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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
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.
The Hospital for Sick Kids, Toronto, Ontario M5G 1X8, Canada.
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,
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
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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
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
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
dihydrostreptomycin; Lyppards, Adelaide, Australia).
Upon recovery from anaesthesia, the ewes were given
analgesia (20 ug/kg, Xylazil; Troy Laboratories,
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.
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
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
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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,
. 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
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
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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
. 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
Table 1. Fetal and maternal arterial blood gases and
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 #
PO2 (mmHg)
PCO2 (mmHg)
saturation (%)
8.4 ± 1.2
9.1 ± 0.3
3.3 ± 0.2 #
1.0 ± 0.2 *, #
1.1 ± 0.2 *, #
0.5 ± 0.1 #
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.
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
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
microscopy) to understand disease pathogenesis and
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
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
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
This article is protected by copyright. All rights reserved.
Accepted Article
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Supporting information
Figure S1. Imaging endogenous NAD(P)H using 740 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.
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.
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