transient increase in caspase-1 activity prior to IL-1ß release, while activation of GSDMD was observed immediately after termination of the stressor. IS also increased mRNA and protein expression of the ESCRTIII protein CHMP4B, which is involved in cellular repair. The present results suggest that exposure to an acute stressor induces the hallmarks of pyroptosis in brain, which might serve as a key cellular process involved in the release of IL-1ß into the extracellular milieu of the brain parenchyma.

X-ray induced photodynamic therapy (PDT) with a mitochondria-targeted liposome delivery system.

In this study, we constructed multifunctional liposomes with preferentially mitochondria-targeted feature and gold nanoparticles-assisted synergistic photodynamic therapy. We systemically investigated the in vitro X-ray triggered PDT effect of these liposomes on HCT 116 cells including the levels of singlet oxygen, mitochondrial membrane potential, cell apoptosis/necrosis and the expression of apoptosis-related proteins. The results corroborated that synchronous action of PDT and X-ray radiation enhance the generation of cytotoxic reactive oxygen species produced from the engineered liposomes, causing mitochondrial dysfunction and increasing the levels of apoptosis.

Polymer brush based fluorescent immunosensor for direct monitoring of interleukin-1ß in rat blood.

A sandwich immunosensor was successfully developed for monitoring of interleukin-1ß (IL-1ß) in rat whole blood. The substrate stainless steel (SS) was first coated with a polydopamine layer and subsequently grafted with poly(ethylene glycol) methacrylate brushes, onto which a sandwich immunosensor was modified for detection of IL-1ß. The device has been successfully applied for monitoring of IL-1ß with a limit of detection of 4.7 pg mL-1, and a linear detection range of 12.5-200 pg mL-1. Good specificity and selectivity for monitoring of IL-1ß in rat macrophage secretion were achieved. Furthermore, this device was validated by detection of IL-1ß in rat whole blood samples with greater concentrations observed in obese rats compared to control, and strong positive correlation between concentrations of IL-1ß and blood glucose. These results suggest this device is feasible for direct detection of target analytes in biological samples.

A novel platform for in vivo detection of cytokine release within discrete brain regions.

Mounting evidence indicates that cytokines secreted by innate immune cells in the brain play a central role in regulating neural circuits that subserve mood, cognition, and sickness responses. A major impediment to the study of neuroimmune signaling in healthy and disease states is the absence of tools for in vivo detection of cytokine release in the brain. Here we describe the design and application of a cytokine detection device capable of serial monitoring of local cytokine release in discrete brain regions. The immunocapture device consisted of a modified optical fiber labeled with a capture antibody specific for the pro-inflammatory cytokine interleukin-1 beta (IL-1ß). Using a sandwich immunoassay method, in vitro data demonstrate that the sensing interface of the modified optical fiber has a linear detection range of 3.9 pg mL-1-500 pg mL-1 and spatial resolution on the order of 200-450 µm. Finally, we show that the immunocapture device can be introduced into a perforated guide cannula for repeated analyte measurements in vivo. An increase in fluorescence detection of spatially localized intrahippocampal IL-1ß release was observed following a peripheral lipopolysaccharide challenge in Sprague-Dawley rats. This novel immunosensing technology represents an opportunity for unlocking the function of neuroimmune signaling.

Functional hyperspectral imaging captures subtle details of cell metabolism in olfactory neurosphere cells, disease-specific models of neurodegenerative disorders.

Hyperspectral imaging uses spectral and spatial image information for target detection and classification. In this work hyperspectral autofluorescence imaging was applied to patient olfactory neurosphere-derived cells, a cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. Taken together, our results demonstrate that multispectral spectral imaging provides a new non-invasive method to investigate neurodegenerative and other disease models, and it paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

A mobility shift assay for DNA detection using nanochannel gradient electrophoresis.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

STUDY QUESTION: Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? SUMMARY ANSWER: Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. WHAT IS KNOWN ALREADY: Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. STUDY DESIGN, SIZE, DURATION: The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. PARTICIPANTS/MATERIALS, SETTING, METHODS: In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. LIMITATIONS, REASONS FOR CAUTION: Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. WIDER IMPLICATIONS OF THE FINDINGS: Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.

Quantification of nanoparticle concentration in colloidal suspensions by a non-destructive optical method.

The estimation of nanoparticle number concentration in colloidal suspensions is a prerequisite in many procedures, and in particular in multi-stage, low-yield reactions. Here, we describe a rapid, non-destructive method based on optical extinction and dynamic light scattering (DLS), which combines measurements using common bench-top instrumentation with a numerical algorithm to calculate the particle size distribution (PSD) and concentration. These quantities were derived from Mie theory applied to measurements of the optical extinction spectrum of homogeneous, non-absorbing nanoparticles, and the relative PSD of a colloidal suspension. The work presents an approach to account for PSDs achieved by DLS which, due to the underlying model, may not be representative of the true sample PSD. The presented approach estimates the absolute particle number concentration of samples with mono-, bi-modal and broad size distributions with <50% precision. This provides a convenient and practical solution for number concentration estimation required during many applications of colloidal nanomaterials.

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 µM), oleic acid (200 µM), and steric acid (75 µM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.