Accepted Article Label-free imaging of redox status and collagen deposition showing metabolic differences in the heart Janna L. Morrison1*, Alexandra Sorvina2, Jack R.T. Darby1, Christie A. Bader2, Mitchell C. Lock1, Mike Seed3, Tim Kuchel4, Sally E. Plush2 and Douglas A. Brooks2 1 Early Origins of Adult Health Research Group and 2Mechanisms in Cell Biology and Disease Research Group, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia, Adelaide, South Australia 5001, Australia. 3 The Hospital for Sick Kids, Toronto, Ontario M5G 1X8, Canada. 4 Preclinical Imaging and Research Laboratories, South Australian Health and Medical Research Institute, Adelaide, South Australia 5086, Australia. * Corresponding and equal last authors. Email: [email protected] and [email protected], Phone: +61 8 830 22166 and +61 8 830 22586. Key words: cardiomyocyte, two-photon excitation fluorescence microscopy, collagen, metabolic activity, proliferation, quiescent/hypertrophic. The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. Here, we have employed twophoton excitation fluorescence microscopy to define the relationship between two critical metabolic co-factors NAD(P)H and FAD, effectively utilising a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two-photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Twophoton excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualise cardiac and other muscle pathologies. Two-photon excitation fluorescence microscopy used to identify different metabolic profiles between a proliferative myocardium reliant on glycolysis as a source of ATP and a quiescent/hypertrophic myocardium with a greater reliance on oxidative phosphorylation. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jbio.201700242 This article is protected by copyright. All rights reserved. Accepted Article 1. Introduction The hearts of the adult zebrafish, neonatal rat, and fetal sheep and human each have cardiomyocytes that are capable of proliferation. This proliferative capacity decreases in late gestation for sheep and humans, and shortly after birth in rats and mice 1-4, at which point the cardiomyocytes ‘switch’ their capacity for cell division and exhibit growth mainly by hypertrophy (Figure 1) 1, 3-5. The timing of this ‘switch’ from proliferative to a quiescent heart that grows via hypertrophy means that the heart of the sheep fetus, neonatal rat and human fetus can, like the adult zebrafish, regenerate and create new cardiomyocytes in response to an insult 6-8. However, following quiescence, this capacity for proliferative regeneration is lost and this results in a limited ability to repair damaged tissue, as is the case in, for example, the human heart 7. Coinciding with this critical transition from a proliferative to a hypertrophic growth profile, there is a change in the main fuel. In the fetus, the heart is mostly dependent on glucose but in the adult the heart is more reliant on fatty acids for ATP production, which provides the energy that supports cardiac function 9. While fatty acid oxidation is more efficient at producing ATP, it requires a relatively high oxygen environment 10, 11. In contrast to the well-oxygenated adult heart, the fetus has a much lower partial pressure of oxygen and is more reliant on glucose and lactate for ATP generation 11, 12. Another factor involved in this metabolic shift is that the placenta has a high capacity for transporting glucose across a concentration gradient, and this effectively creates a ready supply of glucose for ATP production in the fetal heart 13. However, after birth in the sheep and human fatty acids are more readily available 14 and as the lungs begin to function oxygen is no longer a limiting substrate. With the ensuing change in fatty acid supply and changes in oxygenation and growth profile, the metabolic status, reflected in the redox ratio, of the fetal and adult heart are likely to be significantly different. The redox state is determined by a balance of oxidized versus reduced forms of two key metabolic cofactors, nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD), which are found in either the reduced forms NADH/FADH2 or the oxidised forms NAD+/FAD, respectively. For example, the rate of glycolysis is dependent on NAD+ to carry electrons away from the process and as such the redox state is a primary regulator of ATP production from glycolysis (Figure 1) 15. Thus, for glycolysis to proceed the ratio of NADH/NAD+ needs to be low, in order to accept the electrons generated during this metabolic process. If the metabolic environment supports aerobic metabolism, then the pyruvate that is formed during glycolysis can be transported across the mitochondrial This article is protected by copyright. All rights reserved. membrane, where it is metabolised via the Krebs cycle (also known as the tricarboxylic acid cycle or citric acid cycle). As part of the Krebs cycle a molecule of FAD is reduced to FADH2, which is then oxidized, to reduce NAD+ to NADH. NADH is then utilised in oxidative phosphorylation. In contrast, in a more anaerobic environment pyruvate is converted to lactate with an associated conversion of NADH to NAD+. Both NADH and FAD are highly fluorescent and can be imaged by their distinct spectral characteristics 16, 17. A ratio of the fluorescence intensity for these two fluorophores can be used to provide a measure of the redox state 18 in live cells or tissues without the need for exogenous stains 19. Furthermore, two-photon fluorescence microscopy, which utilises near-infrared instead of UV or near-UV light, enables imaging with less damage and deeper tissue penetration 20. Thus, this imaging technology can potentially be utilised to accurately define the redox state in any tissue. Figure 1. Glucose metabolism in a low oxygen environment, which operates in the fetus (anaerobic), and a high oxygen environment as in an adult (aerobic). Another distinguishing feature between the fetal and adult heart is the deposition of collagen. Collagen deposition in the heart increases with age and collagen fibres can also be remodelled to provide different stiffness and elasticity in response to injury. For example, collagen I fibres exhibit higher stiffness, compared to collagen III fibres, which tend to have higher plasticity and are less energy efficient on deformation 21. Interestingly, collagen is another molecule that can be detected by two-photon excitation fluorescence microscopy, and potentially can be used to visualise and define structural differences in the heart. We previously showed that two-photon imaging can be used to measure NAD(P)H, FAD and collagen in cardiac tissue collected from the sheep fetus 22. Herein, we have used two-photon imaging to define the redox state and to visualise collagen deposition in the fetal (proliferative), compared to the adult (quiescent/hypertrophic) heart. Accepted Article 2. Methods analyzer (Radiometer, Copenhagen, calibrated for sheep blood. 2.1 Animal ethics approval and housing conditions 2.4 Post mortem and tissue collection All experimental protocols were reviewed and approved by the Animal Ethics Committee of the South Australian Health and Medical Research Institute (SAHMRI) and abide by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes developed by the National Health and Medical Research Council. The ewes were housed in an indoor facility with a constant ambient temperature of 20-22°C and a 12 hour light/dark cycle. Each ewe was in an individual pen with ad libitum access to food and water and in view of other sheep. All investigators understood and followed the ethical principles outlined in Grundy et al 23. 2.2 Surgical procedures At 107-109 days gestation (term 150 days), 4 ewes underwent fetal catheterization surgery under aseptic conditions. General anaesthesia was induced with diazepam (0.3mg/kg; intravenous) and ketamine (7mg/kg; intravenous) and maintained with inhalation of isoflurane (1-2%) in 100% oxygen. The anaesthesia and health status of the ewe, including oxygen saturation, heart rate and end-tidal carbon dioxide were monitored and recorded by trained staff throughout the protocol. Vascular catheters were inserted into a maternal carotid artery and jugular vein, fetal femoral artery and vein and the amniotic cavity as previously described 24, 25. During surgery, antibiotics were administered to the ewe (153.5 mg of procaine penicillin, 393 mg of benzathine penicillin, 500 mg of dihydrostreptomycin; Lyppards, Adelaide, Australia) and the fetus (150 mg of procaine penicillin, 112.5 mg of benzathine penicillin, 250 mg of dihydrostreptomycin; Lyppards, Adelaide, Australia). Upon recovery from anaesthesia, the ewes were given analgesia (20 ug/kg, Xylazil; Troy Laboratories, Australia). Antibiotics were administered intramuscularly to the ewe for 3 days after the surgery and intra-amniotically to the fetus (500 mg of ampicillin; Lyppards, Adelaide, Australia) for 4 days after the surgery. 2.3 Fetal blood gases Maternal and fetal femoral arterial blood gas samples (0.5 mL) were collected daily for the measurement of PO2, PCO2, pH, oxygen saturation (SO2), and hemoglobin (Hb) at 39°C with an ABL 520 This article is protected by copyright. All rights reserved. Denmark) Four ewes and their fetuses were humanely killed via overdose with sodium pentobarbitone (8 g; Vibrac Australia, Peakhurst, Australia) at 119-121 days gestation (term 150 days). The uterus was removed by hysterotomy, and the fetus was removed and weighed. The maternal and fetal hearts were quickly dissected and weighed; a saturated KCL solution (10mL) was infused into the aorta to ensure that the heart was arrested in diastole. The heart tissue was sectioned perpendicular to the ventricular walls and the tissues transported to the imaging facility within 90 minutes 22. To minimize the variations from sample to sample, fetal and adult tissue samples were prepared at the same time. Tissues were stored in ice-cold PBS and protected from light at all times. Prior to imaging, tissue slices of ~ 5 mm in thickness were prepared using a new scalpel to ensure no damage associated with tissue tearing. Using forceps, the tissue slices were gently moved onto an Ibidi µ-Slide and imaged within 1 hour. The tissue slices were kept moist in sterile PBS during imaging to prevent drying. The spectral data was acquired randomly from fetal and adult tissue samples. Only viable tissue samples were imaged as sample fixation reduces signal from endogenous fluorophores, including NAD(P)H and FAD 26. 2.5 Two-photon excitation fluorescence microscopy Two photon imaging was performed on a Zeiss LSM 710 META NLO inverted microscope (Carl Zeiss, Jena, Germany) supplemented with a twophoton Mai-Tai®, tunable Ti:Sapphire femtosecond pulse laser (710–920 nm; Spectra Physics, Mountain View, CA). Endogenous fluorescence signals from heart tissues were recorded using a polychromatic multichannel detector (META spectral detector), MBS-InVis: MBS 690+, FW1: Rear, excitation wavelengths 740, 840 and 900 nm and emission interval 416–708 nm 22. Images were captured across this emission spectrum in 29.2 nm wavelength intervals. The laser power was 11%. The lateral resolution of single-plane images was 212.34 x 212.34 µm. Images were collected up to a depth of ~ 150 µm. The pinhole was fully opened to 600 µm. The pixel dwell time was set to 1.58 µs, and each image was averaged eight times to increase signal-to-noise ratio. All images were acquired using a LD C-Apochromat 40X/NA 1.1 Water Corr UV-VIS-IR M27 objective (Carl Zeiss, Jena, Germany). Accepted Article The emission of NAD(P)H was determined at 489 nm (474–504 nm interval) and the emission of FAD was detected at 548 nm (533–562 nm interval) by exposure to two-photon illumination 27-29. To avoid crosstalk between these two fluorophores, different excitation wavelengths were used; 740 nm for NAD(P)H and 900 nm for FAD 30. Collagen was visualised at 431 nm emission wavelength (416–445 nm interval), when the samples were illuminated at 840 nm 31. For the preparation of figures, the fluorescence images were processed and collated in Adobe Photoshop CS6 (Adobe Systems Inc., USA). 2.6 Fluorescence intensity measurements Quantitative measurements of fluorescence intensity were made on digital images. The intensity value of a pixel is related to the number of photons present in a field of view, making microscopy data quantitative 32. The mean of NAD(P)H-, FAD- and collagen-fluorescence intensity was defined using Zen 2011 software (Carl Zeiss, Jena, Germany). The total number of fields of view per section for each sample was two, from which the intensity values of NAD(P)H, FAD and collagen were calculated. The redox ratio was calculated for the NADH and FAD signals using the following equation 33: = 2.7 Statistical analysis The difference between group means was assessed by Student's t-test, where the level of significance was P < 0.05 (GraphPad Prism version 7.00 for Windows, GraphPad Software, San Diego California, USA). Data was presented as the mean ± SEM. 3. Results and Discussion Fluorescent images were collected from healthy fetal and adult sheep hearts across an emission spectrum of 416-708 nm, in 29.2 nm intervals, using two-photon excitation at 740 nm and 900 nm (Figure 2). Excitation at 740 nm resulted in the largest quantity of detected fluorescence emission from both fetal and adult hearts. Excitation at this wavelength clearly shows that the morphology of the fetal heart is quite different from the adult; the adult heart shows greater organisation of myosin. Excitation wavelengths of 740 This article is protected by copyright. All rights reserved. nm and 900 nm were chosen to allow for the detection of the endogenous fluorescence from the reduced form of NAD(P)H (λem 489 nm, 474–504 nm interval) and the oxidised form of FAD (λem 548 nm, 533–562 nm interval), respectively, with minimal cross talk 28, 34, 35. Both NAD(P)H and FAD are critical to cellular metabolism and act as electron carriers linking the Krebs cycle with oxidative phosphorylation. It is important to note that the term NAD(P)H has been employed herein as the distinction for cytosolic NADH, as protein bound mitochondrial NADH and NADPH from anabolic pathways are not easily separated using the two-photon imaging techniques employed here 36, 37 . To further explore these changes, future work may use fluorescence-lifetime imaging microscopy (FLIM) to determine levels of FAD in mitochondria and NAD(P)H in the cytosol and mitochondria. A comparison of the spectral profiles from each of the tissues at the excitation wavelengths of 740 nm and 900 nm (Figure 3a, 3b and Figures S1-S2) shows that there is a shift in the emission maxima between the two excitation wavelengths. Excitation at 740 nm results in a single maxima curve with maximum emission recorded between 431-548 nm in fetal and adult hearts. This spectral profile matches that previously recorded for NAD(P)H 22, 38. In comparison, excitation at 900 nm resulted in a ~60 nm bathochromic shift to the wavelength over which the maximal emission was detected (489-635 nm), and this is consistent with the known emission maxima for FAD, which is ~ 520 nm following two-photon excitation 22, 38. Interestingly, a shoulder is present in the spectral profile at ~ 635 nm following excitation at 900 nm. This is not likely to be related to the major metabolic fuels, oxygen availability or proliferative vs hypertrophic growth capacity of the heart because the spectral shape is consistent between fetal and adult sheep hearts at this excitation wavelength. Most importantly, the similarity between the spectral profiles obtained from fetal and adult heart tissue at both 740 and 900 nm respectively, allowed for accurate comparison of quantified emission. Quantification of the emission fingerprinting from these two endogenous fluorophores (Figure 3c, 3d) shows that signal consistent with NAD(P)H accounts for the majority of the signal detected over the range of 431-548 nm (λex 740 nm) in both the fetal and adult hearts. Interestingly, the adult heart tissue has significantly higher levels of NAD(P)H than fetal tissue. The levels of FAD are also elevated in the adult tissue, however, the relative differences between this individual fluorophore in adult and fetal heart tissues are not as large as that observed for NAD(P)H. Accepted Article Figure 2. Imaging endogenous fluorescence in fetal (a) and adult (b) sheep hearts. Representative micrographs of hearts were obtained using a META spectral detector, with sampling emission over the visible spectrum in 29.2 nm wavelength intervals using the excitation wavelengths specified on the y-axis (λex), with the emission wavelength (Em) on the x-axis. Wavelength is in nanometres. Scale bars 50 µm. Figure 3. Histograms showing quantified levels of endogenous fluorescence across the spectrum (a, b) and at 740 nm and 900 nm excitation wavelengths in the fetal and adult sheep hearts showing levels of NAD(P)H (c), FAD (d) and the redox ratio (e). The quantified levels of NAD(P)H and FAD allow for the calculation of a redox ratio from both fetal and adult heart tissues (Figure 3e), with the fetal tissue exhibiting a higher redox ratio (REDOXratio = 0.09 ± 0.01), compared to the adult heart tissue (REDOXratio = 0.06 ± 0.004; P = 0.0356). As the levels of NAD(P)H and FAD are intrinsically linked to the production and use of ATP, the detected differences in endogenous fluorescence between fetal and adult heart tissues and the calculated redox ratios suggest changes in metabolic activity associated with the differing fuel sources, mode of growth and oxygen environment. Interestingly, a recent study shows that mitophagy occurs just after birth in mouse cardiomyocytes and that if this development of adult mitochondria is prevented, there is no switch to fatty acid oxidation as the main source of cardiac fuel 39. The healthy adult heart functions in an oxygen rich environment, which favours the combinatorial approach to ATP synthesis, where glycolysis, the Krebs cycle and oxidative phosphorylation are all utilised. In aerobic This article is protected by copyright. All rights reserved. conditions, the summation of glycolysis and the Krebs cycle produces 10 moles of NAD(P)H per molecule of glucose. Due to the high efficiency of oxidative phosphorylation in the production of ATP (1 mole of NAD(P)H produces 3 moles of ATP), cells with high metabolic potential maintain high levels of NAD(P)H to ensure they can accommodate large increases in ATP production via oxidative phosphorylation. Therefore, the high levels of NAD(P)H fluorescence and low redox ratio in the adult hearts was expected. Despite much higher plasma glucose concentrations in the adult (Table 1), only ~ 10% of ATP production is from glucose in the healthy adult heart 40. Consequently, in healthy adult cardiac cells oxidative phosphorylation accounts for about 70% of ATP production, but as oxygen availability decreases, reliance on glycolysis increases 41. The fetus functions in a low oxygen environment and as the Krebs cycle is heavily reliant on oxygen, it is not a major contributor to the production of ATP (or NAD(P)H by default) production in the fetal heart 11. Cells in anaerobic environments must rely on glycolysis for the production of energy 10. While glycolysis results in the production of NAD(P)H, it is quickly oxidised to NAD. The oxidation of NAD(P)H is afforded by the reduction of the pyruvate produced by glycolysis and this results in the production of lactate. Therefore, higher amounts of lactate production are typically associated with cells exposed to anaerobic conditions. Measurement of plasma lactate in the fetus and adult shows that fetal hearts have significantly higher amounts of lactate (Table 1), suggesting that glycolysis is indeed the major metabolic pathway operating in this tissue during early development. Consequently, approximately 60% of the fetal hearts oxygen consumption is accounted for by lactate oxidation 42 . Furthermore, plasma levels of lactate in the fetus increase with gestational age and thus contribute to more oxygen consumption 43. The reliance on glycolysis for ATP production in fetal cells has a two-fold benefit, not only is glucose more readily available than fatty acids in the fetal circulation, glycolysis also produces key building blocks that are required for mitosis 44. Reduced levels of NAD(P)H were also recorded in infarcted adult hearts 22, Accepted Article where this trauma creates an oxygen deprived environment, requiring reversion to ATP production by glycolysis. Table 1. Fetal and maternal arterial blood gases and metabolites. Fetus (n = 4) 7.364 ± 0.003 * 21 ± 2 * 52 ± 2 * 55.3 ± 4.1 * Adult (n = 4) 7.473 ± 0.022 108 ± 5 34 ± 2 97.9 ± 0.2 # pH PO2 (mmHg) PCO2 (mmHg) Oxygen saturation (%) Hemoglobin 8.4 ± 1.2 9.1 ± 0.3 (g/dL) 3.3 ± 0.2 # Glucose 1.0 ± 0.2 *, # (mmol/L) Lactate 1.1 ± 0.2 *, # 0.5 ± 0.1 # (mmol/L) Results are means ± SEM; * P < 0.05; # n = 3 animals. Collagen deposition in the myocardium increases with age 45, but this is accompanied by increased extracellular matrix breakdown and remodelling in an effort to reduce myocardial stiffening and maintain sufficient contractile function 46. Indeed, only subtle differences in collagen deposition may be observed among healthy adult hearts at different ages, but stark differences in fetal cardiac collagen deposition are expected in comparison to that of the adult (Figure 4a, 4b). Previously, we reported the use of two-photon excitation fluorescence microscopy to assess collagen deposition after a myocardial infarction 22. To assess the amount of this extracellular matrix component (collagen; λex 840 nm) at two distinctly different ages, emission intensity of the endogenous fluorescence was quantified (Figure 4c). As expected the adult heart had significantly higher amounts of collagen compared to the fetal heart (P = 0.0002). Quantification of the endogenous fluorescence, when excited at 840 nm, allows for a more thorough characterization of the myocardium. The lower amounts of collagen visualised in the fetal hearts are consistent with the heart’s relative plasticity during fetal development, and the lower contractile force required to pump blood in a lower pressure system while still attached to the placental/maternal circulation. This article is protected by copyright. All rights reserved. Figure 4. Net collagen deposition in fetal and maternal hearts as determined using two-photon excitation fluorescence microscopy. Histogram showing the quantified levels of endogenous fluorescence at 840 nm excitation wavelength in the fetal and adult sheep hearts (a). Results are means ± SEM (n = 4 animals; P = 0.0002). Two-photon imaging of collagen in fetal (b) and adult (c) sheep hearts at 840 nm excitation wavelength. Scale bars: 50 μm. 4. Conclusion In summary, we have demonstrated that two-photon excitation fluorescence microscopy can be used to quantitatively distinguish between the metabolic profiles of fetal and adult sheep hearts. Adult heart tissues were found to have high amounts of NAD(P)H and this is associated with the reliance on fatty acids and oxidative phosphorylation to provide ATP. In contrast, fetal tissue had significantly lower amounts of NAD(P)H but a higher redox ratio and higher amounts of lactate supporting its reliance on glycolytic pathways for ATP production. An optically derived redox ratio to reflect the amounts of NAD(P)H and FAD have provided insight into the metabolic activity of heart cells with different modes of growth, oxygenation and the metabolic pathways used to fuel ATP production as a source of energy. Structurally there were higher amounts of collagen present in the adult heart compared to the fetus, which is consistent with the adult heart being relatively stiffer than the fetus due to higher contractile demand on the adult heart and higher pressure in the systemic circulation, when compared to the fetus. We concluded that fluorescence imaging can provide critical growth, metabolic and structural information on live tissue, and this will have potential application for assessing a range of different pathologies. Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s website. Acknowledgements JLM was funded by a NHMRC Career Development Fellowship (APP1066916). JRTD and MCL were funded by and Australian government research training program (RTP) scholarships. We acknowledge the assistance of Stacey Holman, Wendy Bonner and Madeline Ragless in surgical procedures and post-operative care of the sheep. Competing Financial Interests Accepted Article The authors declare no competing financial interests. This article is protected by copyright. All rights reserved. Accepted Article Author biographies Professor Janna L. Morrison is Head of the Early Origins of Adult Health Research Group in the Sansom Institute for Health Research at the University of South Australia. Professor Morrison has been funded as a fellow by the Heart Foundation 2004-2013 and is currently a NHMRC Career Development Fellow (2014-2017). Her work focusses on examining the link between low birth weight and heart disease in adulthood. She is a fellow of the Cardiovascular Section of the American Physiological Society (2015). Dr Alexandra Sorvina is a Research Associate in the Mechanisms and Cell Biology of Disease Research Group at the University of South Australia. Her research is focused on the use of imaging techniques (e. g. twophoton and fluorescent microscopy) to understand disease pathogenesis and progression. Jack R.T. Darby is a Ph.D. student in the Early Origins of Adult Health Research Group in the Sansom Institute for Health Research at the University of South Australia. His research focusses on understanding cardiac development and the programming of cardiovascular disease as a result of a suboptimal in utero environment. Dr Christie A. Bader currently works as a Research Associate in the Mechanisms and Cell Biology of Disease Research Group at the University of South Australia. Her research is focused on cell imaging primarily via fluorescence and two-photon excitation fluorescence microscopy; with a view to better understand disease mechanisms through the observation of cellular processes. This article is protected by copyright. All rights reserved. Mitchell C. Lock is a Ph.D. student in the Early Origins of Adult Health Research Group in the Sansom Institute for Health Research at the University of South Australia. His research is focused on the epigenetic regulation of cardiac tissue after heart attack and cardiac regeneration. Dr Mike Seed is the Division Head of Cardiology at the Hospital for Sick Children in Toronto and an Associate Professor of Paediatrics, Medical Imaging and Obstetrics and Gynecology at the University of Toronto. The goal of this research is to develop neuroprotective strategies to mitigate against the adverse effects of abnormal perinatal cardiovascular physiology on the developing brain. Dr Tim Kuchel, is the director of the Preclinical, Imaging and Research Laboratories (PIRL) of the South Australian Health and Medical Research Institute (SAHMRI). It is PIRL which hosts the NIF funded Large Animal Research and Imaging Facility (LARIF). Dr. Kuchel has more than 30 years’ experience in animal research with a special interest in imaging modalities, experimental surgery and comparative anaesthesia. . Dr Sally E. Plush is a Senior Lecturer in chemistry at the University of South Australia. Her research is focused on the use of metal ions in cellular imaging applications (fluorescence microscopy, PET and MRI) to further the understanding of the pathophysiology of major disease states and to develop enhanced diagnostic and therapeutic agents. Accepted Article Professor Douglas A. Brooks is the leader of the Mechanisms in Cell Biology and Disease Research Group at the Sansom Institute for Health Sciences in the School of Pharmacy and Medical Science at the University of South Australia. Professor Brooks has over 30 years of experience in cell biology and research translation, with a focus on disease pathogenesis. This article is protected by copyright. All rights reserved. Accepted Article References  S. S. Jonker, S. Louey, G. D. Giraud, K. L. Thornburg, J. J. Faber Faseb j. 2015, 29, 43464357.  J. H. Burrell, A. M. Boyn, V. Kumarasamy, A. Hsieh, S. I. Head, E. R. Lumbers Anat Rec. 2003, 274A, 952-961.  F. Li, X. Wang, J. M. Capasso, A. M. 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Wavelengths in nanometers (nm). Scale bars: 50 μm. This article is protected by copyright. All rights reserved. Accepted Article Figure S2. Imaging endogenous FAD using 900 nm excitation wavelength in both fetal (a) and adult (b) sheep heart tissues. A META spectral detector, with sampling emission over the visible spectrum, in 29.2 nm wavelength intervals was employed. The wavelength of the emission is specified on the x-axis (Em). Wavelengths in nanometers (nm). Scale bars: 50 μm. This article is protected by copyright. All rights reserved.