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Monitoring spinal muscular atrophy with three-
dimensional optoacoustic imaging
Emmanuel Nedoschill,
Alexandra L. Wagner, Vera
Danko, ..., Regina Trollmann,
Ferdinand Knieling, Adrian P.
Regensburger

adrian.regensburger@uk-erlangen.de

Highlights
Three-dimensional optoacoustic
imaging enables monitoring of
spinal muscular atrophy

The optoacoustic signal correlates

with disease severity and motor
function of patients

Structural and molecular effects of

gene therapy can be assessed in
real time

Nedoschill et al. demonstrate the usability of three-dimensional optoacoustic

imaging for monitoring disease severity in patients with spinal muscular atrophy.
The molecular signal of muscle tissue corresponded with disease severity and was
comparable to healthy controls in a cohort of patients who received gene-
modifying therapy prior to the study.

Nedoschill et al., Med 5, 469–478

May 10, 2024 ª 2024 The Author(s). Published
by Elsevier Inc.
https://doi.org/10.1016/j.medj.2024.02.010
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Resource
Monitoring spinal muscular atrophy
with three-dimensional optoacoustic imaging
Emmanuel Nedoschill,1 Alexandra L. Wagner,2 Vera Danko,1 Adrian Buehler,1 Roman Raming,1
Jörg Jüngert,1 Markus F. Neurath,3 Maximilian J. Waldner,3 Ulrich Rother,4 Joachim Woelfle,1
Regina Trollmann,1 Ferdinand Knieling,1,5 and Adrian P. Regensburger1,5,6,*

SUMMARY CONTEXT AND SIGNIFICANCE

Background: Spinal muscular atrophy is a progressive neuromuscular Spinal muscular atrophy is a
disorder and among the most frequent genetic causes of infant mortal- hereditary neuromuscular
ity. While recent advancements in gene therapy provide the potential to disease. Patients experience
ameliorate the disease severity, there is currently no modality in clinical different degrees of muscle
use to visualize dynamic pathophysiological changes in disease pro- weakness, with some never
gression and regression after therapy. achieving the ability to walk
Methods: In this prospective diagnostic clinical study, ten pediatric unassisted, and the disease is a
patients with spinal muscular atrophy and ten age- and sex-matched frequent cause of infant death.
controls have been examined with three-dimensional optoacoustic Major advancements in recent
imaging and clinical standard examinations to compare the spectral years in the therapy of spinal
profile of muscle tissue and correlate it with motor function muscular atrophy have enabled
(ClinicalTrials.gov: NCT04115475). physicians to restore gene
Findings: We observed a reduced optoacoustic signal in muscle tissue function and ameliorate the
of pediatric patients with spinal muscular atrophy. The reduction in disease in patients. What is
signal intensity correlated with disease severity as assessed by gray- missing in clinical routine care is a
scale ultrasound and standard motor function tests. In a cohort of pa- modality to monitor disease
tients who received disease-modifying therapy prior to the study, the severity and molecular changes
optoacoustic signal intensity was similar to healthy controls. after therapy induction. In this
Conclusions: This translational study provides early evidence that three- study, the authors use three-
dimensional optoacoustic imaging could have clinical implications in dimensional optoacoustic
monitoring disease activity in spinal muscular atrophy. By visualizing imaging, an imaging modality that
and quantifying molecular changes in muscle tissue, disease progres- utilizes laser light to illuminate
sion and effects of gene therapy can be assessed in real time. biological tissue to assess muscle
Funding: The project was funded by ELAN Fonds (P055) at the Univer- tissue of patients with spinal
sity Hospital of the Friedrich-Alexander-Universität (FAU) Erlangen- muscular atrophy. This technique
Nurnberg to A.P.R. could provide a useful tool for
non-invasive evaluation of
neuromuscular diseases.
INTRODUCTION
Spinal muscular atrophy (SMA) is a rare progressive neuromuscular disorder with a
prevalence of 1 in 7,500 to 1 in 1,0000 newborns.1 Biallelic mutations or deletions
in the SMN1 gene and the subsequent loss of the encoded SMN protein cause
degeneration of alpha-motor neurons in anterior horn cells, resulting in muscle atro-
phy and weakness.2 SMA is divided into subtypes (SMA types I, II, III, and IV), which
differ in disease onset and best-achievable motor function of the patient.3 The
phenotype is mainly influenced by the copy number of the highly homologous
SMN2 gene.4 Recent advances in therapy include the development of disease-modi-
fying drugs, such as the antisense oligonucleotide (ASO) nusinersen5 and single-
dose gene replacement therapy.6 By modifying or restoring gene function, these
new therapeutic agents can ameliorate the degeneration of motor neurons and

Med 5, 469–478, May 10, 2024 ª 2024 The Author(s). Published by Elsevier Inc. 469
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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OPEN ACCESS Resource

subsequent muscle atrophy, potentially altering the phenotype of patients with

SMA. This creates the important task for clinicians of monitoring therapeutic efficacy
and disease progression, which is still solely based on the clinical assessment of
best-achievable motor function through standardized scores.7 While the clinical ex-
amination provides important information about the functional status of a patient, it
is dependent on the cooperation of the patient and the experience of the observer
since these scores can have up to thirty different items.8 Standard clinical scores also
do not provide information about structural changes in the muscle tissue of patients,
which is important information when evaluating disease progression and regression
after therapy induction. Although non-invasive imaging modalities like ultrasound
(US) and magnetic resonance imaging (MRI) may allow for muscle assessment,
they are not commonly implemented in the diagnostic workup of SMA. While MRI
is limited by the need for sedation in early childhood, US lacks specific biomarkers
to monitor treatment effects. Molecular-sensitive US technologies, like optoacoustic
imaging (OAI), could close this gap. In OAI, short-pulsed laser light of the near-
infrared spectrum induces thermoelastic expansion in tissue molecules.9 As a result,
these molecules create sound waves, which can be quantitatively attributed to their
specific origin (e.g., hemoglobin).10,11 Uniquely, OAI combines high optical contrast
and deep tissue penetration over several centimeters. While two-dimensional OAI
has previously been used to assess different inflammatory and degenerative dis-
eases,12 three-dimensional OAI may further improve visualization and signal quan-
tification.13,14 In this study, we assessed the imaging capabilities of three-dimen-
sional OAI to resolve muscle degeneration and therapeutic effects in SMA.

RESULTS
Participant characteristics
Pediatric patients with SMA (n = 10) and healthy volunteers (HVs; n = 10) were inves-
tigated. The mean age of patients with SMA was 9.0 (G3.7) years, and the mean age
of HVs was 8.7 (G4.3) years. In both groups, 30% of the participants were female.
The mean BMIs were 16.3 (G5.4) in the SMA group and 16.3 (G3.2) in the HV group.
In the SMA group, 5 out of 10 participants received treatment with nusinersen prior
to or during the study (Table 1). All participants underwent three-dimensional OAI
(Figure 1). No serious adverse events were recorded during the study.

The optoacoustic signal at 800 nm is reduced in muscle tissue of patients with

SMA
Each muscle of each patient was imaged in duplicate, leading to a total of 320
three-dimensional scans (160 scans of patients with SMA and 160 scans of HVs).
The mean optoacoustic signal intensity of all muscle tissue assessed at 800 nm
was lower in patients with SMA than in HVs (1.016 G 0.507 vs. 1.253 G 0.588
a.u., p = 0.0394; Figures 2A–2C). When comparing each muscle region separately, 1Department of Pediatrics and Adolescent
Medicine, University Hospital Erlangen, Erlangen,
the reduction in signal intensity in patients with SMA compared to HVs was most
Germany
significant in the proximal leg (0.7376 G 0.176 vs. 1.026 G 0.256 a.u., p = 0.0195; 2Department of Pediatric Neurology, Center for
Figures 2D and 2E). It was also seen in the proximal arm, albeit not with a statis- Chronically Sick Children, Charité Berlin, Berlin,
tically significant difference (1.195 G 0.76 vs. 1.555 G 0.854 a.u., p = 0.4922). Germany
3Medical Department 1, University Hospital
The distal muscle regions did not show significant differences between groups
Erlangen, Erlangen, Germany
(all p > 0.05; data not shown). 4Department of Vascular Surgery, University
Hospital Erlangen, Erlangen, Germany
The optoacoustic signal at 800 nm correlates with disease severity 5These authors contributed equally
The analysis included patients with type 1 SMA (n = 2), type 2 SMA (n = 4), and type 6Lead contact
3 SMA (n = 4). Volumetric OAI data are illustrated in the supplemental information *Correspondence:
(Video S1). The optoacoustic signal intensity at 800 nm showed the most significant adrian.regensburger@uk-erlangen.de
reduction in the type 1 SMA group compared to HVs (0.862 G 0.241 vs. 1.253 G https://doi.org/10.1016/j.medj.2024.02.010

470 Med 5, 469–478, May 10, 2024

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Table 1. Participant demographics

Characteristics HVs (n = 10) SMA (n = 10)
Age, years 8.7 G 4.3 9.0 G 3.7
Age, months 109.3 G 53.4 114.3 G 44.3
Female sex, n (%) 3 (30) 3 (30)
Height, cm 138.2 G 24.4 133.2 G 20.4
Weight, kg 33.8 G 17.4 30.5 G 15.5
BMI, score 16.3 G 3.2 16.3 G 5.4
SMA type I (%) 0 (0) 2 (20)
SMA type II (%) 0 (0) 4 (40)
SMA type III (%) 0 (0) 4 (40)
Nusinersen, n (%) 0 (0) 5 (50)
Nusinersen treatment duration, months 0G0 15.4 G 11.9
Values are given as mean G standard deviation, adapted from Regensburger et al.15 and Danko et al.16
HVs, healthy volunteers; SMA, patients with SMA; n, number of subjects.

0.588 a.u.). Patients with type 2 SMA showed less reduction in optoacoustic signal
intensity, and patients with type 3 SMA had an optoacoustic signal intensity closest
to the healthy population (Figure 3A). Divided according to muscle regions, signal
intensities in the proximal arm followed the same trend as in the pooled analysis,
albeit not statistically significant (Figure 3B), with patients with type 2 SMA showing
a lower signal intensity (0.9316 G 0.4866 a.u.) than patients with type 3 SMA and pa-
tients with type 1 with SMA showing the lowest signal intensity (0.7025 G 0.2191
a.u.). The same effects have been observed in the distal arm and proximal leg
(Figures 3C and 3D). In the distal leg, more heterogeneous results were observed
(Figure 3E).

Furthermore, disease phenotyping by clinical standard assessment with Hammer-

smith Functional Motor Scale - Expanded correlated best with SWL800 signal in-
tensity of the grouped muscle signal intensity (rs = 0.77, p = 0.021). Medium to
strong correlation was observed for different muscle regions (rs = 0.467–0.667)
(Figure S2).

Clinical assessment by the revised upper limb module correlated with mean
SWL800 at the proximal arm (rs = 0.803, p = 0.012) and the mean value of patients
(rs = 0.812, p = 0.011). All correlations are shown in the supplemental information
(Figure S2).

A multimodality comparison of three-dimensional and two-dimensional OAI,15 US

grayscale values,16 and clinical scoring revealed a greater correlation between
three-dimensional OAI with US and clinical scoring compared to two-dimensional
OAI (Figure 4).

The optoacoustic signal correlates with ASO treatment

50% of the patients with SMA received treatment with ASO prior to or during the
study (n = 5). The optoacoustic signal intensity at SWL800 was significantly higher
in the group of treated patients with SMA compared to non-treated patients
(1.180 G 0.563 vs. 0.852 G 0.393 a.u., p = 0.008). SWL800 signals in treated patients
with SMA were similar to HVs (1.180 G 0.563 vs. 1.253 G 0.588 a.u., p = 0.840; Fig-
ure 5A). Again, when separated by muscle regions, similar trends were observed and
were most prominent in the proximal arm and leg regions (Figures 5B–5E).
SWL800nm showed good AUC (0.74, 95% CI 0.582–0.903, p = 0.009) to discriminate
between treated and non-treated patients (Figure 5F).

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A B C

D E F

Figure 1. Imaging principle

(A) Illustration of handheld three-dimensional optoacoustic muscle imaging in pediatric patients.
(B) Schematic optoacoustic effect in muscle tissue. Short-pulsed laser beams heat endogenous chromophores in the tissue and cause thermoelastic
expansion, which creates sound waves that are registered by the imaging probe.
(C) Absorption spectra of oxygenated and deoxygenated hemoglobin. The isosbestic absorption point is approximately at 800 nm, which was used for
subsequent imaging.
(D) Three-dimensional optoacoustic imaging (OAI) data at 800 nm from the proximal arm of a healthy volunteer (HV), shown as a maximum intensity
projection. The cube’s edges are 15 mm long. A superficial vessel of the skin and the muscle tissue are marked with white arrows.
(E) Illustration of the tissue components shown as OAI imaging data in (D).
(F) Three-dimensional OAI data at 800 nm, shown as x-y, x-z, and y-z planes with cylindrical region of interest, shown in orange. Scale bars represent 5 mm.

DISCUSSION
In this study, three-dimensional OAI has demonstrated the ability to quantify
muscle degeneration in pediatric patients with SMA. A significant reduction of the opto-
acoustic signal intensity was observed in diseased muscles correlating with the respec-
tive extent of disease. These results are consistent with two-dimensional data.15 Howev-
er, the three-dimensional approach allows for the interpretation of volumetric
information and, from a technical perspective, might be more accurate due to the lesser
extent of artifacts and out-of-plane signals.14,17 These volumetric data might aid in
improving the visualization of specific tissue patterns. In this study, the increase in sensi-
tivity already enabled us to differentiate between ASO-treated and non-treated subjects.

Given the new therapeutic options available, a rapid diagnosis of SMA is currently
being established through newborn screening programs. However, especially in
clinical trials, functional muscle testing is still used to assess the true extent of
the disease. For example, in early-onset SMA type I, lack of muscle strength limits
the feasibility of testing because either patients cannot perform the tasks or the
ordinal scale is not sensitive enough to detect small differences.18 In addition,
these tests are, by their very nature, dependent on the motivation and cooperation
of the patients. Therefore, objective and reliable measures of disease status are
urgently required to monitor disease progression and the effects of current and

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Figure 2. Disease phenotyping

(A) Pediatric patients underwent three-dimensional OAI at four muscle regions, two of which are
illustrated here. The volumetric optoacoustic data are shown as maximum intensity projections for
HVs (left) and patients with SMA type I (right) for different signal levels at 800 nm. Muscle tissue is
marked with a dashed line. Scale bars represent 5 mm.
(B) Illustration of the pathophysiology of SMA; depicted is a healthy (HV) and a diseased (SMA)
motoneuron with healthy and degenerating muscle, respectively.
(C) Mean optoacoustic signal intensity for HV and patients with SMA.
(D) Mean signal intensity from the proximal arm.
(E) Analysis of data from the proximal leg. p < 0.05 indicates statistical significance. Data is given as
mean with standard deviation (error bars).

future disease-modifying therapies. This is precisely what has already been tried to
be implemented with MRI, using the fat fraction in thigh muscles as a surrogate
marker.19 However, there are considerable challenges to performing MRI in pa-
tients with SMA, particularly the need for sedation and associated risks. Moreover,
the specific disease characteristics, including scoliosis and contractures, compli-
cate patient positioning and limit investigation times.

In contrast, OAI is non-invasive and does not require sedation or special positioning,
similar to US. Although qualitative and quantitative B-mode US offers valid informa-
tion to interpret muscle affection, it does not provide molecular tissue information
comparable to OAI.16,20 In muscular dystrophies, OAI was capable of detecting
fibrotic tissue transformation,13 enabled by spectral unmixing of multiple

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Figure 3. The optoacoustic signal correlates with disease severity

(A–E) Mean SWL800 levels are compared between HV and SMA type III, type II, and type I groups for (A) mean value of all muscles, (B) proximal arm
muscles, (C) distal arm muscles, (D) proximal leg muscles, and (E) distal leg muscles.
Data is given as mean with standard deviation (error bars).

wavelengths.21 Targeting muscular hemoglobin/myoglobin content through OAI

was also a strategy to aid in classifying peripheral artery disease in adults.22 Howev-
er, a single-wavelength approach bypasses the main confounder of spectrally un-
mixed OAI data, which is called spectral coloring and was shown as promising
biomarker for muscle degeneration in adults.23 Due to the unknown tissue proper-
ties in vivo, light fluence can be corrupted, leading to an increasingly altered spec-
trum with depth.24,25 Novel data analysis algorithms (e.g., blind source spectral un-
mixing) might overcome this issue.26 However, in this study, the wavelength at
800 nm approximately reflects the isosbestic point of hemoglobin,27 the wavelength
where light absorption of oxygenated and deoxygenated hemoglobin is similar,
creating the best contrast-to-noise ratio in three-dimensional OAI data.14 By using
a three-dimensional imaging probe, a single placement for data acquisition is suffi-
cient, which reduces the influence of motion artifacts. Regarding the heterogeneous
nature of different muscular diseases, the volumetric image acquisition utilized in
three-dimensional OAI enables the evaluation of more organ tissue than two-dimen-
sional OAI. This reduces the chances of missing muscle tissue with pathological
structure when the disease is heterogeneous and not every muscle fiber is affected.

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Figure 4. Correlation of three- and two-dimensional OAI data with ultrasound and clinical scores
Mean SWL800 levels are compared between three-dimensional OAI, two-dimensional OAI
(adapted from Regensburger et al. 15 ), ultrasound grayscale values (adapted from Danko et al. 16 ),
the HFMSE, and the revised upper limb module (RULM) clinical score. Patient data are included as
mean values for each patient. Correlations are given as Spearman’s r.

Currently, the three-dimensional probe does not provide B-mode US information,

which would be helpful for anatomical guidance. Such hybrid imaging probes are
currently under development. In our study, muscle tissue was easily identified during
acquisition by adhering to strict anatomical landmarks and characteristic 800 nm OAI
signals.

Limitations of the study

Since SMA is a rare disease, it was a challenge to reach a larger sample size. Given
the small sample size in our study, the results will be subject to confirmation in more
extensive, stratified trials. Regarding the small sample size, it is important to inter-
pret the results carefully, as the p value can have a large sample-to-sample variability
in small cohorts. This imaging technique is not intended to replace clinical examina-
tion in assessing muscular disease but rather is to provide a translational platform
that adds to diagnostic accuracy. Regarding therapeutic effects, a follow-up study
should assess changes in individual patients over time.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the
following:

d KEY RESOURCES TABLE

d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
d METHOD DETAILS
B Study flow
B Three-dimensional optoacoustic imaging
B Two-dimensional optoacoustic imaging
B Ultrasound imaging
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Data analysis
B Statistical analysis

SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.medj.
2024.02.010.

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Figure 5. The optoacoustic signal correlates with nusinersen treatment

(A–E) Mean SWL800 levels are compared between HVs and patients without (not treated) and with (treated) nusinersen therapy for (A) mean value of all
muscles, (B) proximal arm muscles, (C) distal arm muscles, (D) proximal leg muscles, and (E) distal leg muscles.
(F) Receiver operator characteristics of SWL800 levels for discrimination between treated and untreated groups with data from the proximal arm. Data is
given as mean with standard deviation (error bars).

ACKNOWLEDGMENTS
The present work was performed in (partial) fulfillment of the requirements for ob-
taining the degree ‘‘Dr. rer. biol. hum.’’ for E.N. at the Friedrich-Alexander-Universi-
tät Erlangen-Nurnberg (FAU). Figures 1 and 2 and the graphical abstract have been
created with BioRender.com. The project was funded by the Else Kröner-Fresenius-
Stiftung and ELAN Fonds (P055 University Hospital of the FAU Erlangen-Nurnberg)
to A.P.R. F.K. and A.P.R. acknowledge support by the Interdisciplinary Center for
Clinical Research (IZKF) at the University Hospital of the FAU Erlangen-Nurnberg.
F.K. acknowledges funding from Else Kröner-Fresenius-Stiftung (Else Kröner-
Memorial-Stipendium, 2018_EKMS.03). M.J.W. was supported by the Graduate
School in Advanced Optical Technologies of the FAU Erlangen-Nurnberg. M.F.N.
acknowledges funding from the Emerging Fields Initiative (EFI) of the FAU

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Erlangen-Nurnberg. M.J.W. and M.F.N. acknowledge funding from German

Research Foundation (FOR2438 and TRR241). This project has received funding
from the European Union’s Horizon 2020 research and innovation program under
grant agreement no. 830965. F.K. received funding from the European Union
(ERC Starting). The material presented and views expressed here are the responsi-
bility of the author(s) only. The EU Commission takes no responsibility for any use
made of the information set out.

AUTHOR CONTRIBUTIONS
Software, E.N. and A.B.; formal analysis, E.N. and A.B.; data curation, E.N. and A.B.;
writing – original draft, E.N.; writing – review & editing, E.N., A.L.W., R.R., M.F.N.,
M.J.W., U.R., J.W., R.T., F.K., and A.P.R.; visualization, E.N., F.K., and A.P.R.;
conceptualization, A.L.W.; methodology, A.L.W.; investigation, A.L.W., V.D., J.J.,
F.K., and A.P.R.; resources, R.R., F.K., and A.P.R.; supervision, F.K. and A.P.R.; proj-
ect administration, F.K. and A.P.R.; funding acquisition, F.K. and A.P.R. All authors
read and approved the final article and take responsibility for its content.

DECLARATION OF INTERESTS
M.J.W., F.K., and A.P.R. are shared patent holders with iThera Medical GmbH (Mu-
nich, Germany) on the OAI system/software described in the study. F.K. and U.R. are
members of the clinical advisory board of iThera Medical GmbH (Munich, Germany).

Received: December 15, 2023

Revised: February 1, 2024
Accepted: February 26, 2024
Published: March 25, 2024

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Software and algorithms
GraphPad Version 9.5.1
ViewMSOT Version 4.0.3.2
Other
Multispectral Optoacoustic Tomography
System ‘‘Acuity Echo’’
Ultrasound Gel ‘‘Aquasonic CLEAR’’
Ultrasound Gel ‘‘Ultraschallgel’’
Ultrasound System ‘‘Zonare ZS 3’’
GraphPad Software, La Jolla, CA, USA https://www.graphpad.com
iThera Medical GmbH, Munich, Germany https://ithera-medical.com
iThera Medical GmbH, Munich, Germany https://ithera-medical.com
Parker Laboratories Inc., Fairfield, NJ, USA https://www.parkerlabs.com
Medimex GmbH, Limburg, Germany https://www.medimex.de
Zonare Medical Systems Inc., Mountain View, CA, USA https://www.mindray.com

RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources can be directed to the lead contact,
Adrian P. Regensburger (adrian.regensburger@uk-erlangen.de).

Materials availability
This study did not generate new unique reagents.

Data and code availability

This paper does not report original code. The participants data reported in this study
cannot be deposited in a public repository due to participant confidentiality and
privacy concerns. Therefore, data are available upon written request. Any additional
information required to reanalyze the data reported in this paper is available from
the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

This prospective proof-of-concept study aimed to compare the optoacoustic signal
of muscle tissue between pediatric participants with spinal muscular atrophy (SMA)
and healthy volunteers (HV). The participants were enrolled between November 7th,
2019 and January 30th, 2020. The study adhered to ethical standards, including
approval from the local institutional review board (Identifier 168_19B) and the Decla-
ration of Helsinki. It was registered online (clinicaltrials.gov: NCT04115475), where
the full study protocol can be accessed. Informed consent was obtained from the
legal guardians of pediatric participants. The study population has been previously
reported for two-dimensional optoacoustic imaging and ultrasound imaging.15,16
The presented three-dimensional data is entirely distinct and has undergone inde-
pendent analysis.

Eligibility criteria included a genetically confirmed diagnosis for SMA patients and
matching age and sex for HV. Exclusion criteria included pregnancy or a tattoo on
the skin in the region to be examined for all participants, as well as anamnestic or
clinical signs of myopathy for HV. Participants were recruited at the Department of
Pediatrics and Adolescent Medicine of the University Hospital Erlangen. Information
on gender and socioeconomic status was not collected. The pilot nature of the study
prevented the derivation of a sample size prior to enrollment. The primary outcome
measure was comparing the spectral profile of muscle tissue of SMA patients and HV
determined by three-dimensional OAI.

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METHOD DETAILS
Study flow
Clinical assessment was performed using standardized scores as described previ-
ously,15,16 briefly Hammersmith Infant Neurological Examination (HINE), scale
0-26, Hammersmith Functional Motor Scale Expanded (HFMSE), scale 0-66, The
Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP
Intend), scale 0-64, Revised Upper Limb Module (RULM), scale 0-37 or 6-Minute
Walking Test (6-MWT). During clinical assessment, clinical information and the diag-
nosis was available to the assessor, while OAI data was not available. Three-dimen-
sional OAI was performed on muscle tissue of the proximal and distal arm and the
proximal and distal leg in four defined sections. The proximal arm was measured
at the biceps muscle, at two-thirds between the acromion and the cubital fossa.
The distal arm was measured at the forearm flexors, at one-third between the medial
epicondyle and the thumb base. The proximal leg was measured at the quadriceps
muscle, at one-half between the inguinal ligament and the tip of the patella. The
distal leg was measured at the gastrocnemius muscle, at one-third between the
popliteal fossa and the medial malleolus. Three-dimensional OAI was performed
at these four anatomical regions, at the left and right body half and twice per imag-
ing site, resulting in sixteen scans per patient. Clinical assessment and OAI were
performed during a single visit for all patients.

Three-dimensional optoacoustic imaging

Three-dimensional OAI was performed with the multispectral optoacoustic tomog-
raphy system Acuity-Echo (iThera Medical GmbH). The imaging probe for three-
dimensional OAI utilized a hemispherical detector array with 8 MHz center fre-
quency and 100 mm spatial resolution, opposed to an arc-shaped detector with
4 MHz center frequency and 150 mm spatial resolution used in two-dimensional
OAI. Transparent ultrasound gel (‘‘Ultraschallgel’’ Medimex GmbH, Limburg, Ger-
many, or ‘‘AQUASONIC clear’’, Parker Laboratories Inc., Fairfield, NJ, USA) was
used as a coupling medium.

Two-dimensional optoacoustic imaging

Three-dimensional OAI was performed with the multispectral optoacoustic tomog-
raphy system Acuity-Echo (iThera Medical GmbH), as described previously.15 Mean
800 nm signals were used for multimodal comparison.

Ultrasound imaging
Ultrasound imaging was performed on the respective regions with a high-end ultra-
sound system (Mindray, Zonare ZS 3, Zonare Medical System Inc, Mountain View,
CA; Linear probe L14-5w, 12 MHz), as described previously.16 Mean greyscale levels
were used for multimodal comparison.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data analysis
The data analysis process involved the sequential steps of reconstruction, visualiza-
tion and quantification of OAI signals in the regions of interest. Every step was
executed using ViewMSOT software (Version 4.0.3.2, iThera Medical GmbH, Mu-
nich, Germany). All participant data was pseudonymized beforehand and clinical in-
formation or reference test results of participants were not available to the reader of
the OAI data. While two-dimensional OAI produced cross-sectional views, three-
dimensional OAI produced volumetric OAI data, which was quantified by placing
three-dimensional cylindrical regions of interest into the volumetric scans. The
cylinders were standardized with a diameter of 12 millimeters and a height of

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4 millimeters. The cylinders were placed in the center of volumetric OAI scans
regarding the x-y-plane and adjusted in position on the z-axis, orthogonal to the
skin surface, to account for different distances of muscle tissue to the transducer.
Muscle tissue has been identified in OAI data by the 800 nm signal, which was scored
as muscle tissue when it appeared in the depth of tissue after an area without 800 nm
signal, which represented the subcutaneous fat (Figure S1). The data was quantified
as mean pixel intensity in the region of interest and given in arbitrary units (au) multi-
plied by 103.

Statistical analysis
Data was tested for normal distribution using the Shapiro-Wilk test. Inferential
testing was performed using a paired t-test if data was normally distributed. Inferen-
tial testing was performed using the Wilcoxon matched-pairs signed-rank test if data
was not normally distributed. Biological samples have been calculated as means
from two scans per imaging site at the left and right body half. One sample is derived
from four scans, respectively. Due to the heterogeneity of the disease, samples have
been used at the level of muscle groups, not as means per patient. Samples were
analyzed as paired with the respective control. Continuous variables are given as
means and standard deviations. Categorical variables are shown as numbers and
percentages. Correlations are given as Spearmen (rs). All analyses were performed
using GraphPad Prism (Version 9.5.1, GraphPad Software, La Jolla, CA, USA).
P-values < 0.05 were used to indicate statistical significance.

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