Long‐Duration and Non‐Invasive Photoacoustic Imaging of Multiple Anatomical Structures in a Live Mouse Using a Single Contrast Agent

RESEARCH ARTICLE

www.advancedscience.com

Long-Duration and Non-Invasive Photoacoustic Imaging of

Multiple Anatomical Structures in a Live Mouse Using a

Single Contrast Agent

Anjul Khadria, Chad D. Paavola, Yang Zhang, Samuel P. X. Davis, Patrick F. Grealish,

Konstantin Maslov, Junhui Shi, John M. Beals,* Sunday S. Oladipupo,*

and Lihong V. Wang*

Long-duration in vivo simultaneous imaging of multiple anatomical structures

is useful for understanding physiological aspects of diseases, informative for

molecular optimization in preclinical models, and has potential applications

in surgical settings to improve clinical outcomes. Previous studies involving

simultaneous imaging of multiple anatomical structures, for example, blood

and lymphatic vessels as well as peripheral nerves and sebaceous glands,

have used genetically engineered mice, which require expensive and

time-consuming methods. Here, an IgG4 isotype control antibody is labeled

with a near-infrared dye and injected into a mouse ear to enable simultaneous

visualization of blood and lymphatic vessels, peripheral nerves, and

sebaceous glands for up to 3 h using photoacoustic microscopy. For multiple

anatomical structure imaging, peripheral nerves and sebaceous glands are

imaged inside the injected dye-labeled antibody mass while the lymphatic

vessels are visualized outside the mass. The efficacy of the contrast agent to

label and localize deep medial lymphatic vessels and lymph nodes using

photoacoustic computed tomography is demonstrated. The capability of a

single injectable contrast agent to image multiple structures for several hours

will potentially improve preclinical therapeutic optimization, shorten

discovery timelines, and enable clinical treatments.

1. Introduction

Concurrent long-duration live animal imaging of different

anatomical structures, including blood and lymphatic vessels,

A. Khadria, Y. Zhang, S. P. X. Davis, K. Maslov, J. Shi, L. V. Wang

Caltech Optical Imaging Laboratory

Andrew and Peggy Cherng Department of Medical Engineering

California Institute of Technology

Pasadena, CA 91125, USA

E-mail: lvw@caltech.edu

The ORCID identification number(s) for the author(s) of this article

can be found under https://doi.org/10.1002/advs.202202907

This is an open access article under the terms of the Creative Commons

Attribution License, which permits use, distribution and reproduction in

any medium, provided the original work is properly cited.

DOI: 10.1002/advs.202202907

peripheral nerves, and sebaceous glands

can underpin an improved understanding

of disease progression, study effects of ther-

apeutics, and guide molecular optimiza-

tion in preclinical models.

[1–3]

Fluorescence

microscopy has been shown to simultane-

ously image the abovementioned anatom-

ical structures using mice genetically en-

gineered with fluorescent proteins. How-

ever, such methods are expensive, time-

consuming, and are not currently trans-

latable clinically.

[4–6]

The discovery of lym-

phatic vessels in the mouse and human

brain has made the need for long-duration

simultaneous imaging of lymphatic vessels

and other anatomical structures at cellular-

level resolution ever more critical to bet-

ter understand brain-related diseases and

optimize new therapies.

[7,8]

Previous work

involving preclinical imaging of lymphatic

vessels has used standalone dyes such as

Evans blue or indocyanine green (ICG),

which are not photostable, get absorbed

by the blood vessels, and cannot be used

for long-duration imaging.

[3,9,10]

The clear-

ance of these dyes in less than 30–60 min

after injection is well documented in both preclinical and clin-

ical studies.

[10,11]

Deep lymphatic vessels in mice have been

previously imaged using near-infrared fluorescence imaging;

C. D. Paavola, P. F. Grealish, S. S. Oladipupo

Lilly Research Laboratories

Eli Lilly and Company

Lilly Corporate Center

Indianapolis, IN 46285, USA

E-mail: oladipupo_sunday_s@lilly.com

J. M. Beals

Lilly Research Laboratories

Eli Lilly and Company

Lilly Biotechnology Center

San Diego, CA 92121, USA

E-mail: beals_john_m@lilly.com

L. V. Wang

Caltech Optical Imaging Laboratory

Department of Electrical Engineering

California Institute of Technology

Pasadena, CA 91125, USA

Adv. Sci.

2022

, 2202907

2202907 (1 of 9)

www.advancedsciencenews.com

www.advancedscience.com

Figure 1.

a) Schematic of the OR-PAM system. b) Schematic of the linear array-based PACT system with a view from the elevational direction (

-axis)

of the ultrasound probe emphasizing the imaging of the mouse hind limb. c) Scheme of IgG4 isotype control antibody with the available sites (as blue

dots) for the conjugation of sulfo-cy7.5 dye. d) Light absorption spectra of sulfo-cy7.5 dye-labeled IgG4 antibody, oxygenated hemoglobin (HbO

), and

deoxygenated hemoglobin (Hb).

however, those studies did not image the blood vessels simul-

taneously and utilized Evans blue or ICG.

[12,13]

Previous pho-

toacoustic imaging-based studies involving simultaneous imag-

ing of blood and lymphatic vessels have used Evans blue or

ICG, or required up to five wavelengths, which is expensive

and complex.

[3,10,14,15]

Peripheral nerves are an integral part of

the nervous system linking the brain to the rest of the body.

Imaging of peripheral nerves is of utmost importance, for ex-

ample, to study the nervous system to develop strategies to pre-

vent accidental injuries during surgeries.

[16,17]

Previous work on

photoacoustic imaging of peripheral nerves has been limited

to only ex vivo studies.

[18,19]

Studies reporting in vitro and ex

vivo multi-feature photoacoustic imaging have pushed the lim-

its of bioimaging; however, no in vivo studies have been re-

ported that demonstrate simultaneous photoacoustic imaging

of lymphatic vessels, peripheral nerves, and sebaceous glands

along with blood vessels.

[20–22]

Contrast agents that can facili-

tate long-duration and non-invasive in vivo photoacoustic imag-

ing of multiple anatomical structures can benefit several pre-

clinical and potentially clinical physiological studies and diag-

noses such as peripheral neuropathy, lymphoma, vasculitis, and

sebaceoma.

[23–25]

Several photoacoustic-based contrast agents have been re-

ported in recent years; however, most agents are based on tu-

mor imaging using organic or inorganic small molecules or

nanoparticles.

[26–28]

Notably, little or no advancement has taken

place in contrast agents for long-duration photoacoustic imaging

of lymphatic vessels and other anatomical structures such as pe-

ripheral nerves and sebaceous glands.

In this report, we use optical-resolution photoacoustic mi-

croscopy (OR-PAM) (

Figure 1

[29]

to simultaneously image

blood and lymphatic vessels along with peripheral nerves and

sebaceous glands at cellular-level resolution in the mouse ear

skin. We used photoacoustic imaging to visualize blood (label-

free); however, to image lymphatic vessels, sebaceous glands,

and axonal peripheral nerves, we subcutaneously injected the

near-infrared light absorbing sulfo-cy7.5 dye-labeled monoclonal

human IgG4 isotype control antibody. In addition, we injected

the dye-labeled antibody in the mouse hind-paw to observe

its deep medial lymphatic vessel and lymph node through a

Adv. Sci.

2022

, 2202907

2202907 (2 of 9)

www.advancedsciencenews.com

www.advancedscience.com

hand-held photoacoustic computed tomography (PACT) probe

(Figure 1b).

[30]

Dye-labeled antibodies are used widely to fluo-

rescently label and image anatomical structures through epitope

bindings; however, in this report, we use an IgG4 isotype control

antibody, which does not have any specific binding epitope.

[31,32]

2. Results and Discussion

2.1. Dye-Labeling of the IgG4 Isotype Control Antibody

The IgG4 antibody was labeled with the sulfo-cy7.5 dye (Fig-

ure 1c) and characterized by SEC-HPLC and MALDI mass spec-

trometry (see Experimental Section). The labeled antibody was

found to have

≈

4.5 dyes per molecule and exhibited size exclu-

sion behavior consistent with a monomeric, well-behaved anti-

body (Figure S1, Supporting Information). The antibody has no

specific antigen binding and incorporates S228P/L234A/L235A

sequence changes in the Fc region to reduce immune effector

function.

[33]

While the current study does not employ any specific

paratope, antibodies with specific binding could alternatively be

employed to image other anatomical structures offering tissue

specificity advantage as the case may be. We further character-

ized the dye-labeled IgG4 antibody with UV–vis spectroscopy and

found that it has an absorption maximum at 780 nm with a mo-

lar extinction coefficient of around 140000 M

−

,whichis

orders of magnitude higher than that of both oxygenated and de-

oxygenated blood at the same wavelength (Figure 1d). A higher

extinction coefficient at 780 nm will ensure high contrast signals

from the dye-labeled IgG4 antibody.

2.2. Long-Duration OR-PAM of Lymphatic Vessels in Mouse Ear

We performed the OR-PAM at 559 and 780 nm to visualize the

blood and the dye-labeled antibody, respectively, based on their

absorption spectra (Figure 1d). We injected the dye-labeled anti-

body (0.2

L, 20 mg mL

−

) into the mouse ear under anesthesia

and performed photoacoustic imaging using OR-PAM. We rely

on the high molecular weight (

≈

147 kDa) of the antibody to drive

lymphatic absorption at the point of injection since molecules

greater than 20–25 kDa weight are predominantly absorbed by

lymphatic vessels.

[34,35]

Antibodies, due to their large size, have a

slow absorption rate from the subcutaneous injection site, which

we utilize to perform long-duration imaging.

[36,37]

The lymphatic

vessels continuously absorbed the dye-labeled antibody over the

first 3 h of imaging, leading to the visualization of new lymphatic

vessels while the mouse was under anesthesia (

Figure 2

a, and

Movies S1 and S2, Supporting Information). After the first 3 h

of imaging, the mouse was allowed to recover from anesthesia

and kept in an enclosure until the injection site was reimaged 6

h post injection. At 6 h, we did not observe any lymphatic ves-

sels stained with the dye-labeled antibody due to enhanced lym-

phatic clearance from the increased muscle-mediated movement

of the ear in the conscious state.

[38]

To verify that enhanced ab-

sorption of the antibody occurs while the mouse was moving,

we reduced the initial anesthesia time to 60 min and revived

the mouse for only 30 min, then imaged it again under anes-

thesia (Figure 2b). Under these experimental conditions, we did

not observe any lymphatic vessels around the injection site at

90 min.

2.3. Multiple Anatomical Structures Visualizations in Mouse Ear

Our approach to visualizing multiple anatomical features is

based on imaging the peripheral nerves and sebaceous glands in-

side the injected dye-labeled antibody mass while visualizing the

lymphatic vessels outside the mass. In our system, the 780 nm

light that we used to detect the sulfo-cy7.5 dye-labeled IgG4 an-

tibody has a depth of focus of about 300

m, which covers the

thickness of an average mouse ear (200–250

m). The maximum

amplitude projection (MAP) of the complete 3D photoacoustic

image shows only the injected mass of the dye-labeled antibody

and the lymphatic vessels outside the mass (

Figure 3

a). The seba-

ceous glands and peripheral nerves are not clearly visible due to

excess background signals (from the dye-labeled antibody) in the

MAP image. We inspected 2D sections at various depths to avoid

the excess dye-labeled antibody background signals and distinctly

visualize the axonal peripheral nerves and sebaceous glands (Fig-

ure 3b). We further performed image segmentation (see Exper-

imental Section) to digitally label the different structures (Fig-

ure 3c). Peripheral nerves and arteries are aligned together in

mouse skin, a feature that enabled us to identify the nerves in

our images.

[6,39]

The circular structured sebaceous glands were

noticeably visible in the images. The visualization of sebaceous

glands in the mouse ear could be helpful in several types of skin

studies.

[40]

Many biological phenomena or disorders related to se-

baceous glands, such as sebaceous adenoma, sebaceoma, seba-

ceous gland hyperplasia, sebaceous carcinoma, and folliculose-

baceous cystic hamartoma, can be studied by imaging the size

and shape of sebaceous glands as well as their effects on sur-

rounding vessels.

[41]

The major blood vessels in the mouse ear

are also seen in Figure 3b, suggesting that the dye-labeled IgG4

antibody encapsulated the vessels, creating a contrast as the an-

tibody is too large to be absorbed through the pores of the blood

microvasculature.

Upon quantitative comparison of the mean photoacoustic sig-

nal from a single lymphatic vessel that remains visible through-

out the imaging time, we found that the photoacoustic signal

does not fade away significantly (

≈

20% decrease) even 3 h after

the injection under anesthetized conditions (Figure 3d). As men-

tioned above, the widely used lymphatic contrast agents such as

ICG or Evans blue get cleared away in less than 1 h.

[10,11]

These

results validate the efficacy of the large-sized dye-labeled mon-

oclonal antibody for long-duration lymphatic imaging. The pe-

ripheral nerves were also visible for up to 3 h (Figure 3e and Fig-

ure S2, Supporting Information) without a significant decrease

in the mean photoacoustic signal. Note, the signal was detected

after 50 days of initial injection (Figure S3, Supporting Informa-

tion) without observing any local or systemic adverse effects in

the mouse. This is notable because it infers that our dye-antibody

conjugate is not likely to cause any serious, acute toxicity. Mono-

clonal antibodies are widely used as therapeutic drugs and over

100 monoclonal antibodies have been approved by the FDA for

clinical use.

[42,43]

The cyanine-based dyes such as cy7 and cy7.5

and their conjugates with monoclonal antibodies have been used

Adv. Sci.

2022

, 2202907

2202907 (3 of 9)

www.advancedsciencenews.com

www.advancedscience.com

Figure 2.

OR-PAM of lymphatic vessels in mouse ear. a) Visualization of lymphatic vessels for up to 180 min upon injection of sulfo-cy7.5 dye labeled

IgG4 antibody. b) Imaging of the dye-labeled IgG4 antibody performed in the mouse ear while keeping the mouse under anesthesia for a shorter period.

Yellow arrows show lymphatic vessels lighting up. PA: photoacoustic. Scale bars: 500

in multiple preclinical studies without observation of any adverse

effects in the animals.

[44,45]

2.4. Long-Duration Deep Lymphatic Vessel Visualization

We injected the dye-labeled antibody in the mouse hind paw

and performed imaging away from the site of injection through

PACT at 780 and 920 nm in the leg and thigh areas where the

deep medial lymphatic vessel and lymph node are located to ob-

serve them (

Figure 4

a,b and Figure S4 and Movie S3, Supporting

Information).

[12]

Although the pre-injection and post-injection

images at 780 nm were sufficient to establish the dye-labeled an-

tibody uptake by the lymphatic vessel and lymph node, we per-

formed imaging at 920 nm to confirm that no blood leakage oc-

curred during needle insertion while performing the injection.

We chose 920 nm to perform imaging of blood because the ex-

tinction coefficient of hemoglobin in the blood in the NIR region

(without overlapping with the absorption spectrum of the dye-

labeled IgG4 antibody) is highest at around 920 nm, which en-

sures more photoacoustic signal from the blood.

[46]

The images

in Figure 4 were processed by vessel segmentation (see Experi-

mental Section). We performed imaging for up to 3 h following

injection to continuously observe bright signals from the lym-

phaticvesselsatadepthof2–4mmfromthesurfaceofthemouse

skin, thus proving the long-duration efficacy of the method (Fig-

ure 4b and Figure S4, Supporting Information). The dye-labeled

antibody does not absorb any light at 920 nm, but the blood

absorbs significant light. We did not perform any multi-feature

imaging using PACT in deep tissues because of two reasons:

1) The sebaceous glands are present only in the skin for which

high resolution-based OR-PAM is sufficient. 2) We have no pre-

information about the nerves in deep tissues to correctly identify

them such as we had for mouse skin where the peripheral nerves

and arteries are aligned together.

[6,39]

These results show that the dye-labeled IgG4 antibody can be

used for long-duration photoacoustic imaging of superficial and

deep lymphatic vessels. Long-duration imaging of deep lymph

nodes and lymphatic vessels could be a powerful tool for visual-

izing deep tumors during cancer metastasis.

Adv. Sci.

2022

, 2202907

2202907 (4 of 9)