Non-invasive photoacoustic computed tomography of rat heart anatomy and function

Lin et al.

Light: Science & Applications

(2023) 12:12

fi

cial journal of the CIOMP 2047-7538

https://doi.org/10.1038/s41377-022-01053-7

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ARTICLE

Open Access

Non-invasive photoacoustic computed

tomography of rat heart anatomy and function

Li Lin

1,4,5

, Xin Tong

, Susana Cavallero

,YideZhang

,ShuaiNa

,RuiCao

,TzungK.Hsiai

2,3

✉

and

Lihong V. Wang

✉

Abstract

Complementary to mainstream cardiac imaging modalities for preclinical research, photoacoustic computed

tomography (PACT) can provide functional optical contrast with high imaging speed and resolution. However, PACT

has not been demonstrated to reveal the dynamics of whole cardiac anatomy or vascular system without surgical

procedure (thoracotomy) for tissue penetration. Here, we achieved non-invasive imaging of rat hearts using the

recently developed three-dimensional PACT (3D-PACT) platform, demonstrating the regulated illumination and

detection schemes to reduce the effects of optical attenuation and acoustic distortion through the chest wall; thereby,

enabling unimpeded visualization of the cardiac anatomy and intracardiac hemodynamics following rapidly scanning

the heart within 10 s. We further applied 3D-PACT to reveal distinct cardiac structural and functional changes among

the healthy, hypertensive, and obese rats, with optical contrast to uncover differences in cardiac chamber size, wall

thickness, and hemodynamics. Accordingly, 3D-PACT provides high imaging speed and nonionizing penetration to

capture the whole heart for diagnosing the animal models, holding promises for clinical translation to cardiac imaging

of human neonates.

Introduction

Cardiovascular disease remains the leading cause of

morbidity, responsible for 16% of the world

’

s total

deaths

. Diagnostic imaging and therapeutic targets war-

rant preclinical investigations of animal models

understand the cardiac disease mechanisms. Over the last

several decades, non-invasive imaging of small animal

models provides in vivo insights into the structural and

functional phenotypes with physiological and clinical

relevance. The current non-invasive imaging modalities

allow from a wide range of vertebrate animal models,

including light-sheet

fl

uorescent microscopy

, echo-

cardiography

, magnetic resonance imaging (MRI)

, x-ray

computed tomography (CT)

, positron emission tomo-

graphy (PET)

, and/or single-photon emission computed

tomography (SPECT)

, and a combination of these ima-

ging techniques

–

. Each imaging technique provides

the unique degree of tissue penetration, resolution, and

contrast to image the speci

fi

c animal models from zeb-

fi

sh to mouse to swine models, and the combination of

these complementary techniques allows for addressing

the spatial and temporal resolution,

fi

eld of view (FOV),

and relative phenotypes in response to the particular

imaging needs. For example, echocardiography is a por-

table tool to assess cardiac function by interrogating the

contracting/relaxing heart chambers and open/closure of

the valves in real-time. In contrast, CT and MRI are bulky

but offer larger FOV and/or

fi

ner spatial resolution nee-

ded for cardiac anatomy and vascular system

Complementary to the well-established imaging mod-

alities, photoacoustic (PA) computed tomography

(PACT) is an emerging imaging technique that combines

functional optical contrast from light illumination and

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Correspondence: Tzung K. Hsiai (

THsiai@mednet.ucla.edu

)or

Lihong V. Wang (

LVW@caltech.edu

)

Caltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department

of Medical Engineering, Department of Electrical Engineering, California

Institute of Technology, Pasadena, CA, USA

Department of Bioengineering, UCLA, Los Angeles, CA, USA

Full list of author information is available at the end of the article

These authors contributed equally: Li Lin, Xin Tong

1234567890():,;

high spatial resolution from acoustic detection. Compared

with echocardiography, PACT offers rich optical contrasts

that are physiologically relevant

and suffers a lower level

of speckling artifacts

. As compared with MRI, PACT

usually provides a higher imaging speed and a more

portable imaging platform. Unlike X-ray CT, PACT uses

nonionizing illumination to image cardiac vasculature,

avoiding injection of contrast agents. Like PET, PACT can

reveal molecular information

with

fi

ner spatial resolu-

tion obviating the need for ionizing radiation.

Despite these unique advantages, the majority of the

published PACT prototypes have not been demonstrated

for non-invasive cardiac imaging of the detailed anato-

mical or functional phenotypes due to suboptimal illu-

mination and detection schemes. This limitation has been

recognized by the previous PACT studies, including

in vivo imaging of mouse hearts with suboptimal clarity

for anatomical/functional details

and ex vivo imaging

of excised/perfused heart with no intracardiac

fl

dynamics

. There are 3 challenges for in vivo cardiac

PACT. (1) Ribs and lungs surrounding the heart partially

block and disturb PA signals. The same limitation exists

in echocardiography; however, PACT only suffers from

one-way acoustic disturbance since the PA signals are

generated in hearts. (2) The high concentration of

hemoglobin and myoglobin renders the myocardium

highly absorptive to light. This property may produce high

PA signal amplitudes; however, it reduces optical pene-

tration into the heart. (3) The periodic heartbeat requires

real-time imaging or motion-correction mechanisms to

give the imager

’

s spatial resolution full play for dynamic

cardiac structures. For example, the time-gating is widely

used in CT

, MRI

, and PET

, dividing every cardiac

cycle into multiple phases and collating all the data from a

speci

fi

c phase for imaging reconstruction.

In this context, we seek to overcome these challenges

and enhance PACT for pre-clinical and physiological

applications. Here, we modi

fi

ed a recently developed

three-dimensional PACT (3D-PACT) platform

with

regulated illumination and detection schemes to image

the whole rat heart in vivo. This 3D approach allows for a

hemispherical detection mesh with an extensive view

aperture, detecting PA signals from the heart semi-

panoramically to alleviate the acoustic blockage caused by

ribs and lungs. In addition, the 1064-nm light suffers less

scattering in the biological tissue

than the wavelengths

that are commonly used by PACT within the near-

infrared window (680

–

950 nm). During the scanning, we

synchronized the PACT measurement with cardiac cycles

by gating with the electrocardiogram (ECG). The 3D-

PACT scanned the rat heart in 10 s and reconstructed a

series of cardiac images based on a time-gating strategy.

The 3D-PACT along with the ECG-aided synchroniza-

tion largely resolves the challenges from the previous

PACT approaches and reveals the whole rat heart

anatomy non-invasively. Speci

fi

cally, the 3D-PACT sig-

fi

cantly enhances the image clarity and captures the

dynamic changes in the cardiac structure between obese

and control (i.e., lean) rats; namely, chamber size, myo-

cardial wall thickness, and intracardiac

fl

ow. Moreover,

the 3D-PACT is capable of recording the distinct blood

fl

ow in various sizes of the vascular system from healthy,

obese, and hypertensive rats, including the aortas, pul-

monary arteries, left and right coronary arteries. Notably,

the 3D-PACT shows the near-term potential to provide

cardiac image quality comparable to that of MRI

, deli-

vering optical contrast for cardiac structure and function,

and providing physiological parameters for blood

fl

ow.

Results

3D-PACT synchronization with ECG-gating for 4D (3D

space

+

time domain) non-invasive cardiac imaging

In the 3D-PACT platform (Fig.

a), we applied light

illumination with a pulse repetition rate of 50 Hz to the

subject through an optical window (Figs.

a and S1). Four

arc-shaped ultrasonic transducer arrays rotated coaxially

around the optical window for 90° to form a hemi-

spherical detection matrix (Fig. S2 and Methods section).

We covered the imaging aperture with a transparent

membrane to separate the imaging system from the

subject (rat), which was placed in a prone position over

the imaging aperture with chest close to the center

(Fig.

b and Methods section). During the 10-s imaging

session, the arrays were scanned for 500 steps (see

Methods section), and the heart was periodically beating

at about 5 Hz

. Due to the motion artifacts induced by

the heart beats, the reconstructed heart image was largely

blurred without motion correction (Fig. S3). Therefore,

we applied time-gated motion correction guided by the

synchronized ECG measurement (see Methods). For

example, to reconstruct the heart image when ventricles

are at maximum dilation (end of the ventricular diastole),

we only used the photoacoustic data acquired at the

scanning positions gated to the R waves of the ECG sig-

nals (Fig.

c). Thus, the 3D approach reveals the time-

dependent changes in cardiac structure, including the left

vs. right ventricle, free wall vs. septum, pulmonary artery

vs. aortic arch, and left vs. right carotid arteries.

3D-PACT interrogation of rat cardiac anatomy and vascular

system

To address acoustic distortion from the chest ribs and

lung air sacs that encompass the heart, we demonstrate a

large acoustic detection aperture to collect PA signals

from numerous view angles (Fig.

d). To further improve

the PACT reconstruction performance of cardiac anat-

omy, we synchronized the 3D-PACT with ECG, scanning

the heart with ~11 steps (i.e., 11 laser pulses) during each

Lin et al.

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(2023) 12:12

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Imaging aperture

Ultrasonic

array housing

Four arc-shaped

ultrasonic arrays

Optical window

0.5

Time (s)

-0.2

0.2

Voltage (mV)

Norm. PA amp.

Signal from skin

Signal from heart

1.76

solid angle

0.45

solid angle

0.22

solid angle

Normalized PA amplitude

5 mm

4×256-channel

DAQs

Nose cone

with tooth bar

Air bleeds

Mounting

post

Heating pad

Expanded

laser beam

Heart

Ultrasonic

array

1550

1590

1630

1670

1710

DAQ sampling points

Fig. 1 3D-PACT synchronized with ECG for cardiac imaging. a

Representative sketch of the 3D-PACT platform with a close-up view of the

imaging aperture.

Experimental setup for the rat heart imaging. The rat was mounted in a prone position, and the expanded light beam was

directed towards the rat

’

s chest from the bottom.

Example of the signal synchronization between the 3D-PACT (top) and ECG (bottom)

measurements. The 3D-PACT signal was collected from one transducer element on one array at multiple scanning positions (i.e., on the same

latitude). In the photoacoustic signal diagram, the horizontal axis represents the scanning steps (i.e., time), and the vertical axis stands for the

time-of-

fl

ight signal (data acquisition sampling at 20 MHz). The P, R, and T waves in the ECG signals correspond to atrial contraction, ventricular contraction,

and ventricular relaxation, respectively.

The rat heart images reconstructed with different ultrasonic apertures, showing the impact of a sizeable

ultrasonic detection aperture on image reconstruction. The red arcs on the top right corner of each image represent the cross-sectional view of the

ultrasonic detection aperture

Lin et al.

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cardiac cycle. Accordingly, we divided every cardiac cycle

T into 11 phases and reconstructed a volumetric image

for each phase (Fig.

a and Supplementary Movie 1).

Since the image of Phase 1 (i.e., Time 0) was recon-

structed using the data acquired at the R waves of the

ECG signals, the heart was close to the start of systole.

During the

T period, the heart was in systole

but started to relax from

T. Such a heartbeat pattern

complies with the Wiggers diagram. We further mapped

the

fl

uctuations of PA signals across the heart (Fig. S4) to

take in the heartbeat as dynamic contrast, showing more

spatial movements in the apex of the heart than in the top

regions.

To reveal the internal cardiac structure, we further

showed the cross-sectional images on the sagittal (Fig.

and Supplementary Movie 2) and coronal planes (Fig.

and Supplementary Movie 3). In the cross-sectional

images, we can identify the dynamic changes in atria

and ventricles during cardiac contraction (systole) and

relaxation (diastole). In Fig.

c, the end ventricular dia-

stolic volume was observed near Time 0, whereas the end

atrial diastolic volume was near

T. We further encoded

the imaging depth by colors and peeled away the

structures from the chest wall towards the posterior

region of the heart, showing cardiac structures at different

depths (Fig.

d). The imaging contrast of the cardiac

anatomy acquired by the 3D-PACT is from optical

absorption of myoglobin from the myocardium and

hemoglobin from the blood

3D-PACT interrogation of hypertrophic hearts in the obese

rats

It is widely recognized that overweight and obesity are

cardiometabolic risk factors for developing acute coronary

syndromes and stroke

. Obesity-associated increase in

cholesterol levels and vascular in

fl

ammation are recog-

nized to cause ventricular remodeling and to reduce blood

perfusion to the myocardium

. Here, we demonstrate to

capacity of 3D-PACT to interrogate obesity-induced

cardiac enlargement or known as hypertrophic abnorm-

alities in cardiac anatomy and function by non-invasively

imaging Zucker obese/lean rats of a similar age (Table S1).

Compared with the lean rats, whose cardiac cycles typi-

cally have 11 phases (11 phases/50 Hz

=

0.22 s, i.e., 4.55-Hz

cardiac rate), the three obese rats we imaged have 9

phases for each cardiac cycle (i.e., 5.55-Hz cardiac rate),

Time 0

Normalized PA amplitude

Depth from the chest wall

0 cm

5 mm

1.8 cm

5 mm

PuA

SVC

Aorta

ITV

z = 0 – 1.8 cm

z = 0.5 – 1.8 cm

z = 0.9 – 1.8 cm

Posterior

Fig. 2 Rat heart anatomy acquired by the 3D-PACT. a

Front view of the heart within a cardiac cycle. The heart is identi

fi

ed by a magenta circle at

T. BA, brachiocephalic artery; ITV, internal thoracic vessels; IV, intercostal vessels.

Cross-sectional images of the heart on the sagittal plane. Each

image is a maximum amplitude projection (MAP) of a slice marked by the green dashed lines in (

) (Time 0).

Cross-sectional images of the heart on

the coronal plane. Each image is an MAP of a slice marked by the yellow dashed lines in (

) (Time 0). LA, left atrium; LV, left ventricle; PuA, pulmonary

artery; RA, right atrium; RV, right ventricle; SVC, superior vena cava.

The same data (at Time 0) shown with color-encoded depths. Shallower

structures were peeled away in the lower images to show the posterior anatomy

Lin et al.

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indicating a higher cardiac rate for augmented cardiac

output to compensate for the increased metabolic

requirements

The 3D imaging capability enables the measurement of

cardiac anatomy and function. By sectioning the volu-

metric images on the coronal plane (Fig.

a, b), we

observed concentric hypertrophy

in the obese rat

’

s heart

with increased free wall thicknesses in both left and right

ventricles compared to the lean rat (Fig.

c). For thickness

measurement, we selected the middle slice of the heart on

the coronal plane and quanti

fi

ed the full-width-at-half-

maximum of the photoacoustic signals across the wall

section with a length of about 1.3 mm near the middle

(Fig. S5). Ex vivo measurements were performed by

splitting the dissected hearts on the coronal plane near the

middle after imaging, showing concordance with the

in vivo measurements (Fig. S6).

In addition, measurements of the changes in cardiac

volumes enabled us to assess both the systolic and dia-

stolic function. We divided each heart image into ten

sections with speci

fi

c thicknesses (1.04 mm) on the cor-

onal plane and segmented ventricular and atrial regions to

evaluate the volume variations (Fig. S5). To reduce the

individual difference within each group (

=

3), we nor-

malized the volumes of ventricles and atria to their values

at Phase 1 (at the end of diastole). The relative changes in

the cardiac function between the lean and obese rats

(Figs.

d and S7) were characterized in terms of the end-

systolic ventricular volume and end-diastolic atrial

volume of the cardiac cycle. Probably due to the reduced

strain of ventricles or well-recognized diastolic

dysfunction in the hypertrophic hearts

, we observed

slightly lower changes in the average of normalized left

ventricular volumes in obese rats. Similarly, the left atria

in the obese rats developed slightly lower variations in the

normalized volume.

3D-PACT interrogation of cardiovascular hemodynamics

Given PACT

’

s high sensitivity to the hematogenous

contrast, the 3D-approach enabled periodic hemody-

namic interrogation of the cardiovascular system. In a

cardiac image acquired by 3D-PACT (Fig.

a), the aorta,

pulmonary artery, right coronary artery, and left coronary

artery were captured. To measure the intravascular

hemodynamics, we selected a region of interest (ROI)

slightly larger than the vessels

’

cross-sections and aver-

aged the photoacoustic signal amplitude within a section

of 10-voxel length (1.3 mm) along the vessel. Since oxy-

hemoglobin dominates the optical absorption at 1064 nm

(i.e.,

μ

(oxyhemoglobin)

≈

10 ×

μ

(deoxyhemoglobin)

;

μ

absorption coef

fi

cient)

, the changes in spatially averaged

PA signals within the ROI mainly re

fl

ect the variations in

the amount of oxyhemoglobin, which is determined by

the blood volume (or vascular diameter) and oxygen

saturation (sO

The measurements of hemodynamics in the coronary

arteries introduce distinct myocardial perfusion among

healthy, hypertensive, and obese rats. The averaged PA

signals in the aorta and pulmonary artery, peaked during

ventricular systole (0.4

–

0.5 T), showed slightly higher

variations in hypertensive hearts (blue dashed lines in

Fig.

b) than in the control group, yet with insigni

fi

cant

Depth from the chest wall

0 cm

1.8 cm

5 mm

Left ventricle

Normalized volume

Right ventricle

Time in one cardiac cycle

Aorta

Lean

Obese

Left ventricle

Left atrium

0.6

2.2

<0.01

0.4

0.8

1.0

1.8

1.4

1.0

00.5TT

Lean

Wall thickness (mm)

Normalized volume

Obese

Lean

Obese

Fig. 3 Differences in cardiac anatomy and function between the Zucker obese and lean rats. a

Cardiac anatomy of a Zucker obese rat. Left

panel: Color-encoded depth-resolved image of the heart; middle panel: Cross-sectional image of the heart; Right: Longitudinal sectioning of the

corresponding hypertrophic heart. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.

Cardiac anatomy of a Zucker lean rat (control

group).

Free wall thickness measurements of the left ventricle (top:

< 0.01,

=

3) and right ventricle (bottom:

< 0.01,

=

3) between the obese

and lean rats.

Relative volume variations of the left ventricles and atriums between the obese and lean rats. The shadow behind curves represents

the standard deviation across multiple rats (

=

Lin et al.

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statistical differences. This observation could be sup-

ported by measuring relative volume changes of the left

ventricles (Fig.

c). In comparison, we noticed more sig-

fi

cant

fl

uctuations in the relative PA signal amplitudes

from the left coronary arteries in hypertensive hearts

within the cardiac cycle (Fig. S8). We speculate that the

abnormal hemodynamics are related to the accumulated

fat in the damaged arteries, which may affect blood

perfusion

Similarly, we measured and plotted the hemodynamics

of cardiac vessels in obese rats. Probably due to the aug-

mented cardiac output and obesity-induced arterial

hypertension

, we observed higher variations in the

averaged PA signals from the four vessels (red dashed

lines in Fig.

b). However, only the right coronary arteries

and aortas in the obese rats showed signi

fi

cant statistical

differences from the control group (Fig. S8). The com-

bined measurements in cardiac anatomy and hemody-

namics depict a potential to provide diagnosis of

cardiovascular diseases in individuals with hypertension

or obesity.

Discussion

For decades, the photoacoustic

fi

eld has faced chal-

lenges in cardiac imaging, including acoustic disturbance

from ribs and lungs, optical attenuation, and motion

artifacts from the periodic heartbeat. Nevertheless, pre-

clinical research and clinical practice are in need of

innovative imaging methods with complementary

advantages to provide high-dimensional physiological

information. In this study, we have demonstrated non-

invasive 3D-PACT of cardiac anatomy and function in

rats, providing substantial improvements in anatomical

image clarity and cardiovascular function measurement.

To reveal the cardiac anatomy, 3D-PACT employs a

hemispherical acoustic detection aperture with large view

angles to mitigate the in

fl

uences induced by ribs and

lungs; The 1064-nm light and uniform illumination also

facilitate deep penetration of the entire heart (Fig.

b);

although this study has not provided snapshot or real-

time imaging, the periodic heartbeat allows the ECG-

guided time-gating method to reduce motion-induced

artifacts during a 10-second scan. Clear anatomical ima-

ging further enables measurements of the cardiac func-

tions, including the chamber volume variations and

cardiovascular hemodynamics. For example, the changes

in photoacoustic signals from the cardiac vessels indicate

the variations in blood volume and sO

, showing differ-

ences in hemodynamics of the hypertensive, hypertrophic,

and normal control hearts. In summary, the optical ima-

ging contrast in 3D-PACT depicts the distribution of

myoglobin in the myocardium and hemoglobin in the

blood, revealing cardiac anatomy and function with phy-

siological relevance

While the study was performed based on the mod-

fi

cation of a recently published imaging system

, addi-

tional technical innovations are critical to producing clear

cardiac images: (1) The ECG-guided time-gating strategy

Norm. PA amp.

Aorta

Pulmonary

artery

RCA

LCA

Right coronary artery

Aorta

Pulmonary artery

Left coronary artery

0.5T

00.5TT

1.0

0.6

1.4

Norm. PA amp.

1.0

0.6

1.4

Norm. PA amp.

Control

Hypertensive

Obese

Time in one cardiac cycle

Norm. volume

Time in one cardiac cycle

1.0

0.4

0.6

0.8

0.5T

Fig. 4 Cardiac hemodynamics in control (i.e., healthy), hypertensive, and obese hearts. a

A heart image acquired by the 3D-PACT shows the

aorta, pulmonary artery, right coronary artery (RCA), and left coronary artery (LCA).

PA signal

fl

uctuations in the four cardiac vasculatures among

the control, hypertensive, and obese hearts (

=

3 for each group).

Relative changes in ventricular volume of the control (black solid line),

hypertensive (blue dashed line), and hypertrophic (red dashed line) hearts during a cardiac cycle (

=

3 for each group)

Lin et al.

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(2023) 12:12

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synchronized the 3D-PACT system with the ECG mea-

surement by sending a spike signal to one of the ECG

’

electrodes when the system starts to run (see Methods).

(2) Regulated laser illumination and gated spatial sam-

pling are synchronized to image the heart dynamics

during multiple cardiac cycles (see Methods), providing

an FOV large enough for whole-heart imaging in 3D

space

. (3) In PACT, the light illumination, acoustic

detection, and imaging object comprise a comprehensive

signal excitation and detection system. Therefore, animal

positioning that can reliably match the illumination and

detection is usually overlooked but critical for high-

quality imaging (see Methods section).

Based on the improvements in this study, PACT of the

heart can be further developed towards the following

directions: (1) Using ultrasonic arrays with a higher center

frequency (e.g., 5

–

10 MHz) could help reveal detailed

cardiac anatomy in small animals with a higher resolution.

However, the unwanted effects from acoustic attenuation

and rib disturbance will be more signi

fi

cant. (2) To

maintain the lateral FOV with an improved imaging speed,

rearrangement of the ultrasonic transducers (e.g., eight arc

arrays, each with 128 elements) would be implemented to

provide an equally dense azimuthal sampling with less

scanning steps. (3) The data ampli

fi

cation and acquisition

circuits can be modi

fi

ed to integrate ultrasonography,

offering well-interpreted imaging contrast (i.e., informa-

tion) in coregistered images for reference.

3D-PACT is an open imaging platform for preclinical

research and clinical translation by simultaneously pro-

viding cardiac anatomy and function. Compared with

established imaging modalities, PACT of the heart offers

additional physiology-related information with high ima-

ging speed and resolution, yet without the need for

ionizing radiation or invasiveness, making PACT a pro-

mising tool for cardiac imaging of human neonates.

Materials and methods

Optical illumination and acoustic detection

For optical illumination, PACT requires a relatively uni-

form energy deposition within the

fi

eld of view. While for

acoustic detection, high-quality imaging requires a large

view angle (i.e., synthetic aperture) and dense sampling in

time and space. In 3D-PACT, we directed a 1,064 nm laser

beam (DLS9050, Continuum; 50 Hz pulse repetition rate;

–

9 ns pulse width) to the optical window mounted on the

scanning axis. The optical window is composed of an

engineered diffuser (EDC-15, RPC Photonics Inc.) and a

condenser lens (ACL25416U-B, Thorlabs Inc.), expanding

the beam to a diameter of ~4.8 cm (Fig. S2). The illumi-

nation area is slightly larger than the imaging object (i.e.,

heart) to lower the requirement of precise animal posi-

tioning. Because the heart is

fi

lled with blood (i.e., high

optical absorption), we used 1064-nm light in this study for

less optical attenuation in biological tissues to achieve deep

penetration. The optical

fl

uenceonthetissuesurface

(~20 mJ/cm

at 50 Hz) was limited by the American

National Standards Institutes

’

safety standards

The acoustic detection module comprises four ultrasonic

arc arrays with a separation of 90 degrees. Each array

has 256 transducer elements with a central frequency of

2.25 MHz, and each element has a dimension of

0.6 × 0.7 mm

, generating a divergence angle (i.e., far-

fi

eld

acoustic diffraction) around 60 degrees covering the ima-

ging object semi-panoramically. Here, we applied a hemi-

spherical detection matrix to enlarge the view angle so the

system can acquire acoustic signals propagating in most

independent directions. In a

ddition, the 2.25-MHz center

frequencyofthearrayalsoallowsthedetectionofsuf

fi

ciently low frequency signals that suffer less distortion from

the ribs. Notably, this acoustic detection scheme would not

avoid distortions from bones and lungs, but would reduce

their effects on the reconstructed images. Connecting to the

ultrasonic arrays, 1024-channel pre-ampli

fi

cation and

analog-to-digital conversion circuits (PhotoSound, Inc.)

provide one-to-one mapped data acquisition

ECG-guided time gating and spatial sampling

The data acquisition modules and mechanical scanner

of the 3D-PACT system were triggered by the laser

’

“

Lamp Sync Out

”

trigger output. At the same time, the

computer controlled the trigger signal transmission via an

electronic relay in the system

’

s control box. We turned on

the laser and ECG systems shortly before closing the relay,

which coordinated the data acquisition, laser pulses, and

mechanical scanning. We further directed a coaxial cable

from the system

’

s control box to one of the ECG probes

(left or right arm) and output a spike signal much higher

than the ECG spikes when the relay was closed (Fig. S9).

Accordingly, we pinpointed the start time of the photo-

acoustic data acquisition in the ECG signal, achieving the

data synchronization between ECG and 3D-PACT.

We then grouped the PACT data into multiple heart-

beat phases based on the synchronized ECG cardiac cycle

and reconstructed a volumetric heart image for each

phase (Fig.

c). Since the heart rate was around 5 Hz and

the pulsed laser illuminated at 50 Hz, each cardiac cycle

can be divided into 9

–

11 phases, enabling the recon-

struction of 9

–

11 volumetric images to depict the beating

heart. The variations in cardiac cycle duration were

caused by differences in weight (e.g., obese versus regular

rats) and anesthesia (i.e., light versus deep anesthesia).

However, the ECG signal shows that the heart rate was

relatively stable during the 10-second scan.

For spatial sampling, each of the four arrays was scan-

ned for 500 steps (50 Hz × 10 s) during the 10-second

imaging, providing 2000 azimuthal sampling positions.

Accordingly, each heart image is reconstructed by ~200

Lin et al.

Light: Science & Applications

(2023) 12:12

Page 7 of 9

(2000/10 phases) azimuthal positions times 256 polar

positions (i.e., each arc array has 256 elements), which

could provide an effective FOV with a lateral diameter of

15 mm

and elevational radius of 38 mm.

Animal preparation

Four strains of rats were imaged in this study (Table S1):

(1) Hsd:Sprague Dawley SD rats (120

–

150 g body weight),

(2) Zucker obese rats (200

–

250 g), (3) Zucker lean rats

(120

–

150 g), (4) Spontaneously hypertensive inbred SHR/

NHsd rats (120

–

150 g). We

fi

rst imaged the SD rats to

optimize the experimental setup and demonstrate the

imaging capability (Fig.

). The Zucker lean rats were

imaged as the control group to compare the cardiac

anatomy and function with the obese rats (Fig.

). We

compared the cardiovascular hemodynamics in the

hypertensive, obese, and SD rats (Fig.

The imaging aperture was covered by a transparent

membrane (disposable plastic wrap) that separated the rat

from the imaging system and served as a support for the rat.

We then covered a feedback-controlled heating pad with a

zip bag for waterproo

fi

ngandplaceditabovethemem-

brane to maintain the animal

’

s body temperature (Fig.

b).

Before imaging, we removed the hair on the chest and

placed the rat above the heating pad. A 3D-printed nose

cone and tooth bar were positioned to provide air with 2%

vaporized iso

fl

urane. During imaging, we placed two cross

lines above the imaging aperture to mark the center posi-

tion of the FOV. We further bent the rat

’

sfrontlimbs

towards the back of the body so that the heart would be

prominent towards the chest wall. After the imaging session

was completed, we euthanized the rat, collected the heart,

and obtained a photo of the dissected heart for reference.

All the animal experiments followed the protocol approved

by the Institutional Animal Care and Use Committee

(IACUC) of the California Institute of Technology.

Image processing and visualization

We used the dual-speed-of-so

und back-projection algo-

rithm

implemented in C

++

to reconstruct all images

with mitigated artifacts induced by the acoustic inhomo-

geneity between the biological tissue and the coupling

fl

uid

O). Each volumetric image w

as reconstructed with a

voxel size of 0.13 × 0.13 × 0.13 mm

. We then processed

each image to improve the contrast through the following

steps: (1) A depth compensation (e

0.81×depth (cm)

)method

was applied to enhance the PA amplitude in deep tissue. (2)

We then used a high-pass

fi

lter to suppress the low-

frequency background. (3) The high-passed image was

further denoised using sparse 4D transform-domain colla-

borative

fi

ltering

. (4) To enhance the contrast of blood

vessels, a Hessian-based Frangi vesselness

fi

lter

was

applied to the denoised image. (5) Finally, we added the

vesselness-

fi

ltered images (self-norm

alized) with a weighting

factorof0.5backtothehigh-passedimages(witha

weighting factor of 0.5) and obtained the presented images.

The color-encoded images in Figs.

and

were acquired

using a specialized 3D visualization software package,

namely 3D PA Visualization Studio

Acknowledgements

This work was sponsored by the United States National Institutes of Health

(NIH) grants R35 CA220436 (Outstanding Investigator Award) and U01

NS099717 (BRAIN Initiative).

Author details

Caltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department

of Medical Engineering, Department of Electrical Engineering, California

Institute of Technology, Pasadena, CA, USA.

Department of Bioengineering,

UCLA, Los Angeles, CA, USA.

Division of Cardiology, Department of Medicine,

UCLA, Los Angeles, CA, USA.

Present address: College of Biomedical

Engineering and Instrument Science, Zhejiang University, Hangzhou, China.

Present address: The First Af

fi

liated Hospital, Zhejiang University School of

Medicine, Hangzhou, China

Author contributions

L.V.W. and L.L. conceived and designed the study. L.L. constructed the

hardware system. S.N. and L.L. developed the control program. Y.Z. and R.C.

modi

fi

ed the control program for ECG synchronization. L.L. and X.T. performed

the experiments. L.L., X.T., and Y.Z. analyzed the data. L.V.W., T.K.H., and S.C.

supervised the study. All authors wrote the manuscript.

Data availability

All data are available within the Article and Supplementary Files, or available

from the authors upon request.

Con

fl

ict of interest

All the authors declare no competing interests. L.V.W. has a

fi

nancial interest in

Microphotoacoustics, Inc., CalPACT, LLC, and Union Photoacoustic

Technologies, Ltd., which, however, did not support this work.

Supplementary information

The online version contains supplementary

material available at

https://doi.org/10.1038/s41377-022-01053-7

Received: 11 July 2022 Revised: 29 November 2022 Accepted: 1 December

2022

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