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Theranostics 2016; 6(13):2394-2413. doi:10.7150/thno.16715 This issue Cite

Review

Recent Advances in Photoacoustic Imaging for Deep-Tissue Biomedical Applications

Sheng Wang^1,2,3*, Jing Lin^1*, Tianfu Wang¹, Xiaoyuan Chen³ , Peng Huang¹

1. Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, Department of Biomedical Engineering, School of Medicine, Shenzhen University, Shenzhen 518060, China;
2. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China.
3. Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States.
* S.W. and J.L. contributed equally to this work.

✉ Corresponding authors: shawn.chengov, peng.huangedu.cn.

Abstract

Photoacoustic imaging (PAI), a novel imaging modality based on photoacoustic effect, shows great promise in biomedical applications. By converting pulsed laser excitation into ultrasonic emission, PAI combines the advantages of optical imaging and ultrasound imaging, which benefits rich contrast, high resolution and deep tissue penetration. In this paper, we introduced recent advances of contrast agents, applications, and signal enhancement strategies for PAI. The PA contrast agents were categorized by their components, mainly including inorganic and organic PA contrast agents. The applications of PAI were summarized as follows: deep tumor imaging, therapeutic responses monitoring, metabolic imaging, pH detection, enzyme detection, reactive oxygen species (ROS) detection, metal ions detection, and so on. The enhancement strategies of PA signals were highlighted. In the end, we elaborated on the challenges and provided perspectives of PAI for deep-tissue biomedical applications.

Keywords: Photoacoustic imaging

1. Introduction

Optical imaging techniques, due to their high sensitivity, high temporal resolution and short acquisition time, have received tremendous attention in the field of biomedical applications [1-4]. However, one major challenge of optical imaging techniques is the strong light absorption/scattering of skin, tissue, blood, and so on, leading to limited tissue penetration [5-7]. Conventional optical imaging does not allow for the diagnosis of deep-seated lesions. Therefore, it is highly desirable to develop a novel optical imaging technique to overcome the above bottleneck for deep-seated tissues.

Near-infrared (NIR) optical imaging techniques (e.g. diffuse optical tomography, NIR fluorescence imaging) show great potential to overcome the limitation of tissue penetration for deep tissues imaging because NIR light (700-2,500 nm) can pass through biological tissues (e.g. skin) more efficiently than visible light [8-14]. Particularly, photoacoustic imaging (PAI), which is based on photoacoustic effect, is a promising biomedical imaging modality for deep-tissue applications [15-17]. So far, PAI has been employed to visualize hierarchical biostructures from organelles, cells to organs. As shown in Figure 1, in principle, when laser pulses are applied, some of the delivered optical energy will be absorbed by contrast agents or biological tissues and converted into heat, then a broadband ultrasonic emission will be generated due to the heat-induced transient thermoelastic expansion, which can be detected by an acoustic detector and analyzed to reconstruct PA images. PAI combines the high selectivity of optical imaging and the deep-tissue penetration of ultrasonic imaging, so it could overcome the limitations of conventional optical imaging techniques [18].

In this paper, we will introduce recent advances of contrast agents, applications, and signal enhancement strategies for PAI. The challenges and outlooks of PAI for deep-tissue biomedical applications are also included.

2. PA contrast agents

2.1 Endogenous PA contrast agents

Some endogenous substances with special light absorption such as hemoglobin (Hb) [19], melanin [20], and lipid [21], have been used as PA contrast agents for biomedical applications (e.g. anatomical, functional and metabolic studies) [22-25]. For example, Hb is an iron-containing metalloprotein in red blood cells that carries oxygen throughout the body. According to the different light absorption spectra of oxyhemoglobin and deoxyhemoglobin, PAI is able to measure the total Hb concentration (C_Hb) and oxygen saturation (sO₂) [26]. Melanin, a group of natural pigments occurring in the hair, skin, and iris of the eye in most organisms, is a classical endogenous PA contrast agent with strong light absorption in both UV/vis and NIR regions [27]. Besides these agents, genetically encoded probes can also provide good PA contrast by expressing NIR-absorbing proteins, which endows PAI the ability to monitor and image gene expression [28].

2.2 Exogenous PA contrast agents

The use of exogenous PA contrast agents promises PAI with high sensitivity, specificity and signal-to-background ratio over endogenous PA contrast agents. To date, a variety of inorganic/organic PA contrast agents with strong NIR absorbance and high photothermal conversion efficiency have been explored for deep-tissue PAI. According to their structures, they are mainly categorized into inorganic and organic PA contrast agents (Table 1).

 Figure 1 

Schematic illustration showing the process of PAI: (1) non-ionizing laser pulses are delivered into biological tissues; (2) the delivered energy is absorbed by endogenous or exogenous contrast agents; (3) the absorbed energy is converted into heat, leading to transient thermal expansion; (4) the thermal expansion causes the generation of ultrasonic emission; (5) the generated ultrasonic emission is detected by ultrasonic transducers and (6) then analyzed for image formation.

2.2.1 Inorganic PA contrast agents

Metallic and semimetallic nanomaterials [29-33], especially gold nanomaterials [34], can achieve high light-to-heat conversion through localized surface plasmon resonance (LSPR), which occurs when the frequency of incident photons matches the frequency of electrons on the surface of these nanomaterials, leading to a strong optical absorption. By controlling the size/morphology of gold nanomaterials, their LSPR absorbance can be engineered into the NIR region. Moreover, gold nanomaterials have the following merits of relative inertness, prominent biocompatibility, and excellent plasmonic property. So far, various gold nanostructures (nanorods [35-38], nanostars [39, 40], nanocages [41, 42], nanoshells [43], nanovesicles [44], nanoflowers [45, 46]) with LSPR peaks in the NIR region have been explored as contrast agents for PAI as well as theranostics. However, some gold nanomaterials with suboptimal photothermal stability could be melted under pulsed laser irradiation and thus lose their NIR absorbance.

Carbon-based nanomaterials, such as carbon nanotubes [47, 48], graphenes [49-55], carbon dots [56, 57], have been widely used in biomedical applications. Carbon nanomaterials usually show broad absorbance from UV to NIR regions. In addition, a large amount of compounds/materials are easy to integrate with carbon-based nanomaterials for PAI and other functions [58-61]. However, one disadvantage of carbon-based nanomaterials is their heterogeneity. For example, carbon nanotubes are a mixture of nanotubes with different lengths and diameters.

Transition metal chalcogenides (TMC)-based nanomaterials, such as copper sulfide (CuS), tungsten sulfide (WS₂), molybdenum sulfide (MoS₂) and so on, due to energy band transitions, are a class of semiconductor nanocrystals with strong NIR absorption [62-69]. Among them, CuS nanoparticles, due to their good photothermal effect and low cost, have been extensively studied for PAI [70-73]. Despite the high photothermal conversion efficiency and good photothermal stability, TMC-based nanomaterials usually contain heavy metal elements, which limits their potential for clinical translation.

Although a lot of inorganic PA contrast agents exhibited excellent PAI performance in animal experiments, the intrinsic non-biodegradability of these agents may lead to relative long retention time and potential long-term toxicity, which limits their clinical translation [74]. Recently, black phosphorus (BP) nanomaterials with broad absorption in both UV and NIR regions, due to its excellent biodegradable property, has been used in PAI [75]. Compared with other inorganic PA contrast agents, BP nanomaterials are composed of element phosphorus without any heavy metals. Furthermore, BP nanomaterials can be fully degraded into biocompatible ions, which can be absorbed, metabolized, and cleared by the body [76].

2.2.2 Organic PA contrast agents

Besides inorganic PA contrast agents, various NIR-absorbing organic agents including porphyrin- and cyanine-based dyes, perylene-diimide (PDI) derivatives and semiconducting polymers, due to their good biodegradability and biocompatibility, have been widely developed for PAI [77-79].

In the past decades, a plethora of small molecule organic dyes have been synthesized for bioimaging [80-82]. Compared with inorganic nanomaterials, these dyes show good biocompatibility and biodegradability. Among them, indocyanine green (ICG), has been approved by the US Food and Drug Administration (FDA) and used in the clinic for a long time [83]. However, most of organic dyes suffer from poor aqueous solubility, poor photothermal stability and short bloodstream circulation half-life. To address these issues, many nanocarriers including micelles, liposomes and proteins, have been employed as the vehicles for delivering organic dyes [79, 84-86].

A series of conducting and semiconducting polymers with conjugated structures, such as polypyrrole, polyaniline, polydopamine and so on, have also been developed as PA contrast agents [87-91]. Compared with small molecule organic dyes, polymer-based PA contrast agents showed much better photothermal stability. During the synthesis process of these polymers, many functional moieties, such as drugs (doxorubicin, sorafenib), photosensitizers (chlorin e6), contrast agents (⁶⁴Cu, Fe₃O₄, gadolinium, tantalum oxide), and so on, can be integrated for PAI with other imaging modalities and therapeutics [91-97]. Many in vitro and in vivo studies have demonstrated biocompatibility of these polymer-based nanomaterials [98, 99], but till now their biodegradation behaviors are still not studied in details.

3. Biomedical applications of PAI

Owing to the merits of rich contrast, high resolution and deep penetration depth, PAI has received tremendous attention as a promising noninvasive and nonionizing technique which enables multiscale and multicontrast visualization in a wide range of biomedical applications. In this section, we will summarize the biomedical applications of PAI.

3.1 Deep tumor imaging and therapy monitoring

Recently, a phosphorus phthalocyanine dye with intense absorption at 1000 nm was employed as PA contrast agent for deep PAI beyond 10 cm of chicken breast phantom and through a 5 cm human arm [100]. These results suggested that NIR light-induced PAI is a promising technique for deep-tissue diagnosis. For tumor imaging, the PA contrast agents should be tumor specific either through passive targeting or active targeting. The leaky tumor vasculature combined with poor lymphatic drainage of tumors may allow nano-sized PA contrast agents (< 100 nm) to effectively accumulate in the tumor tissue in a passive targeting manner through the so-called enhanced permeability and retention (EPR) effect [101]. In addition, with the conjugation of targeting ligands (e.g. antibodies, peptides, aptamers), PA contrast agents can actively bind to overexpressed receptors on the tumor cell membranes [102]. In a recent study, Cheng et al. developed perylene-diimide (PDI)-based nanoparticles as highly efficient PA contrast agents for in vivo deep brain tumor imaging [18]. As shown in Figure 2A, PDI molecules were encapsulated into the micelles composed of amphiphilic DSPE-mPEG. The resulting PDI-based nanoparticles with an average particle size of 48 nm were then used for detection of deep orthotopic brain tumor with high PA contrast and sensitivity (Figure 2B). Furthermore, by constructing the 3D PA image, the spatial distribution of PDI-based nanoparticles in tumor was clearly mapped. As shown in Figure 2C, the PDI-based nanoparticles were localized around the injection position of tumor cells.

Besides tumor imaging, PAI also can be used to evaluate the therapeutic efficacy of chemotherapy, photodynamic therapy, radiation therapy and antiangiogenic therapy [103-106]. For example, Hasan et al. used PAI to monitor the progress of photodynamic therapy (PDT) [104]. PDT is a clinical technique which exploits photosensitizers and light excitation. Upon laser irradiation, photosensitizers consume oxygen to generate cytotoxic reactive oxygen species (ROS), thus causing changes in _SO₂ [107]. By measuring the level of oxyhemoglobin and deoxyhemoglobin with PAI, the _SO₂ can be monitored and used as a marker for predicting PDT treatment efficacy. On PDT treatment, the drug-light-interval (DLI, defined as the interval between photosensitizer administration and light irradiation) plays an important role. The previous studies demonstrated that PDT with 1 h DLI was more effective when compared to PDT with 3 h DLI [108]. As shown in Figure 3, in the 1 h DLI group, clear hypoxic areas can be observed at both 6 and 24 h post-PDT; while no statistical change in _SO₂ value in the 3 h DLI group. These results demonstrated that PAI can be used to monitor the response to PDT by measuring _SO₂ change.

3.2 Metabolic imaging

PAI is also suitable for anatomical, functional and metabolic imaging, by measuring the contents of hemoglobin concentration (C_Hb), oxygen partial pressure (pO₂) and oxygen saturation (sO₂) [19, 22, 23]. For example, as shown in Figure 4A, the PA image of C_Hb reveals the vascular anatomy down to single capillaries in a living mouse ear [19]. The in vivo real-time oxygen unloading process can also be monitored by measuring the sO₂. As shown in Figure 4B, the sO₂ map of a capillary loop in a human finger cuticle indicated that most oxygen is unloaded from hemoglobin at the tip of the loop [24]. Based on the measurements of vessel diameter, total C_Hb, sO₂ and blood flow velocity, the metabolic rate of oxygen can be computed, which is helpful to understand metabolism-related pathogenic mechanisms (Figure 4C) [25]. With the help of a NIR dye-labeled 2-deoxyglucose (IRDye800-2DG), PAI can be used for in vivo tumor glucose metabolism imaging [109]. As shown in the PA images of orthotopically implanted 786-O kidney tumors (Figure 4D), the tumor-bearing kidney of mice displayed higher IRDye800-2DG uptake than the health kidney. At the most metabolically active site in the tumor, the tumor-to-normal kidney contrast for IRDye800-2DG uptake was approximately 3.3.

3.3 Imaging tumor microenvironment

As we know, the pH change, enzyme activity, reactive oxygen species (ROS) level, and metal ion concentration, play very important roles in life science. Aberrant behaviors of these parameters are associated with many diseases including cancer, inflammation, cardiovascular diseases, Alzheimer's disease, Wilson disease, and so on [110-113]. In the past decades, many fluorescent probes have been developed for in vivo detections [114-118]. Encouraged by the absorption changes of some PA contrast agents at different conditions, PAI show great potential to monitor or track the environment changes based on the readout intensity of PA signals [119-126]. Compared to traditional detection methods (e.g. fluorescence imaging), PAI promises in vivo quantitative detection with deep tissue penetration.

 Figure 2 

(A) Schematic illustration showing the structure of perylene-diimide (PDI) nanoparticles and the in vivo PAI process of brain tumor. (B) The ultrasonic (US), PA and their overlay images of brain coronal sections at 2 d postinjection of PDI nanoparticles. (C) Photographic transverse image (left) and PA sagittal 3D image (right) of tumor-bearing brain. The red dot is the injection point of cells. Reproduced with permission [18]. Copyright 2015 Wiley-VCH.

 Figure 3 

(A) PA images of blood oxygen saturation (StO₂) in 1 h drug-light-interval (DLI) group (up) and 3 h DLI group (down) at various time points (pre-PDT, post-PDT, 6 h and 24 h post-PDT). Blue and red signals represent hypoxic and oxygenated regions, respectively. (B) Mean StO₂ values in 1 h DLI group and 3 h DLI group. Error bars indicate standard deviation. Reproduced with permission [104]. Copyright 2015 Ivyspring International Publisher.

 Figure 4 

(A) In vivo PA image of relative C_Hb in a living mouse ear, revealing the vascular anatomy. Reproduced with permission [19]. Copyright 2011 Optical Society of America. (B) In vivo PA images of C_Hb and sO₂ in human finger cuticle. Reproduced with permission [24]. Copyright 2013 Biophysical Society. (C) In vivo PA images of C_Hb, sO₂, and blood flow speed in a nude mouse ear. Reproduced with permission [25]. Copyright 2015 Optical Society of America. (D) In vivo PA images of orthotopically implanted 786-O kidney tumors. Left: anatomical image; middle: total hemoglobin concentration (HbT) image; right: IRDye800-2DG concentration (DG) image; HK: healthy kidney; HM: hypermetabolic. Reproduced with permission [109]. Copyright 2012 SPIE

 Figure 5 

(A) Illustration showing the mechanism of albumin-BPOx-IR825 complex for pH detection. (B) The structure change of BPOx under different pHs. PA images (C) and signal intensity ratios (D) in response to various pH values. PA images (E) and PA signals (F) of a mouse with local administration of HSA-BPOx-IR825 in its tumor (left side) or muscle (right side). (G) The quantitative analysis of pH value in the tumor and muscle. Reproduced with permission [119]. Copyright 2015 Wiley-VCH.

3.3.1 pH detection

Since many diseases such as cancer are always accompanied by pH change at the microenvironment [110], the in vivo real-time pH detection may be a potential indicator for early recognition, diagnosis, monitoring, and prognosis of diseases. In order to detect pH in vivo, recently, some organic dyes with pH-responsive NIR absorption shift have been exploited as PA contrast agents for PA pH detection [119-121]. For example, Liu et al. developed a pH-responsive nanoprobe based on albumin-benzo[a]phenoxazine (BPOx)-IR825 complexes (HSA-BPOx-IR825) for in vivo ratiometric PA pH imaging (Figure 5A) [119]. Along with the decrease of pH, the IR825 absorbance peak at 825 nm showed no obvious change; however, the absorbance at 670-680 nm significantly increased through the protonation and intramolecular charge-transfer of BPOx (Figure 5B). Therefore, the PA signal intensity at 680 significantly increased while PA signal intensity at 825 nm showed negligible change (Figure 5C). In the pH range of 5.0-7.0, the ratiometric PA signal (PA_{680 nm}/PA_{825 nm}) showed excellent pH dependence, which is suitable for PA imaging of tumor microenvironment (Figure 5D). In vivo pH detection of tumor and muscle was then carried out on a mouse model via the local administration of HSA-BPOx-IR825. As shown in Figure 5E and F, in the tumor region, the PA intensity at 680 nm was much stronger than that at 825 nm, resulting in relatively high PA signal ratio (PA_{680 nm}/PA_{825 nm} = 2.35). In contrast, the PA signal ratio in the muscle region (PA_{680 nm}/PA_{825 nm} = 1.25) was obviously lower than that in the tumor. As shown in Figure 5G, the pH values of tumor and muscle were determined to be ≈6.7 and >7.0 based on the standard calibration curve in Figure 5D, which demonstrated that the HSA-BPOx-IR825 could be used as a PA contrast agent for pH detection in vivo. In another study, as shown in Figure 6A, Pu et al. developed an activatable PA nanoprobe (SON), which consists of a semiconducting oligomer (SO, inert PA matrix) and a boron-dipyrromethene dye (pH-BDP, pH indicator) [122]. Under tumor acidic environment, the hydroxyl group of pH-BDP can undergo protonation, thus leading to the absorption changes of SON. With the pH decrease, the absorption peak at 750 nm decreased significantly, while the absorption peak at 680 nm remained unchanged (Figure 6B). Consequently, the PA intensity at 750 nm also dropped along with the decrease of pH (Figure 6C and D). Therefore, the ratiometric PA signals (PA_{680 nm}/PA_{750 nm}) were used to quantify pH (Figure 6E). For in vivo pH detection, SON was locally injected into tumor and muscle in living nude mice. Then PA images were recorded under the excitation of 680 and 750 nm lasers, respectively. As shown in Figure 6F, relatively high PA signals at both 680 and 750 nm were detected in the muscle; while in the tumor site, the PA intensity at 750 nm was obviously lower than that at 680 nm. To minimize the interference of endogenous PA contrast agents (e.g. hemoglobin), the PA intensity increments (PA intensity after injection of samples subtracted by the tissue background intensity before injection) at 680 and 750 nm (ΔPA_{680 nm} and ΔPA_{750 nm}) were used to evaluate the in vivo pH. Quantification of the ratiometric PA intensity increments (ΔPA_{680 nm}/ΔPA_{750 nm}) showed that the ΔPA_{680 nm}/ΔPA_{750 nm} in the tumor was higher than that in the muscle, suggesting a lower pH value in the tumor region (Figure 6G).

 Figure 6 

(A) Illustration showing the mechanism of activatable PA nanoprobe (SON) for pH detection. Absorption spectra (B), PA images (C), PA spectra (D) and ratiometric PA signals (680/750) (E) of SON at different pH. In vivo PA images (F) and ratiometric signals (G) of muscle and tumor with local injection of SON. Reproduced with permission [122]. Copyright 2016 Wiley-VCH.

3.3.2 Enzyme detection

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptides that degrade proteins in the extracellular matrix, not only take action in tumor angiogenesis, but also are involved in multiple signaling pathways [127]. To detect MMPs in vivo, Liu et al. conjugated NIR-absorbing CuS nanoparticles with a black hole quencher 3 (BHQ3) via a MMP-cleavable peptide linker (Figure 7A) [124]. As shown in Figure 7B, the as-prepared CuS-peptide-BHQ3 (CPQ) showed PA signals at 680 nm (owing to BHQ3 absorbance) and 930 nm (owing to CuS absorbance). In the presence of MMPs, the BHQ3 would be released from the CuS nanoparticles due to the cleavage of peptide linker, leading to the decrease of absorption peak at 630 nm (Figure 7C). In the tumor microenvironment, the small molecule BHQ3 would be rapidly metabolized after cleavage, while the CuS nanoparticles would be retained in the tumor. The ratiometric PA signals (PA_{680 nm}/PA_{930 nm}) were used as an in vivo indicator of MMPs activity (Figure 7D). Xing et al. developed a MMP₂ antibody conjugated gold nanorod (AuNR-Abs), which can specifically target MMP₂ to achieve PAI detection of MMP₂ in atherosclerotic plaques [123]. The results demonstrated the feasibility of quantitative PA detection of MMP₂ in atherosclerotic plaques.

3.3.3 ROS detection

Recently, Rao et al. reported an in vivo real-time PA ROS detection method [125]. As shown in Figure 8A, IR775S, a cyanine dye that can sense specific ROS-mediated oxidation [128], was loaded into a semiconducting polymer nanoparticle (SPN) composed of poly(cyclopentadithiophene-alt-benzothiadiazole) (SP1). The PA spectrum of obtained ratiometric photoacoustic probe (RSPN) showed three PA peaks at 700, 735 and 820 nm, respectively. In the presence of ONOO^- and ClO^-, the PA peak of IR775S at 820 nm decreased significantly due to the ROS-mediated decomposition of IR775S (Figure 8B); while the peak of SP1 at 700 nm had negligible change (Figure 8C). Thus the ROS level can be evaluated according to the change of ratiometric PA signals (PA_{700 nm}/PA_{820 nm}) (Figure 8D). In order to build an in vivo model, zymosan, a structural polysaccharide that can simulate the generation of ROS, was injected intramuscularly into the thigh of living mice. After 20 min, RSPN was injected into the same location and the PA signals at both 700 nm and 820 nm were monitored simultaneously. As shown in Figure 8E, for the mice without zymosan treatment, the PA signals at both 700 nm and 820 nm remained nearly unchanged over time. In contrast, the PA signal at 820 nm for the mice with zymosan treatment significantly decreased over time, resulting in an increased PA_{700 nm}/PA_{820 nm} value (Figure 8F). Thus, RSPN can be used as a PA probe for in vivo effective detection of ROS.

 Figure 7 

(A) Mechanism of CuS-peptide-BHQ3 (CPQ) for MMP detection by using PAI. (B) Absorption spectra of CuS nanoparticles and CPQ. (C) Time-dependent absorbance spectra of CPQ incubated with matrix metalloproteinase-13 (MMP-13). (D) In vivo PA images showing CPQ response to MMP-13. Reproduced with permission [124]. Copyright 2014 Ivyspring International Publisher.

 Figure 8 

(A) Mechanism of ratiometric photoacoustic probe (RSPN) for reactive oxygen species (ROS) detection. (B) The structure change of IR775S in response to ROS. (C) PA spectra of RSPN in the absence and presence of ROS. (D) Ratio of PA amplitude (PA₇₀₀/PA₈₂₀) after ROS treatment. (E) PA images of saline (i) and zymosan (ii) treated thigh regions in a murine model of acute oedema. (F) Ratiometric PA signals (700/820) as a function of time post-injection of RSPN. Reproduced with permission [125]. Copyright 2014 Nature.

3.3.4 Metal ion detection

PAI also can be used to detect metal ions, such as copper (Cu²⁺) and lithium (Li⁺). For example, Chan et al. developed a PA probe for the chemoselective visualization of Cu²⁺, which is a crucial metal ion in chronic nervous diseases (e.g. Alzheimer's disease) [126]. As shown in Figure 9A, the acoustogenic probe for Cu²⁺-2 (APC-2) contains a 2-picolinic ester sensing module that can specifically respond to Cu²⁺. In the absence of Cu²⁺, the PA signal of the probe at 767 nm will be lower than that at 697 nm, leading to a small ratiometric PA₇₆₇ nm/PA₆₉₇ nm. In the presence of Cu²⁺, the sensitive module hydrolyzed, a stronger PA signal at 767 nm and a weaker PA signal at 697 nm will be detected, resulting in a large ratiometric PA₇₆₇ nm/PA₆₉₇ nm (Figure 9B). Based on the ratiometric PA₇₆₇ nm/PA₆₉₇ nm change, the Cu²⁺ can be detected (Figure 9C). Due to the high selectivity and the deep-tissue penetration of PAI, this PA probe shows great potential for in vivo Cu²⁺ detection. In another study, Clark et al. developed a PA nanosensor for in vivo Li⁺ detection [129]. As shown in Figure 9D, the nanosensor is mainly composed of a lithium selective crown ether ionophore, lithium ionophore VI (L), and a chromoionophore (CH⁺). Li⁺ is recognized and extracted into the hydrophobic polymer core of the nanosensor, leading to the deprotonation of CH⁺, which changes the optical properties of the nanosensor. As a result, the photoacoustic amplitude at 515 nm increase and the photoacoustic amplitude at 660 nm decreases (Figure 9E). For in vivo detection of Li⁺, the nanosensor was injected into the skin of mice and imaged with PA tomography (Figure 9F). Upon intraperitoneal administration of lithium (38 mg/kg), the ratiometric PA_{515 nm}/PA_{660 nm} of the nanosensor increased by 25% in 14 min (Figure 9G). To date, many organic dyes that can respond to metal ions and realize absorbance change have been developed for fluorescence detection [130-132], which could be utilized for PA metal ions detection.

 Figure 9 

(A) Mechanism of PAI for Cu²⁺ detection. (B) Normalized absorbance spectra of acoustogenic probe for Cu²⁺-2 (APC-2) and its hydrolyzed product (20). (C) PA signal responses of APC-2 to various metal ions. Reproduced with permission [126]. Copyright 2015 American Chemical Society. (D) The principle of PAI for Li⁺ detection. (E) PA signal responses of nanosensor to Li⁺. (F) In vivo dual wavelength PA images of mice. (G) Ratiometric PA signals (515/660) as a function of time post-injection of Li⁺. Reproduced with permission [129]. Copyright 2015 American Chemical Society.

 Figure 10 

(A) Illustration of an indocyanine green (ICG) coated single-walled carbon nanotubes (SWNTs) and PA image of agarose phantoms containing U87MG cells exposed to SWNT-ICG-RGD and SWNT-RGD. Reproduced with permission [136]. Copyright 2010 American Chemical Society. (B) Illustration showing the synthesis process of graphene oxide (GO)-coated gold nanorods (GO-AuNRs) and reduced graphene oxide (rGO)-coated gold nanorods (rGO-AuNRs). In vitro PA images (C) and PA intensities (D) of AuNRs, GO-AuNRs, rGO-AuNRs (1), and rGO-AuNRs (2). Reproduced with permission [135]. Copyright 2015 American Chemical Society. (E) Illustration and TEM image of rGO-loaded gold nanorod vesicle (rGO-AuNRVe). In vitro PA images (F) and PA intensities (G) of samples. Reproduced with permission [137]. Copyright 2015 American Chemical Society.

4. Strategies to enhance PA signals

The PA signal enhancement strategies are critical for improving the sensitivity of both diagnosis and detection. Different strategies for PA signal enhancement, such as synergistic effect, plasmonic coupling effect, self-assembly, photoinduced electron transfer, as well as background signal deduction will be summarized in this section.

4.1 Synergistic effect

A simple way to amplify the PA signal is the combination of two or more PA contrast agents together to achieve higher absorbance. Various nanocomposites have been developed for the amplification of PA signals, such as dye-graphene [133], gold-graphene [134, 135]. The PA signal amplification mechanisms of these nanocomposites mainly include absorption superposition [136], interaction of different agents [137] and reduced heat transfer resistance [135]. For example, Gambhir et al. developed a family of composites based on single-walled carbon nanotubes (SWNTs) and organic dyes for highly sensitive PAI [136, 138]. As shown in Figure 10A, compared with plain SWNTs, the indocyanine green (ICG)-coated SWNTs exhibited a 20-fold higher absorbance due to the absorption superposition of both dyes and SWNTs. Thus the ICG-coated SWNTs showed higher PA contrast and can easily detect ∼20 times fewer cancer cells. Lim and co-workers reported a hybrid PA contrast agent (rGO-AuNR), composed of gold nanorods and reduced graphene oxide (rGO), for sensitive PAI [135]. GO-coated gold nanorods (GO-AuNRs) were prepared through simple electrostatic interactions. After chemical or optical reduction, rGO-AuNRs were successfully prepared (Figure 10B). In this case, rGO can not only enhance NIR light absorption via absorption superposition, but also reduce heat transfer resistance from the gold nanorods to the ambient signal-generating medium. The results demonstrated that the rGO-AuNRs exhibit significantly amplified PA signal intensity when compared to AuNRs and GO-AuNRs (Figure 10C and D). Chen et al. reported a hybrid rGO-loaded gold nanorod vesicle (rGO-AuNRVe) for sequential drug release and enhanced photothermal and PA effect (Figure 10E) [137]. Compared with the simple mixture of AuNRVe and rGO, the rGO-AuNRVe showed remarkably amplified PA signal at the same OD₈₀₈ value (Figure 10F and G). It is believed that the cavity of gold nanorod vesicle can concentrate the electromagnetic radiations, resulting in the absorption efficiency enhancement of the encapsulated rGO [139].

4.2 Plasmonic coupling effect

Plasmonic nanostructures, such as gold nanomaterials, have been actively studied as PA contrast agents. Since the size of the nanomaterials are much smaller than the wavelength of light, the lateral and vertical finite spacing will lead to strong interaction between the neighboring elements. Compared to an individual element, the optical properties of plasmonic nanostructures will be changed substantially [140]. With the distance decrease of adjacent gold nanoparticles, the plasmonic coupling effect would be significantly enhanced, which promises the PA signal amplification. For example, Chen et al. developed a plasmonic biodegradable gold nanovesicle (BGV) by the self-assembly of polymer-tethered gold nanoparticles [141]. As shown in Figure 11A, in the formation process of BGV, the distance (d) between adjacent gold nanoparticles reduced, leading to an ultrastrong plasmonic coupling effect. Plasmonic coupling between gold nanoparticles can generates enhanced electromagnetic field, resulting in enhanced photothermal conversion efficiency and thus amplified PA signals (Figure 11B and C). In a more recent study, they further demonstrated that the chain gold vesicle, self-assembled through a stepwise hierarchical self-assembly process, had stronger plasmonic coupling effect than the non-chain vesicle, and therefore achieved PA signal amplification (Figure 11D and E) [142]. Based on the same principle, the developed plasmonic gold bellflowers (GBFs) which also amplified the PA signals because of the presence of strong plasmonic coupling effect among the multiple-branched petals (Figure 11F) [45]. The PA signal intensities of GBFs were much stronger than that of gold nanorods (GNRs) and gold nanostars (GNSs) at the same OD₈₀₈ value (Figure 11G). This PA enhancement strategy is suitable for plasmonic metal nanomaterials, such as gold, silver, and so on.

4.3 Self-assembly

A J-aggregate is a type of nanoassembly when dye molecules aggregate under the influence of certain conditions. Compared to dye monomer, J-aggregate shows red-shifted absorption wavelength and higher absorption coefficient [143]. Therefore, the use of a self-assembly nanostructure is another strategy to amplify the PA signals of organic dyes [144-146]. For example, Chen et al. reported an in situ tumor-specific supramolecular self-assembly (ICG doped nanofiber) for enhanced PAI [147]. The mixture of ICG and an alkaline phosphatase (ALP)-responsive peptide forms micelles, which can efficiently self-assemble into nanofibers in the ALP-rich tumor macroenvironment (Figure 12A). As shown in Figure 12C and D, the PA signal of nanofibers in tumor site was much stronger than that of ICG. This excellent performance of nanofibers in PAI was mainly attributed to their unique in situ self-assemble features: (i) the J-aggregate structure of ICG in nanofibers induced an amplified PA signal (Figure 12B), and (ii) the assembly induced enhanced tumor retention. Based on a similar strategy, Wang and co-workers designed an enzyme-responsive small-molecule precursor 1, which had three functional segments: purpurin 18 (P18) as the NIR dye, enzyme-responsive peptide (PLGVRG) as the linker, and RGD peptide as the targeting ligand [144]. As shown in Figure 12E, in the presence of gelatinase, which is overexpressed in tumor, the PLGVRG linkers was cleaved, and then the residues self-assembled into nanofibers due to the enhanced hydrophobicity and reduced steric hindrance. Compared to 2 (P18-PMGMRGRGD without responsiveness) and 3 (P18-PLGVRGRDG without targeting), 1 showed a significantly amplified PA signal, which was attributed to the assembly induced signal amplification and assembly induced retention effect (Figure 12F). This similar strategy was also used for bacterial infection detection [145]. This self-assembly PA enhancement strategy is suitable for organic dyes by forming ordered arrangement, such as J-aggregates.

 Figure 11 

(A) Illustration showing the formation process of biodegradable gold nanovesicles (BGVs). (B) Photothermal conversion efficiencies of BGVs, gold nanovesicles (GVs), gold nanorods (GNRs) and gold nanostars (GNSs). (C) PA signals of BGVs and GNRs as a function of OD₈₀₈ values. Reproduced with permission [141]. Copyright 2013 Wiley-VCH. (D) Illustration showing the formation of chain and non-chain vesicles. (E) PA images of mouse tissues before and after the injection of chain vesicles (a, b) or non-chain vesicles (c, d). Reproduced with permission [142]. Copyright 2015 Wiley-VCH. (F) The formation process and TEM image of gold bellflowers (GBFs). (G) PA images and signals of GBFs at different OD₈₀₈ values. Reproduced with permission [45]. Copyright 2014 American Chemical Society.

4.4 Photoinduced electron transfer (PET)

PET is a transfer process of excited state electron, in which excited electron is transferred from donor to acceptor. This strategy is able to enhance the nonradiative heat generation, which can be used for PA signal amplification [148]. Pu et al. developed a fullerene doped semiconducting polymer nanoparticle (SPN-F) that can amplify the PA signal through the intraparticle photoinduced electron transfer [148]. As shown in Figure 13A, the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) are -3.5 and -4.9 eV for semiconducting polymer and -4.3 and -6.5 eV for fullerene, respectively. Therefore, the semiconducting polymer and fullerene play their roles as electron donor and acceptor, respectively, leading to quenched fluorescence and enhanced nonradiative heat generation upon light irradiation, ultimately achieve amplified PAI signal. As shown in Figure 13B and C, with increasing amount of fullerene doped (SPN-Fx, x: the weight percentages of fullerene), the PA intensity obviously increased. The amplified PA signals of SPNs have also been demonstrated in vivo. As shown in Figure 13D and E, compared to SPN-F0 injected mice, the mice injected with SPN-F20 exhibited much higher PA signals in the tumor at all the time points.

4.5 Background signal deduction

Besides the above mentioned strategies for PA signal amplification, the strategy of PA background signal deduction is another way to indirectly enhance the PA signal. For example, Wang et al. reported a reversibly switchable bacterial phytochrome based PA probe for in vivo tumor detection with substantially decreased background signals [149]. As shown in Figure 14A, upon light illumination, rhodopseudomonas palustris (BphP1) can be reversibly switched between Pfr (ON-state) and Pr (OFF-state). Because the PA background signal of vascular absorbers remains relatively constant, this switchable property of PA probe allows to subtract the two obtained PA images under individual states, and completely remove the background signals (Figure 14B and C), thus achieving enhanced detection sensitivity and improved spatial resolution (Figure 14D). In another study, Gao et al. reported iron oxide and gold-coupled core-shell nanoparticles (MNP-Au) for magnetomotive PAI (mmPAI) [150]. As shown in Figure 14E, a pulsed magnetic field is applied during PA signal acquisition. MNP-Au particles move and create a moving source when the field is on, and then return to their original positions when the field is off. In contrast, the non-magnetic background PA sources do not move during this process. Therefore, coherent motion processing of a PA image sequence can identify sources related to MNP-Au and reject all diffuse and localized background signals. Then both conventional PAI and mmPAI were performed using gold nanorod (Au NR) to mimic strong background tissue signals. As shown in Figure 14F, compared to conventional PAI, the Au NR mimicking a strong background signal is almost completely suppressed in mmPAI.

 Figure 12 

(A) Illustration showing the in situ formation of nanofibers. (B) PA intensity changes of ICG (1) and nanofiber (5) at different ICG concentrations. PA images (C) and PA intensities (D) of tumor tissues after intravenous injection of samples. Reproduced with permission [147]. Copyright 2015 American Chemical Society. (E) Illustration showing the structure of compound 1 and the in situ formation of nanofibers. (F) PA signal intensities of tumor sites after intravenous injection of samples. Reproduced with permission [144]. Copyright 2015 Wiley-VCH.

 Figure 13 

(A) Illustration showing the mechanism of photoinduced electron transfer-induced amplified PA signal. In vitro PA images (B) and PA intensities (C) of fullerene doped semiconducting polymer nanoparticle (SPN-F) solutions. (D) PA images of tumor tissues after injection of SPN-F0 and SPN-F20. (E) PA intensities of SPN-F0, SPN-F20, or saline as a function of postinjection time. Reproduced with permission [148]. Copyright 2016 American Chemical Society.

5. Conclusion and outlook

PAI, as a promising real-time imaging technique with high spatial resolution and tissue penetration depth, has been widely studied. This paper briefly summarized recent advances in PAI for biomedical applications, such as deep tumor imaging, therapy monitoring, metabolic imaging, pH detection, enzyme detection, ROS detection, metal ions detection, and so on. To improve PAI sensitivity, various exogenous contrast agents, such as metallic nanomaterials, semimetallic nanomaterials, carbon-based nanomaterials, transition metal chalcogenides, organic dyes, polymer-based nanomaterials, have been developed for PAI in recent years. In order to enhance PA signals, different strategies which are based on synergistic effect of two or more PA contrast agents, plasmonic coupling effect of plasmonic metal nanomaterials, self-assembly of organic dyes, photoinduced electron transfer and background signal deduction have been reported. The exploitation of these strategies not only can improve the PA sensitivity, but also can reduce the dose of PA contrast agents.

Although various PA contrast agents have demonstrated their high PA efficiency and unnoticeable toxicity in pre-clinical studies, they have not been approved for clinical use. In the future, the following directions should be taken into account: 1) Fabrication of activatable PA contrast agents that sense specific stimuli of interest followed by PA signal change in a predictable manner, such as a shift in the absorption peak. By analyzing the differential PA signals, the surrounding PA noises can be reduced and then the selectivity and sensitivity of PAI can be improved. 2) Optimization of targeting PA contrast agents to actively target the tumor, improve the tumor accumulation rate, and reduce the reticuloendothelial system (RES) retention by using specific biomarkers (such as peptides, antibodies, and so on). 3) Development of new strategies that can further enhance the PA signals of PA contrast agents. The current strategies to amplify the PA signals mainly focus on the improvement of NIR adsorption and photothermal effect by exploiting plasmonic coupling effect, self-assembly or photoinduced electron transfer. 4) Development of PA-based multi-modal contrast agents that can provide complementary information by combining information from various imaging modalities and overcome the weaknesses of each imaging modality. 5) Systematic assessment of the stability, toxicity, biocompability, biodegradability, immunogenicity and pharmacokinetics of PA contrast agents that would be of great help to accelerate their clinical translation for PAI.

 Figure 14 

(A) Molar extinction spectra of BphP1 in the ON-state (Pfr) and OFF-state (Pr). (B) In vivo PA images of a nude mouse at 1 week after injection of BphP1-expressing tumor cells. (C) Normalized PA signal profiles along the white dashed line in B. (D) An overlay of the tumor (in color) and the blood-dominated OFF-state image (in grayscale). Hb: hemoglobin. Reproduced with permission [149]. Copyright 2016 Nature. (E) Illustration showing the structure of MNP-Au and principle of mmPAI. (F) Conventional PAI and mmPAI of Au NR, MNP-Au and MNP. Reproduced with permission [150]. Copyright 2010 Macmillan Publishers Limited.

Acknowledgements

This work was supported by the National Science Foundation of China (81401465, 51573096), and the Intramural Research Program (IRP) of the NIBIB, NIH.

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding authors: shawn.chengov, peng.huangedu.cn.

Received 2016-7-4
Accepted 2016-8-16
Published 2016-10-7

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