HDBME_9823268 1..17 - 9823268.pdf

Review Article

Recent Advances in Photoacoustic Tomography

Lei Li

and Lihong V. Wang

Caltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department of Medical Engineering, Department of

Electrical Engineering, California Institute of Technology, 1200 East California Boulevard, Mail Code 138-78, Pasadena,

CA 91125, USA

Correspondence should be addressed to Lihong V. Wang; lvw@caltech.edu

Received 31 December 2020; Accepted 29 April 2021; Published 16 June 2021

2021

Lei

and Lihong V. Wang. Exclusive Licensee Suzhou Institute of Biomedical Engineering and Technology,

CAS. Distributed under a Creative Commons Attribution License (CC BY 4.0).

Photoacoustic tomography (PAT) that integrates the molecular contrast of optical imaging with the high spatial resolution of

ultrasound imaging in deep tissue has widespread applications in basic biological science, preclinical research, and clinical trials.

Recently, tremendous progress has been made in PAT regarding technical innovations, preclinical applications, and clinical

translations. Here, we selectively review the recent progresses and advances in PAT, including the development of advanced

PAT systems for small-animal and human imaging, newly engineered optical probes for molecular imaging, broad-spectrum

PAT for label-free imaging of biological tissues, high-throughput snapshot photoacoustic topography, and integration of

machine learning for image reconstruction and processing. We envision that PAT will have further technical developments and

more impactful applications in biomedicine.

1. Introduction

Biomedical imaging has played a signi

cant role in modern

medicine for diagnosing diseases, monitoring therapy, and

providing biological insights into lives [1, 2]. Photoacoustic

tomography (PAT), also known as optoacoustic tomography,

is an emerging biomedical imaging modality that provides

cross-sectional or three-dimensional (3D) imaging of an

object based on the photoacoustic (PA) e

ect

—

a physical

phenomenon that converts absorbed light to sound [3].

Although discovery of the photoacoustic e

ect dates back

to 1880 [4], PAT has enjoyed a rapid growth since early

2000s following the development in ultrasonic detectors,

computations, and lasers [5]. Since 2010, PA imaging has

become one of the largest research areas in biophotonics

and still enjoys the rapid growth [6].

Figure 1 illustrates the principle of PAT. Typically, PAT

employs nonionizing laser pulses directed to the object for

excitation. When using microwave or radiofrequency pulses

for illumination, it is referred to as thermoacoustic tomogra-

phy (TAT) [7, 8]. The delivered photons are absorbed by the

molecules in the object, elevating the molecules from the

ground state to the excited state. Another photon or heat is

emitted, when the excited molecule relaxes to the ground

state. The absorbed photons can be converted to heat

through nonradiative relaxation [9]. Via thermoelastic

expansion, a pressure rise is induced by the heat [10]. The

pressure rise propagates (with a speed of

1500 m/s) inside

biological tissue as an acoustic wave, also mentioned as a

PA wave [11, 12]. By detecting the PA waves, we can form

images that map the optical absorption of the object. Based

on the image formation methods, PAT has two major imple-

mentations: PA microscopy (PAM) and PA computed

tomography (PACT) [6]. PAM forms images by raster scan-

ning the focus of light and sound across the object, while

PACT yields images by inverse reconstruct the detected sig-

nals induced by wide-

eld illumination.

PAT is a hybrid imaging method that harvests both opti-

cal and acoustic energies. Thus, PAT inherits the advantages

of both optical and ultrasound imaging, o

ering rich optical

contrast and high spatial resolution inside deep tissue [13].

PAT is directly sensitive to molecules

’

optical absorption

[14]. By exciting molecules at preferred wavelengths accord-

ing to the spectrum signatures, PAT achieves multicontrast

imaging of molecules based on their chemical compositions

[15]. Till now, PAT has spectroscopically imaged numerous

endogenous molecules, including oxy- and deoxy-hemoglo-

bin, oxy- and reduced myoglobin, melanin, cytochrome,

DNA/RNA, bilirubin, lipid, and water, which enables PAT

for anatomical, functional, metabolic, and histologic imaging

AAAS

BME Frontiers

Volume 2021, Article ID 9823268, 17 pages

https://doi.org/10.34133/2021/9823268

(Figure 2(a)) [16

–

27]. Thanks to the strong optical absorp-

tion of exogenous contrast agents, e.g., micro/nanoparti-

cles, organic dyes, and genetically encoded proteins, PAT

enjoys superb sensitivity in deep tissue and o

ers molecular

imaging (Figure 2(b)) [27

–

33]. Increasing the optical

uence

improves the detection sensitivity, as long as the temperature

increase is within the safety limit. To guarantee the safety,

typically, the laser exposure to the skin surface and eye is reg-

ulated by the American National Standards Institute (ANSI)

standard. Thanks to the PA e

ect, PAT detects ultrasonic

waves induced by excitation photons, including both ballistic

and scattered ones; thus, PAT achieves much deeper penetra-

tion than conventional optical microscopy relying on ballistic

photons. Moreover, acoustic waves are orders of magnitude

less scattered inside soft tissues; therefore, PAT provides far

better spatial resolution than pure optical imaging methods

in deep tissues (

2 mm) [34]. With a skin surface radiant

exposure of 21 mJ/cm

at 1064 nm, which is only one-

fth

of the ANSI limit (100 mJ/cm

), PAT demonstrated an imag-

ing thickness up to 7 cm

in vivo

with double-sided light illu-

mination [35]. Because of the acoustic detection, PAT

’

spatial resolution and penetration depth are scalable with

the detected acoustic frequency. The spatial resolution of

PAT improves as the acoustic central frequency and band-

width increase at the expense of penetration depth [36].

PAT has uniquely shown a capability of multiscale imaging

using a consistent optical contrast, ranging from organelles,

cells, and tissues to whole-body small animals and human

organs, as shown in Figure 2(c) [25, 37

–

39]. Previously,

preclinical small-animal imaging and clinical applications

typically employ nonoptical imaging modalities, including

magnetic resonance imaging (MRI), X-ray computed tomog-

raphy (X-ray CT), positron emission tomography (PET),

single-photon emission computed tomography (SPECT),

and ultrasound imaging (USI), which all can provide deep

penetration [40]. However, those approaches still face

challenges. For example, MRI necessitates a long data-

acquisition time, not suitable for capturing fast dynamics

[41, 42]. PET and SPECT have a low spatial resolution to

resolve detailed structures. X-ray CT, PET, and SPECT

employ ionizing radiation, impeding longitudinal monitor-

ing [43]. Conventional USI does not reveal molecular con-

trasts outside blood vessels [44]. PAT, as a noninvasive

approach, achieves high-resolution imaging in deep tissues

with optical contrasts, providing a complementary approach

for preclinical research and clinical translations.

In recent years, PAT has an even more rapid develop-

ment, including technical innovations, various biomedical

applications, and clinical translations. In this paper, we selec-

tively review some of the recent progresses and advances in

PAT, including small-animal whole-body imaging, novel

molecular imaging, rapid assessments of brain functions,

broad-spectrum imaging of tissues, human organ imaging,

snapshot photoacoustic topography, and integration of

machine learning for advanced image reconstruction. We

envision that PAT will have more impactful applications in

fundamental science, preclinical research, and clinical trials.

2. Whole-body PACT of Small Animals

Small-animal whole-body imaging plays an indispensable

role in preclinical study [56]. With optical contrasts and high

spatiotemporal resolution, small-animal imaging can provide

physiological understandings of biological processes and

dynamics, advancing fundamental biology, preclinical study,

and clinical translation [57]. A recent development in PACT

for small-animal whole-body imaging, termed single-

impulse panoramic PACT (SIP-PACT), permits anatomical,

functional, and molecular whole-body imaging with excep-

tional quality and speed [58]. The schematic of SIP-PACT

Optical absorption

High

Low

Tissue

Chromophores

Temperature rise

Pressure rise

and propagation

Image reconstruction

Ultrasonic

transducers

Pressure

waves

Laser beam

Excited

state

Ground

state

Heat

Non-radiative

relaxation

Diffused

photons

Temperature rise

PA amplitude

High

Low

High

Low

Figure

1: The principle of PAT.

BME Frontiers

is shown in Figure 3(a). SIP-PACT employed full-ring ultra-

sonic detection (512 elements, 5 MHz central frequency,

over 90% one-way bandwidth) with parallel ampli

cation

and digitization, maximizing the detection signal-to-noise

ratio (SNR) and speed. The light illumination and acoustic

detection are aligned confocally to optimize the detection

sensitivity (Figure 3(b)). It takes 50

μ

s for SIP-PACT to

acquire a 2-dimensional (2D) image of a cross-section

in vivo

. The in-plane panoramic acoustic detection of SIP-

PACT o

ered an isotropic resolution (

125

μ

m) within the

whole cross-section and full-view

delity. Moreover, to bet-

ter reveal detailed structures of internal organs, a half-time

dual-speed-of-sound universal back-projection algorithm

was developed to account for the acoustic inhomogeneity

between the animal tissue and the surrounding coupling

medium (water). Representative small-animal whole-body

images, acquired at 1064 nm, from SIP-PACT are shown in

Figures 3(c)

–

3(f) [58].

Recently, a hemispherical transducer array-based PACT

system (Figure 3(g)) has been built to image the whole body

Whole-body small

animals/human (PACT)

Spatial resolution (m)

–3

–5

–6

–4

–7

Penetration limit (m)

–2

–1

–3

–4

–5

Organelles

(PA nanoscopy)

Single cells

(optical-resolution PAM)

Tissues

(acoustic-resolution

PAM)

(a)

(b)

(c)

–13

–4

–3

–2

–1

iRFP

GNC

SWNT

GNB

GNR

mCherry

IRDye800

Microbubble

Melanin

HbR

HbO

DNA

RNA

Bilirubin

Water

MbO

Lipid

MbR

EGFP

RFP

ICG

–12

–11

–10

Noise equivalent molar concentration (M)

Molar extinction coefficient (cm

–1

/M)

Absorption coefficient (cm

–1

)

Wavelength (nm)

200

400

600

1000800

1200

–9

–8

–7

–6

–5

–4

–3

Figure

2: Multicontrast and multiscale PAT. (a) Absorption spectra of endogenous molecules at normal concentrations

in vivo

[27].

Bilirubin: 12 mg L

-1

in blood; DNA/RNA: 1 g L

-1

in cell nuclei; HbO

: oxyhemoglobin; HbR: deoxyhemoglobin, 2.3 mM in blood; MbO

oxymyoglobin; MbR: reduced myoglobin, 0.5% mass concentration in skeletal muscle; melanin: 14.3 g L

-1

in the skin; lipid: 20% volume

concentration in tissue; water: 80% volume concentration in tissue. (b) Noise equivalent molar concentrations of some widely used

exogenous contrast agents, based on reported values from the literature [27]. Illumination

uence is not compensated. EB: evens blue [45];

EGFP: enhanced green

uorescent protein [46]; GNB: gold nanobeacon [47]; GNC: gold nanocage [48]; GNR: gold nanorod [49]; ICG:

indocyanine green [50]; IRDye800: near-infrared Dye800 [51]; iRFP: near-infrared red

uorescent protein [52]; MB: methylene blue [53];

mCherry: monomeric cherry protein [46]; microbubble [54]; RFP: red

uorescent protein [52]; SWNT: single-walled nanotube [55]. The

dashed curve is power function

tting

:

−

, where

is the noise equivalent concentration in molars and

the molar extinction

coe

ffi

cient in cm

-1

. (c) Multiscale PAT and representative images. Organelles and PA nanoscopy of a single mitochondrion (scale bar,

500 nm) [37]. Single cells, optical-resolution PAM of red blood cells (scale bar, 20

μ

m) [38]. Tissues, acoustic-resolution PAM of human

skin (scale bar, 500

μ

m) [25]. Whole-body small animals and whole-body PACT of a nude mouse

in vivo

(scale bar, 4 mm) [39].

BME Frontiers

of small animals [59]. By spiral scanning the hemispherical

transducer array (256 elements, 4 MHz central frequency,

100% one-way bandwidth) around the animal, volumetric

whole-body imaging can be achieved (Figure 3(g), the

whole-body tissue is shown in gray; illumination wavelength,

800 nm). By parking the array at a given position and acquir-

ing data continuously, high-speed imaging of the dynamics

inside an organ can be realized. For example, the high imag-

ing speed reveals cardiac dynamics

in vivo

in detail, as shown

in Figure 3(h) (the beating heart is shown in color) [59].

The recent progress in small-animal PACT promises

more extensive preclinical studies, particularly when high

spatiotemporal resolution and high sensitivity are necessary.

For instance, tracking immune cells can visualize immune

responses during cancer progression and treatment, advanc-

ing our understanding of immune system dynamics. Overall,

the development of small-animal PACT enables real-time

imaging of biological processes in basic life science research

and ultimately in clinical applications.

3. Molecular PACT

Because endogenous contrast agents often lack speci

city or

sensitivity, scientists usually rely on exogenous contrast

Mirror

Prism

Diffuser

USTA

DAQ

Computer

Pre-amp

MBS

Ti-Sa laser

1064 nm laser

Illumination beam

Acoustic focus

30 mm

Prism

Diffuser

Membrane

(a)

(b)

(c)

(d)

(e)

(f)

(h)

(g)

= 110 ms

= 90 ms

= 0

= 30 ms

DAQ

Laser

Anesthesia

휙

Trigger

Breathing

mask

Water

tank

Counter

balance

Mouse

Focal point

Spherical array

Figure

3: Whole-body PACT of small animals [58, 59]. (a) Schematic of the SIP-PACT system for trunk and brain (blue dashed boxed inset)

imaging [58]. Dual-wavelength illumination is used. BC: beam combiner; CL: conical lens; DAQ: data acquisition system; MBS: magnetic base

scanner; OC: optical condenser; USTA: (full-ring) ultrasonic transducer array; WT: water tank. (b) Close-up of the green dashed line in (a),

showing the confocal design of light illumination and acoustic detection. (c

–

f) Representative cross-sectional images of the brain (c), the liver

(d), the upper abdominal cavity (e), and the lower abdominal cavity (f) in a live mouse, acquired by SIP-PACT [58]. Scale bar: 5 mm. (g)

Layout of the spiral scanning PACT system for small-animal whole-body imaging [59]. DAQ: data acquisition unit. (h) Representative 3D

whole-body images of a live mouse. Each image overlays the beating mouse heart (color) onto a whole-body anatomical image (gray) of

the same mouse [59]. Scale bar: 5 mm.

BME Frontiers

agents that can visualize biological phenomena with high

sensitivity and speci

city to study biological activities.

Exogenous contrast agents for deep-penetrating PAT hold

two key advantages: (1) they are optimized for high optical

absorption at near-infrared (NIR) wavelengths for high

detection sensitivity inside deep tissues; (2) they are specif-

ically designed to conjugate with targeting moieties to

selectively bind to receptors to achieve high speci

city.

Exogenous contrast agents, such as organic dyes [60

–

62],

genetically encoded proteins [63

–

66], and micro/nanoparti-

cles [67

–

71], have been extensively explored for PA molecular

imaging. With the innovations and advances in exogenous

contrast agents, molecular PAT has recently enjoyed a pros-

perous development (Figure 4).

A tyrosinase-based reporter that triggers human cells to

express eumelanin has been developed to provide a strong

PA contrast [64]. Combining the tyrosinase-based reporter

and a Fabry-Perot-based ultrasound sensor (22 MHz band-

width), PACT has imaged transgenic cancer cells expressing

eumelanin (Figure 4(a), illumination wavelength: 600 nm)

and monitored tumor development

in vivo

[64]. An

in vivo

pH mapping technology, consisting of a photoacoustic pH

indicator, SNARF-5F, has been developed [72]. Facilitated

by multiwavelength PACT, the pH levels inside a tumor have

been quantitatively measured. Here, a linear transducer

array-based PACT system (128 elements, 11.25 MHz central

frequency, and 75% one-way bandwidth) was used. The

SNARF-5F-based nanoparticles were injected via the tail vein

Spleen

Intestine

Left kidney

Right kidney

Spinal cord

Norm. decay constants

0.01

0.85

Norm. lock-in amplitude

PA amplitude

Max

5.5

Depth (mm)

(a)

(b)

(c)

(d)

(e)

(f)

5.2

Max

Min

PA amplitude

Norm. PA amplitude

Figure

4: Representative images of molecular PACT. (a)

In vivo

PA images of Tyr-expressing K562 cells after subcutaneous injection into the

ank of a nude mouse (vasculature is color-coded for depth; K562 cells are false-colored yellow). Scale bar, 1 mm [64]. (b) Quantitative PACT

of pH

in vivo

. Functional PA image in pseudo-color is superimposed on the gray-scale ultrasound image. Scale bar, 2 mm [72]. (c)

In vivo

image of a 4T1 tumor-bearing mouse, given a single injection of 150

μ

g of OMV

Mel

via the tail vein. The image was acquired at 3 h

postinjection, showing the accumulation of OMV

Mel

in tumor tissue, where the OMV

Mel

is in color and the background tissue is in gray

[73]. (d)

In vivo

multicontrast PACT of two types of tumor cells in the liver. Two types of tumors expressing di

erent photoswitchable

proteins are separated by their decay characteristics. The tumors are shown in color, and the background tissues are shown in gray.

Norm.: normalized [74]. (e) PA image of a hydrodynamic-transfected liver. The photoswitching signals are shown in color, con

rming the

existence of reconstituted DrSplit induced by protein-protein interactions. The background tissues are shown in gray [74]. (f) PA image of

the microrobots in the intestines

in vivo

. The migrating microrobots are shown in color, and the mouse tissues are shown in gray. The

yellow arrows indicate the direction of migration [76].

BME Frontiers

into a tumor-bearing mouse; multiwavelength PACT of the

mouse at 75 min postinjection has mapped the pH distribu-

tion inside the tumor

in vivo

(Figure 4(b), illumination wave-

lengths: 565 nm and 600 nm) [72]. Bacterial outer membrane

vesicles (OMVs) have been explored for delivering drugs,

vaccines, and immunotherapy agents. Recently, bioengi-

neered OMVs for contrast-enhanced PA imaging have been

investigated [73]. OMVs encapsulating biopolymer-melanin

(OMV

Mel

) were produced using a tyrosinase-expressing bac-

terial strain, which provides strong optical absorption at NIR

wavelengths. Here, an arc-shaped transducer array-based

PACT (270

in-plane detection angle, 256 elements, 5 MHz

central frequency, and 90% one-way bandwidth) was used.

Using multiwavelength PACT, tumor-associated OMV

Mel

distribution has been monitored

in vivo

(Figure 4(c), illumi-

nation wavelengths: 680

–

900 nm and 10 nm interval) [73].

Genetically encoded photoswitchable proteins (RpBphP1,

DrBphP-PCM, etc.) that e

ectively improve the PA imaging

sensitivity and speci

city have been reported recently [31,

32, 63, 65, 66, 74, 75]. Here, a 512-element full-ring transducer

array-based PACT system (5MHz central frequency, 90%

one-way bandwidth) was used. By analyzing the unique decay

characteristics of di

erent photoswitchable proteins, quan-

titative multicontrast imaging has been achieved to di

er-

entiate multiple types of tumors in deep tissue

in vivo

(Figure 4(d), illumination wavelength: 780 nm) [65, 74].

In addition, a split version of DrBphP-PCM has been

engineered, providing the

rst bimolecular PA comple-

mentation reporter to detect protein-protein interactions

in deep tissue

in vivo

(Figure 4(e)) [74].

Substantial progress in the development of micro/-

nanorobots has been accomplished for biomedical applica-

tions in recent years. However, present micro/nanorobot

platforms are ine

ective for imaging and motion control in

deep tissue

in vivo

. Recently, a PACT-navigated microrobotic

system that achieved controlled propulsion and prolonged

cargo retention

in vivo

was reported, where a full-ring

transducer array (512 elements, 5 MHz central frequency,

90% one-way bandwidth) was used for acoustic detection

[76, 77]. Thanks to the molecular contrast and high spatio-

temporal resolution at depths, PACT can locate and navigate

the microrobots

in vivo

. As shown in Figure 3(f) (illumina-

tion wavelength: 750 nm), PACT visualizes the migration of

microrobots in the intestines in real time

in vivo

[76]. The

integration of PACT and the newly engineered microrobotic

system allows deep tissue imaging and accurate control of the

microrobots

in vivo

and paves a new path for precision

medicine.

4. PAT of the Brain

Studying how the brain works will directly bene

t basic sci-

ence and help us to better understand and treat neurological

disorders, such as Alzheimer

’

s and Parkinson

’

s diseases [78].

Thanks to optical contrast and deep penetration, PAT pro-

vides a powerful tool for multiscale functional brain imaging.

Based on the endogenous molecules, including hemoglobin,

lipid, and cytochrome, label-free PAT has resolved whole-

brain vasculature and structures. Taking advantage of the

high resolution and high sensitivity, PAM has mapped corti-

cal vasculature and quanti

ed oxygen saturation of hemoglo-

bin (sO

) at the single capillary level with the skull intact

(Figure 5(a), illumination wavelength: 532 nm) [79]. After

mitigating the blood signals from the brain, label-free PACT

has imaged detailed internal brain structures with MRI image

quality. Based on the lipid and cytochrome contrast, a full-

ring transducer array-based PACT (512 elements, 5 MHz

central frequency, and 90% one-way bandwidth) identi

brain structures, including neocortex, corpus callosum, hip-

pocampus, inferior colliculus, and cerebellum (Figure 5(b),

illumination wavelength: 600 nm) [80]. Employing NIR light

illumination, a hemispherical transducer array-based PACT

(512 elements, 140

angular tomographic coverage, 5 MHz

central frequency, and 100% one-way bandwidth) has

imaged a mouse brain with structural details revealed in 3D

ex vivo

(Figure 5(c), illumination wavelength: 740 nm) [81].

A full-ring transducer array-based PACT (512 elements,

5 MHz central frequency, and 90% one-way bandwidth) has

also monitored the whole rat brain resting-state hemody-

namics and mapped the whole-brain functional connectivity

(Figure 5(d), illumination wavelength: 1064 nm) [58].

The deep penetration and high spatiotemporal resolution

enable PACT to monitor large-scale neural activities in detail.

As a demonstration, a hemispherical transducer array-based

PACT (512 elements, 5 MHz central frequency, and 100%

one-way bandwidth) has imaged rapid calcium responses to

electrical stimulations in a GCaMP6s expressing mouse brain

in vivo

(Figure 5(e), illumination wavelength: 488 nm) [82].

In addition, a linear transducer array-based PACT (256 ele-

ments, 21 MHz central frequency, and 52% one-way band-

width) has also visualized the epileptic wave propagation

across the whole brain during a seizure (Figure 5(f), illumina-

tion wavelength: 1064 nm) [83]. With the capability to mon-

itor neuronal activities and hemodynamics across the whole

brain, PAT has displayed encouraging potentials in studying

various brain disorders and diseases, such as traumatic disor-

ders, brain cancer, stroke, Alzheimer

’

s disease, and seizures

of various etiologies.

5. Broad Spectrum PAM

Any molecule has a unique absorption spectrum and has a

uorescence quantum yield lower than 100%, providing

contrast for PAT [27]. By tuning the wavelength from

ultraviolet (UV) to mid-infrared (MIR), spectral PAT has

detected endogenous biological molecules, including cyto-

chromes, DNA/RNA, hemoglobin, myoglobin, melanin,

protein, lipids, and water [12, 19, 20, 84

–

88].

Breast-conserving surgery is aimed at excising all cancer

cells. However, no intraoperative device that can quickly

examine the lumpectomy specimen at a microscopic scale is

available. Thus, up to 60% of patients have to undertake

second surgeries to reach clear margins. PAM employing

UV illumination (UV-PAM) can speci

cally highlight cell

nuclei, yielding microscopic images with a similar contrast

as hematoxylin-labeled histological images [20]. A fast

UV-PAM system was recently developed to provide

high-resolution histology-like images of unprocessed and

BME Frontiers

unlabeled breast tissues [88]. Figure 6(a) shows UV-PAM

images of a

xed, unprocessed breast tumor specimen with

eld of view (FOV) of

10 mm × 4

:

2mm

(illumination

wavelength, 266 nm). The close-up images (Figures 6(b)

and 6(c)), corresponding to the red and yellow dash boxed

regions in Figure 6(a), reveal detailed structures of the carci-

noma. As shown in Figure 6(d) (a zoomed-in image of the

magenta dash boxed region in Figure 6(a)), it is clear UV-

PAM can resolve individual cell nuclei. Label-free UV-PAM

with histology-like imaging capability has demonstrated its

potential as an intraoperative margin assessment tool for sur-

geons and pathologists to identify tumor margins [88].

Conventional optical microscopy is fundamentally limited

by a lack of chemical speci

city or by cellular phototoxicity;

Max

5 mm

V2MM

CA1

D3V

ZID

SNr-VTA

IFN

OA (a.u.)

훥

OA/OA (%)

Norm. PA

amplitude

0.9

0.6

1.0

Correlation

coefficient

2.4 mm

5.5 mm

9.7 mm

4 mm

1.0

Oxygen saturation

0.4

Max

Min

PA amplitude

0 ms

240 ms

480 ms

960 ms

25 s

45 s

35 s

65 s

2 mm

Norm. PA amplitude

Fractional change (%)

2 mm

(a)

(c)

(e)

(b)

(f)

(d)

2 mm

1 mm

Figure

5: Multiscale PAT of the brain. (a) PAM of oxygen saturation of hemoglobin in a mouse brain [79]. (b) A cross-sectional PACT image

of a saline-perfused mouse brain (horizontal plane) at 2.8 mm depth, showing internal structures of the brain clearly. Nc: neocortex; CC:

corpus callosum; Hp: hippocampus; Cb: cerebellum; IC: inferior colliculus [80]. (c) 3D PACT image of a mouse brain

ex vivo

Illumination wavelength: 740 nm; V2MM: secondary visual cortex, medio-medial; CA1: hippocampal CA1 area; DG: dentate gyrus; D3V:

dorsal third ventricle; ZID: zona incerta dorsal; SNr: substantia nigra reticulate; VTA: ventral tegmental area; IFN: interfascicular nucleus

[81]. (d) Functional mapping of the resting-state connectivity in a rat whole brain (coronal plane), showing a clear correlation between

corresponding regions across the left and right hemispheres [58]. (e) PACT of GCaMP6s responses to electrical stimulation of the right or

left hind paw. First from the left, maximum amplitude projection along the depth direction of the 3D images of a GCaMP6-expressing

mouse; second to last, relative increases in PA signal with respect to the baseline for a slice at

1 mm depth at di

erent time points

following the stimulation pulse for the GCaMP6s-expressing mouse [82]. (f) PACT images of epileptic activities during a seizure at

erent times. The fractional changes (color) in the PA amplitude are overlaid on the anatomical image (gray, Bregma:

−

1.0 mm). The

arrow indicates the injection site, and the dashed green arrows indicate the epileptic wave propagation direction [83].

BME Frontiers

thus, label-free optical imaging cannot directly reveal bio-

molecule dynamics in living cells [89, 90]. Recently, MIR-

PAM, based on chemically speci

c vibrational excitation by

MIR absorption, has been developed to provide label-free

bond-selective metabolic imaging in live cells [91]. MIR-

PAM enables spatiotemporal pro

ling of lipids, proteins,

and carbohydrates in cells and tissues. As a demonstration,

MIR-PAM monitored lipid and protein dynamics during

lipolysis in live cells (Figure 6(e), illumination wavelength:

3500 nm), proving its capability of imaging of biomolecular

dynamics in living cells without labeling [91].

However, the long MIR wavelength fundamentally limits

the spatial resolution of MIR-PAM due to optical di

raction

[91]. In addition, the high water content in fresh samples

severely reduces the imaging contrast due tot its strong

MIR absorption. A recent innovation that employs MIR PA

imaging localized with a pulsed UV illumination (266 nm)

successfully overcomes the above limitations. This technol-

ogy, termed ultraviolet-localized MIR PAM (ULM-PAM),

has achieved high-resolution MIR imaging of fresh samples

without water background [85]. ULM-PAM employed a

focused mid-MIR laser pulse to excite the sample. A confo-

cally aligned UV laser pulse photoacoustically detected the

MIR-induced temperature rise, thereby revealing MIR

absorption contrast. ULM-PAM

’

s lateral resolution, de

ned

by the UV wavelength, is over 10-fold higher than conven-

tional MIR microscopy. Moreover, most biomolecules in

living cells, including lipids, proteins, and nucleic acids,

have strong absorption of UV light (200

–

230 nm). Mean-

while, UV light is transmissive in water, which signi

cantly suppresses the water background in ULM-MIR.

Illuminating formalin-

xed 3T3 mouse

broblast cells at

wavelengths of 3420 nm and 6050 nm, respectively, ULM-

PAM mapped detailed distribution of lipids and proteins

in living cells (Figures 6(f) and 6(g)) [85]. As a comparison,

the MIR-PAM images of lipids (Figure 6(h)) and proteins

(Figure 6(i)) display a high water background and low spatial

resolution [85]. As a demonstration, ULM-PAM has imaged

neonatal (Figure 6(j)) and mature (Figure 6(k)) cells, show-

ing high-resolution, high-contrast imaging of lipids (blue),

proteins (green), and nucleic acids (red) [85]. Therefore,

ULM-PAM permits label-free imaging of biological samples

at high resolution with high contrast.

6. PACT of Human Breasts

Breast cancer is the number two cause of cancer death glob-

ally (11.6%), with a worrying mortality rate of 6.6% [92, 93].

Recently, substantial progress has been made for noninvasive

breast cancer diagnosis utilizing PAT. A technical advance,

1 mm

100

휇

100

휇

IDC

DCIS

Min 86

Min 0

ROI 1

ROI 2

Lipid

Iso

Min 257

Min 172

IDC

Tumor

Normal

DCIS

(a)

(b)

(f)

(h)

(e)

(i)

(k)

(g)

(j)

(c)

(d)

Mature

Neonatal

ULM

lipids

ULM

proteins

MIR

lipids

MIR

proteins

휇

Figure

6: Broad-spectrum PAM of tissues and cells. (a) PAM image of a

xed, unprocessed breast tumor. Illumination wavelength, 266 nm

[88]. (b

–

d) Zoomed-in PAM images of the red, yellow, and magenta dashed regions in (a), respectively. IDC: invasive ductal carcinoma;

DCIS: ductal carcinoma in situ; CN: cell nuclei [88]. (e) Monitoring intrinsic lipid contrast during lipolysis in di

erentiated 3T3-L1

adipocytes at 2,857 cm

−

. Two regions of interest (ROIs) enclosing individual adipocytes are marked: green dashed circle for ROI 1 and

red dashed circle for ROI 2. The white arrow follows the process of lipid droplet remodeling in a single adipocyte enclosed in ROI 1 [91].

(f, g) ULM-PAM images of lipids (f) and proteins (g) [85]. MIR-PAM images of lipids (h) and proteins (i), imaged at 3,420 nm and

6,050 nm, respectively [85]. Composite images of cells formed by overlaying the images of lipids (blue), proteins (green), and nucleic acids

(red) in di

erent color channels at neonatal (j) and mature (k) stages [85].

BME Frontiers

termed single-breath-hold PACT (SBH-PACT), can obtain a

3D image of a whole breast within a single breath hold (

15 s)

[94]. SBH-PACT employed a full-ring transducer array (512

elements, 2.25MHz central frequency, and 95% one-way

bandwidth) for acoustic detection and 1064nm light for exci-

tation. The high detection sensitivity of SBH-PACT allows

detecting breast tumors in detail, promising wide applications

in clinical breast care. By detecting local angiogenesis, SBH-

PACT can di

erentiate lesions from normal tissues. Highly

correlated with the tumor sites indicated in X-ray mammo-

grams (Figure 7(a)), SBH-PACT can identify a tumor by

revealing its higher blood vessel densities (Figure 7(b)) [94].

By further examining a tumor-containing slice (marked by

white dashed lines in Figure 7(b)), the same tumor, displaying

higher PA signals, can be visualized at the corresponding loca-

tion (Figure 7(c)) [94]. Furthermore, a vessel density map of

the breast was computed, where the breast tumor was

highlighted due to its high vessel density (Figure 7(d)) [94].

A PACT system with spiral scanning of a hemispherical

transducer array (512 elements, 2 MHz central frequency,

and 90% one-way bandwidth) was recently reported for

human breast imaging [95]. The dense spatial sampling and

the isotropic 3D resolutions yielded high-quality imaging of

a healthy breast (Figure 7(e), illumination wavelength:

795 nm). By illuminating the breast tissue at two wavelengths

of 756 nm and 797 nm, respectively, hemoglobin oxygen sat-

uration (sO

) was also evaluated using an approximate

parameter, termed S-factor, computed from measurements

obtained at the two wavelengths [96]. The S-factors can be

evaluated on neighboring vessels, such as adjacent arteries

and veins, assuming that the optical

uence in the neighbor-

ing region is the same. As shown in Figure 7(f), arteries and

veins in a healthy breast can be clearly distinguished in the

S-factor image [96]. Figure 7(g), a fusion of the ultrasound

(red color) and S-factor images, shows an example of the

results obtained from a breast cancer lesion. Notably, the

detailed vasculature surrounding the tumor is clearly visible.

In addition, the arterioles and venules show clustering [96].

Recent advances in PACT for breast imaging hold the

potential to complement X-ray mammography for breast

1 cm

20 mm

100

Depth (mm)

20 mm

Vessel density (mm

–2

)

0 cm

4 cm

Elevational distance from nipple

(b)

(e)

(f )

(g)

(d)

(c)

(a)

Norm.

PA a mp.

Norm. PA amp.

Figure

7: PACT of human breasts. (a) X-ray mammograms of an a

ected breast [94]. (b) Depth-encoded angiogram of the a

ected breast

acquired by SBH-PACT. The breast tumor is identi

ed by a white circle; the nipple is marked by a magenta circle [94]. (c) Maximum

amplitude projection (MAP) images of the thick slice in sagittal planes marked by white dashed lines in (b) [94]. (d) Automatic tumor

detection on vessel density maps. Tumors are identi

ed by green circles. Background images in gray scale are the MAP of vessels deeper

than the nipple [94]. (e) Depth-encoded 3D PACT image of a healthy breast [95]. (f) Evaluation of the S-factor in a healthy breast, where

the color represents the measured S-factor [96]. (g) A fusion image of the S-factor and 3D-US images (red color) [96].

BME Frontiers

cancer diagnosis and treatment monitoring. Unlike X-ray

mammography, PACT uses light for excitation, which is non-

ionizing and safe. In the meantime, it provides su

ffi

cient pene-

tration in the breast. Moreover, the optical absorption provides

a much higher soft tissue contrast than the X-ray mammogra-

phy. Further, PACT can noninvasively monitor breast tumor

responses to chemotherapy as treatment proceeds.

7. PACT of Human Extremities

Vascular disease is the leading cause of death in the United

States (

30%) [96]. And vascular disease most commonly

presents in appendicular regions. In addition, peripheral

blood vessel examination can identify the visceral disease

and estimate an individual

’

s lifestyle [97]. Therefore, angio-

graphic imaging of extremities can o

er signi

cant insights

into the health conditions of patients, especially for hyperten-

sive, diabetic, and hyperlipidemic patients [96]. Thanks to its

high sensitivity of blood, deep tissue penetration, and nonin-

vasiveness, PACT becomes a promising modality for imaging

the vasculature of extremities.

A newly designed PACT system equipped with a hemi-

spherical transducer array (1024 elements, 3.34 MHz central

frequency, and 85% one-way bandwidth) was developed to

image the vasculature of human limbs [98]. After scanning

the detector array, it can image a

eld of view up to

180 mm × 270 mm

within 10 minutes, providing high-

quality images of human limbs. As shown in Figures 8(a)

–

8(d) (illumination wavelength, 797 nm), various extremities,

including palm (Figure 8(a)), back of the hand (Figure 8(b)),

forearm (Figure 8(c)), and lower thigh (Figure 8(d)), have

been imaged, revealing detailed vasculature [98].

Recently, a PACT system with a Fabry-Perot interfero-

metric ultrasound sensor (30 MHz bandwidth, -3 dB points)

was built [99]. A volumetric image (

14 × 14 × 14

) can

be acquired within 90 s at a 30 Hz laser repetition rate (illumi-

nation wavelength, 750 nm). Fingertips were imaged before

and after a thermal stimulus. As shown in Figure 8(e), fewer

vessels were depicted after cold water immersion, showing

thermally induced peripheral vasoconstriction [99].

These experiments demonstrated the capability of PACT

to image peripheral vessels and their responses to vasomotor

changes, promising the diagnosis of peripheral vascular

diseases.

8. Photoacoustic Topography through an

Ergodic Relay

In previous sections, we review the advanced technical inno-

vations and their widespread applications of both PAM and

PACT. However, till now, PAM still requires serial detection

using a focused detector for data acquisition, which limits the

throughput, while PACT employs parallel detection using

multiple detection elements and multichannel ampli

ers

and digitizers, which is complex and expensive. Recently, a

high-throughput PA imaging technique based on an ergodic

relay (ER) has been developed, which employs a single-

element detector to obtain snapshot wide-

eld images. This

technology is termed as photoacoustic topography through

an ER (PATER) [100]. The ER, a critical component in

PATER, is a waveguide that permits acoustic waves originated

from any input point to reach any other output point with dis-

tinctive reverberant characteristics [101]. Here, the ER e

ec-

tively encodes each one-dimensional depth image into a

unique temporal sequence. Because of the uniqueness of each

temporal signal, PA waves from the whole volume through the

ER can be recorded in parallel. Finally, we can decode them

mathematically to reconstruct 2D projection images.

The mechanism of PATER has been illustrated in

Figures 9(a)

–

9(f). PATER has two imaging modes: the

2 mm

Depth (mm)

5 mm

(a)

(b)

(c)

(d)

(e)

Figure

8: PACT images of human extremities. (a

–

d) PACT images of various extremities, including palm (a), back of the hand (b), forearm

(c), and lower thigh [98]. (e) MAP images of human

ngertips after cold (left-hand panels) and warm (right-hand panels) water immersion,

color-coded for depth, while arrows show the same vessels in each imaging condition [99].

BME Frontiers