Light in and Sound Out: Emerging Translational Strategies for Photoacoustic Imaging

Published OnlineFirst February 10, 2014; DOI: 10.1158/0008-5472.CAN-13-2387

Cancer Review Research

Light In and Sound Out: Emerging Translational Strategies for Photoacoustic Imaging

S. Zackrisson1,2,3,4,5, S.M.W.Y. van de Ven1,2,3,4, and S.S. Gambhir1,2,3,4

Abstract Photoacoustic imaging (PAI) has the potential for real-time molecular imaging at high resolution and deep inside the tissue, using nonionizing radiation and not necessarily depending on exogenous imaging agents, making this technique very promising for a range of clinical applications. The fact that PAI systems can be made portable and compatible with existing imaging technologies favors clinical translation even more. The breadth of clinical applications in which photoacoustics could play a valuable role include: noninvasive imaging of the breast, sentinel lymph nodes, skin, thyroid, eye, prostate (transrectal), and ovaries (transvaginal); minimally invasive endoscopic imaging of gastrointestinal tract, bladder, and circulating tumor cells (in vivo ﬂow cytometry); and intraoperative imaging for assessment of tumor margins and (lymph node) metastases. In this review, we describe the basics of PAI and its recent advances in biomedical research, followed by a discussion of strategies for clinical translation of the technique. Cancer Res; 74(4); 979–1004. 2014 AACR.

Introduction In this review, we will first describe the basics of PAI, Photoacoustic imaging (PAI), also referred to as optoacous- followed by a comprehensive overview of its clinical applica- tic imaging, is an emerging new imaging technique with tions. We will then discuss strategies for future translation to significant promise for biomedical applications. The key clinical practice. strength of PAI is its ability to collect functional and molecular information from most tissues. One of the major advantages of Principles of Photoacoustic Imaging this technique over other imaging modalities is that PAI has the PAI relies on the photoacoustic effect, first described by Bell ability to provide tissue information without using an exoge- (1). In short, a pulsed nanosecond-long red-shifted laser beam nous molecular imaging agent, by making use of endogenous is used to illuminate the tissue(s) of interest causing photons to contrast alone. Additional advantages over other imaging propagate diffusely inside. The absorption of these photons modalities are that PAI can provide images: (i) in real-time; leads to a slight localized heating of the tissue causing thermo- (ii) at clinically relevant depths; (iii) with relatively high spatial elastic expansion. This transient thermoelastic tissue expan- resolution; (iv) and without the use of ionizing radiation. PAI sion generates pressure waves (ultrasound), which can then be can be performed either by (i) relying on intrinsic tissue detected by broadband ultrasonic transducers and converted contrast alone (e.g., mapping endogenous chromophores such into images. These basic principles are illustrated in a potential as melanin, hemoglobin, and lipids) or by (ii) using exogenous clinical application (see Fig. 1). The shared detector platform molecular imaging agents that can either target specific molec- (the detector array) facilitates a natural integration of PAI and ular processes (targeted agents) or extravasate due to leaky ultrasound imaging creating a hybrid imaging technique that vasculature and the enhanced permeability and retention combines functional (PAI) and structural (ultrasound) infor- (EPR) effects found in tumor tissue (nontargeted agents). mation, which is favorable for clinical translation (Fig. 1). In addition, the fact that PAI is similar to ultrasound in technology and handling—it can also itself be real-time, portable, Authors' Affiliations: Departments of 1Radiology, 2Bioengineering, and relatively cheap, and safe (nonionizing radiation)—makes it 3 4 Materials Science & Engineering, Molecular Imaging Program at Stan- even more adoptable/integrable for clinicians. ford, Stanford University School of Medicine, Stanford, California; and 5Diagnostic Radiology, Department of Clinical Sciences in Malmo,€ Lund The combined use of light and sound in PAI has an impor- University, Lund, Sweden tant advantage over other imaging techniques, such as optical Note: Supplementary data for this article are available at Cancer Research imaging, ultrasound, MRI, computed tomography (CT), posi- Online (http://cancerres.aacrjournals.org/). tron emission tomography (PET), and single-photon emission S. Zackrisson and S.M.W.Y. van de Ven contributed equally to this work. computed tomography (SPECT), namely the unique scalability of its spatial resolution and depth penetration across both Corresponding Author: S.S. Gambhir, Stanford University School of Medicine, The James H. Clark Center, 318 Campus Drive, East Wing, 1st optical and ultrasonic dimensions. In optical imaging, scatter- Floor, Stanford, CA 94305-5427. Phone: 650-725-2309; Fax: 650-724- ing of light hinders penetration depth, whereas in PAI the 4948; E-mail: [email protected] detection of acoustic signal, which has much lower tissue doi: 10.1158/0008-5472.CAN-13-2387 scattering, allows for superior depth penetration. Spatial reso- 2014 American Association for Cancer Research. lution can be very high (microscopic level) at higher ultrasound

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Figure 1. Principles of PAI presented for a potential clinical application: diagnostic breast imaging by integrated real-time photoacoustic/ultrasound imaging. The laser sends nanosecond pulses of NIR light through the transducer into the tissue. This light is then absorbed inside the tissue (at different levels for each tissue type/component) causing a localized transient thermoelastic expansion. This expansion leads to the emission of pressure waves (ultrasound), which can be detected by the array in the transducer. Finally, a photoacoustic image is calculated and displayed in real-time. At the same time, the ultrasound system can be used in its b-mode to provide structural information about the tissue, in addition to the functional/molecular information obtained by PAI, and both images can be displayed on the viewing screen together (either next to each other or integrated).

frequencies, but with lower depth penetration, as a trade-off, and microscopic level (11), although so far most systems have due to the increasing attenuation of ultrasound with frequency. been designed to image specifically at one or the other of these Photoacoustic microscopy (PAM) generates lateral resolutions levels. Although spatial resolution of PAI may be superior over down to 45 mm at a depth of 3 mm when using ultrasound other imaging techniques, such as PET and SPECT (12), we frequencies of 50 MHz (2–5), and this can even improve to 200 have to keep in mind that these modalities do not suffer from nm (subcellular level) in optical-resolution PAM, but only at depth limitations (as attenuation of g rays can be corrected imaging depths of up to 1 mm (6). Macroscopic PAI systems for), which makes them suitable for whole-body imaging. Most allow for deeper tissue imaging by using lower ultrasound likely PAI will be used in adjunct to clinically established frequencies of 4 to 8 MHz and typically generate resolutions of imaging techniques, or in combination with them in a multi- 0.5 to 5 mm at reported imaging depths of approximately 3 to 7 modality setting. More information on these multimodality cm (3, 6–10). A summary of the largest depths reached with imaging approaches in which anatomic and molecular infor- macroscopic PAI systems is presented in Table 1. The unique mation are combined, can be found under the organ-specific scalability of spatial resolution and imaging depth may afford a sections of this review. wide breadth of imaging ranging from the microscopic to the Another key advantage of PAI, mentioned earlier, is that the macroscopic scale, something that is very challenging to do optical component of the technique allows for molecular using most other imaging modalities. For example, comparing imaging even without the use of an imaging agent, by using optical microscopy images with the CT images is difficult multiple wavelengths (multispectral imaging of endogenous because the image contrast is obtained in entirely different contrast). Different tissue components have unique optical ways (optical absorption vs. X-rays). Thus, PAI could open up scattering and absorption properties for each wavelength. inimitable opportunities to study complex biologic systems In Fig. 2, the absorption spectra for the main light-absorbing and pathways in the same organism from the subcellular level tissue components (oxy- and deoxyhemoglobin, lipid, water, to the organ level, using the same optical absorption contrast. and melanin) are shown. As seen from this figure, total light Some PAI systems can already image both at the macroscopic absorbance in these tissue components is lowest in the

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Translational Strategies for Photoacoustic Imaging

Table 1. Summary of the largest imaging depths reported for macroscopic PAI in experimental and clinical studies

Depth reached (mm) Photoacoustic system Subjects Target Imaging agent Author Experimental studies 10 PAT Canine prostate Prostate pseudo lesions — Wang and in vivo with fresh canine colleagues (112) blood 20 LOIS-P (laser Dog prostate in vivo Blood rich–induced — Yaseen and optoacoustic imaging prostate lesions colleagues (113) system for the prostate) 32 Photoacoustic Phantom Breast lesion-mimicking Ecoline 700 black Manohar and mammoscope objects water color dye colleagues (95) prototype 50 Hand-held Phantoms/chicken SLNs Methylene blue Kim and photoacoustic/ breast and rats colleagues (186) ultrasound probe 52 PAT Phantoms/chicken Lesion-mimicking Blood/ICG Ku and Wang (3) breast objects 52 Hand-held Phantoms/chicken SLNs Methylene blue Kim and photoacoustic/ breast and rats colleagues (7) ultrasound probe 60 Optoacoustic Phantoms Breast cancer cells, Gold nanoparticles Copland and tomography HER2 conjugated to colleagues (52) HER2-antibody 70 TAT and PAT Phantoms Breast lesion-mimicking Black India ink Pramanik and objects colleagues (99) Clinical studies 28a PAT Patients (n ¼ 26) Breast cancer — Kitai and colleagues (93) 30 LOIS-64 (laser Patients (n ¼ 27) Breast cancer — Ermilov and optoacoustic imaging colleagues (31) system for the breast) 30 Photoacoustic Patients (n ¼ 12) Breast cancer and cysts — Heijblom and mammoscope colleagues (94) 40 PAT Healthy subject (1) Breast vasculature — Kruger and colleagues (9) 60 Photoacoustic Patients (n ¼ 13) Breast cancer — Manohar and mammoscope colleagues (8)

aDepth reported for one case only.

wavelength range of 600 to 1,000 nm, referred to as the near-IR photoacoustic CT as exemplified under the organ-specific (NIR) or "optical window." Because the absorbance is lowest in sections that follow. In short, PAI systems can be categorized the NIR range, light of these wavelengths is used to obtain as linear array systems (13–16) or tomographic systems sufficient tissue-penetration depth (a few centimeters). Infor- (9, 17). Tomographic systems detect photoacoustic waves mation on tissue composition, that is, relative concentrations emanating from the "entire" perimeter of the subject of of (de)oxyhemoglobin, water, lipid, or an injected imaging interest using either a single element or an array transducer. agent, can be calculated by combining photoacoustic data In linear array systems, an ultrasound array is used to detect acquired at multiple wavelengths. Numerous relevant clinical photoacoustic waves from "limited angles" around the parameters can be assessed this way, for example hypoxia, object. The limited field of view results in an image quality perfusion, and neoangiogenesis. inferior to that of tomographic systems. However, linear Many different PAI platforms for clinical use already exist arraysystemsaremoreportableandcanbeintegratedinto or are under development including handheld probes and already existing clinical ultrasound systems, enabling the

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Figure 2. Absorption spectra of the main light absorbing tissue components: melanin, oxy- and deoxyhemoglobin, lipid, and water. The total light absorbance in these components is lowest in the wavelength range from 600 to 1,000 nm. The best tissue penetration depth can thus be reached in this "optical window." Data were obtained from http://omlc.ogi.edu/spectra.

visualization of organs less accessible with tomographic normal surrounding tissue. PAI based on endogenous contrast systems (e.g., via endoscopes or catheters). alone has been studied for various diseases (e.g., atheroscle- During the last few decades, major progress has been made rosis, breast cancer, and prostate cancer; refs. 30–32). However, in source and detector development, allowing for instance for not all absorption spectra for different tissues are known. faster (real-time) imaging at more wavelengths and greater Multiwavelength PAI makes it possible to image several tissue depth penetration (18). Light propagation models and image components in the same image, although the need for a tunable reconstruction algorithms have also improved significantly pulsed laser source increases the costs of the system signifi- (18–23), which is critical to image quantitation. Obtaining cantly. Because some tissue components have very similar truly quantitative images is only possible when correcting for absorption spectra within the NIR range, there is sometimes a the amount of light delivered to an actual tissue depth, and this need for exogenous molecular imaging agents to enhance the remains challenging because light attenuation depends on contrast. Moreover, exogenous imaging agents can potentially several factors, not only the imaging depth but also the tissue facilitate deeper tissue imaging. type in which the light propagates. The abovementioned progress in technology plus the Exogenous imaging agents inherent advantages of PAI are paving the way for clinical Exogenous imaging agents can greatly enhance the PAI translation of the technique. A summary of potential clinical contrast. One could use nontargeted imaging agents that applications of PAI in different organ systems is illustrated mostly extravasate from the blood stream due to increased in Fig. 3. More detailed descriptions of the principles of vascular permeability and the EPR effect in tumorous tissue, or photoacoustics can be found in book chapters (24, 25) and targeted imaging agents that target a specific molecular – other articles covering this topic (17, 26 28). process, that is, a certain receptor, protein, or enzyme. The advantage of targeted imaging agents is that they can provide Imaging Contrast specific information on molecular or cellular processes in Endogenous contrast the tissue. There is a great variety of imaging agents that can PAI takes advantage of the fact that the body tissues contain be used for PAI (see Table 2 and Fig. 4). The selection of a a variety of endogenous chromophores with different absorp- particular imaging agent will depend upon the application for tion spectra, such as melanin, water, fat, HbO2 (oxyhemoglo- which it will be used and one should thoroughly consider bin) and Hb (deoxyhemoglobin), as shown in Fig. 2. Many various characteristics, such as the size, shape, toxicity, sta- diseases cause changes in tissue composition, such as neovas- bility, surface chemistry, and targeting moieties of the imaging culature in cancer development (29) and vascular fat deposi- agent. Besides a few imaging dyes that have been approved for tion in atherosclerotic plaques (30). PAI has the potential to human use [indocyanine green (ICG), Evans blue, and meth- visualize and quantify these changes in comparison with the ylene blue], the rest of the imaging agents are not yet approved,

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Figure 3. Overview of potential clinical applications of PAI. For each organ system, the main possible applications are listed. Some of these applications are male-specific (left, blue line) and some are female-specific (right, red line). More detailed information on these clinical applications, the research progress, and challenges can be found in the text under the organ-specific sections. and have only been evaluated in phantoms or animal models. designed by our laboratory especially for PAI, using activatable So far, not much information is available on the mass range cell-penetrating peptide as its peptide platform with a NIR that is needed for any of the potential imaging agents to chromophore (quencher or fluorophore) on either side of this perform their respective PAI studies. This is an important platform to enable dual-wavelength imaging. The intact probe issue and the toxicity of these imaging agents will have to be is photoacoustically silent through subtraction of the images determined relative to the mass needed to be injected to obtained at the two wavelengths, but after activation by matrix perform a given study. It is likely that higher masses will be metalloproteinases (MMP), specifically MMP-2, the cleaved necessary for PAI studies compared with very sensitive tech- probe shows a change in photoacoustic signal because now niques such as PET and SPECT. Mass ranges will have to be only the cell-penetrating peptide portion of the probe (carrying assessed for each specific agent and its applications. one of the chromophores) accumulates in the cells and the rest of the probe (carrying the other chromophore) diffuses away Small-molecule dyes (35). This platform is generalizable and can be tailored to the There is a range of small-molecule dyes that absorb in the target proteases of interest, but its potential was recently NIR window and can thus be used for PAI. Many of these dyes shown in mice in vivo for PAI of thyroid carcinoma (36). In are fluorescent and are commonly used in optical fluorescence addition, Razansky and colleagues showed that the commer- imaging. The difference with PAI is that for this technique only cially available activatable fluorescent probe MMPSense 680 the absorption part of the spectrum is used and not the can be used for molecular imaging with PAI systems as well fluorescent emission part, making it more efficient to have a (37). MMPSense 680 also undergoes changes in light absorp- low quantum yield (i.e., a low ratio of photons emitted to tion after activation by MMPs. These changes can then be photons absorbed, as more of the absorbed energy can be spectrally resolved over the inactivated probe and the back- converted into photoacoustic signal). The dyes can be used as ground by acquiring the photoacoustic signal over a range of agents that are not targeted, but can also be functionalized to wavelengths, compared with the two-wavelength imaging used enable molecular targeting. For example, a dye can be directly for the previously described activatable photoacoustic probe. conjugated to a targeting antibody such as trastuzumab to The advantages of the small-molecule dyes (1 nm in size) image HER2 expression (33) or to a caspase-binder to image are their biocompatibility and their fast and complete clear- apoptosis (34). Furthermore, an activatable probe has been ance from the body due to their small size. This makes them

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2014 American Association for Cancer Research. 984 Downloaded from acrRs 44 eray1,2014 15, February 74(4) Res; Cancer Table 2. Overview of exogenous photoacoustic molecular imaging agents al. et Zackrisson

Absorption wavelength, Imaging agent Type Size nm Evaluation Applications References cancerres.aacrjournals.org Small-molecule dyes ICGa Fluorescent dye <2 nm 800 Rat model Brain imaging Wang and colleagues Published OnlineFirstFebruary10,2014;DOI:10.1158/0008-5472.CAN-13-2387 (angiography), lymph (39), Ku and Wang (3), node imaging Kim and colleagues (38) Evans bluea Fluorescent dye <2 nm 550 Mouse and rat Imaging microvasculature Yao and colleagues model and lymph nodes (187), Song and colleagues (41), Li and colleagues (42) IRDye800CW (ICG Fluorescent dye <2 nm 774 Mouse model Brain tumor imaging, Li and colleagues (43) derivative) integrin targeting on September 25, 2021. © 2014American Association for Cancer Research. Alexa Fluor750 Fluorescent dye <2 nm 752 In vitro Cancer imaging, HER2 Bhattacharyya and targeting colleagues (33) Methylene bluea Fluorescent dye <2 nm 677 Rat model Lymph node imaging Song and colleagues (40) MMPSense 680 Fluorescent dye <2 nm 620; 680 Human Imaging vulnerability of Razansky and endarterectomy atherosclerotic plaques, colleagues (37) specimens activated by MMPs NIR caspase-9 probe Fluorescent dye <2 nm 640 Mouse model Monitoring cancer cell Yang and colleagues (34) apoptosis BHQ3 Quencher <2 nm 672 In vitro and in vivo Cancer imaging, thyroid Levi and colleagues in mice tumors, activated by (35, 36) MMPs QXL680 Quencher <2 nm 680 In vitro Cancer imaging, activated Levi and colleagues (35) by MMPs Nanoparticles—nonplasmonic SWNT conjugated with Graphene cylinders 1–2by50–300 nm 690 Mouse model Cancer imaging, integrin De la Zerda and targeting peptide targeting colleagues (69), Xiang and colleagues (188) SWNT conjugated with ICG Graphene cylinders 1–2by50–300 nm 780 Mouse model Cancer imaging, integrin De la Zerda and and/or QSY and targeting targeting colleagues (68, 189) peptide SWNT conjugated with ICG Graphene cylinders 1–2by50–300 nm 820 Rat model Lymph node imaging Koo and colleagues (70) Perﬂuorocarbon Fluorescent dyes 210–230 nm 790 Rat model Imaging lymph nodes Akers and acrResearch Cancer nanoparticles loaded with encapsulated in colleagues (190) NIR dyes perﬂuorocarbon particles ICG-embedded PEBBLEs Fluorescent dye 100 nm 790 Mouse model Cancer imaging, HER2 Kim and colleagues (44) encapsulated in ormosil targeting spheres (Continued on the following page) Downloaded from www.aacrjournals.org Table 2. Overview of exogenous photoacoustic molecular imaging agents (Cont'd )

Absorption wavelength, Imaging agent Type Size nm Evaluation Applications References ICG encapsulated in Fluorescent dye 30 nm 760–820 Phantoms PAI Gupta and cancerres.aacrjournals.org virus-mimicking encapsulated in protein colleagues (45) nanoconstructs shell, purified from virus Published OnlineFirstFebruary10,2014;DOI:10.1158/0008-5472.CAN-13-2387 Quantum dots Fluorescent 2–10 nm 640 In vitro PAI Shashkov and colleagues (72) Copper sulfide nanoparticles Copper sulfide spheres 11 nm 1,064 Mouse and rat Brain and lymph node Ku and colleagues (73) model imaging Nanoparticles—plasmonic Gold nanocages Silver cubes coated with a 40 nm 800 Mouse and rat Brain imaging Skrabalak and layer of gold model (angiography), colleagues (54), Yang melanoma imaging and colleagues (55), on September 25, 2021. © 2014American Association for Cancer Research. Kim and colleagues (53) Gold nanoshells Spherical particles with silica 100–200 nm 800 Mouse and rat Cancer imaging, colon and Xie and colleagues (47), core and gold shell model brain cancer, integrin Li (48) targeting Gold nanorods Solid gold rice-shaped 10 by 40–60 nm 650–1,100 Mouse model Cancer imaging, HER2 and Eghtedari and colleagues particles EGFR targeting (49, 50), Copland and colleagues (52), Li (51), Bayer and colleagues (46), Jokerst and colleagues (57) Silica-coated gold nanorods Solid gold rods coated with 60–70 by 665 Mouse model Mesenchymal stem cell Jokerst and silica layer 80–90 nm imaging colleagues (56) Imaging Photoacoustic for Strategies Translational Gold nanospheres Solid gold spheres 20–50 nm 520–530 Mouse model Cancer imaging Zhang and colleagues (191) Hollow gold nanospheres Spheres with hollow core and 50 nm 800 Mouse model Brain vasculature and Lu and colleagues

acrRs 44 eray1,2014 15, February 74(4) Res; Cancer gold shell tumor imaging, (192–194) melanoma targeting Gold nanobeacons Liposomes containing 100–200 nm 520–1,100 Mouse model Cancer imaging, integrin Pan and plasmonic nanoparticles targeting, lymph node colleagues (195, 196) imaging Gold nanoclusters 4-nm gold spheres 50–100 nm 700–900 Phantoms Imaging with high clearance Yoon and colleagues (62) connected with biodegradable polymer SWNT with gold coating Gold-coated grapheme 11 by 100 nm 850 Mouse model Imaging lymphatic vessels Kim and colleagues (71) cylinders (Continued on the following page) 985 986 Downloaded from acrRs 44 eray1,2014 15, February 74(4) Res; Cancer Table 2. Overview of exogenous photoacoustic molecular imaging agents (Cont'd ) al. et Zackrisson

Absorption wavelength, Imaging agent Type Size nm Evaluation Applications References – cancerres.aacrjournals.org Fluorescent nanodiamonds Diamond nanocrystallites 150 250 nm 530, 565 In vitro PAI Zhang and

conjugated with gold linked to gold particles colleagues (65) Published OnlineFirstFebruary10,2014;DOI:10.1158/0008-5472.CAN-13-2387 nanoparticles Radiation-damaged Nanodiamonds irradiated 70 nm 820 Mouse model PAI Zhang and nanodiamonds with 40 keV Helium ions colleagues (66) Silver nanoplates Triangular silver plates 10–30 by 550–1,080 Mouse model Cancer imaging, targeting Homan and 25–250 nm EGFR colleagues (197) Multimodality imaging agents MPR nanoparticle Gold core with Raman active 120–180 nm 540 Mouse model Imaging brain tumors with Kircher and layer and silica shell coated three modalities: MRI, PAI, colleagues (86)

on September 25, 2021. © 2014American Association for Cancer Research. with gadolinium and Raman imaging Photoacoustic nanodroplets Droplets of perﬂuorocarbon 200 nm 520–1,200 Mouse model Optically triggered Wilson and loaded with optically dual-contrast imaging for colleagues (84) absorbing nanoparticles photoacoustic and ultrasound imaging Radiolabled gold nanorods Gold nanorods labeled 10 by 40–60 nm 650–1,100 Rat model Dual mode PAI and SPECT Agarwal and with 125 I in rat tail joints colleagues (85) Pearl necklaces Gold nanorods decorated 90 nm 785 In vitro Dual mode cancer imaging Wang and with iron oxide spheres with PAI and MRI colleagues (80) Nanostars Iron oxide cores with gold 120 nm 800–900 Rat model Imaging lymphatic vessels Kim and colleagues (58) spikes and nodes Nanoroses Clusters of iron oxide cores 20–80 nm 700–800 In vitro and rabbit Cancer imaging, EGFR Ma and with thin knobby gold model targeting; colleagues (59, 60) coating (atherosclerosis) atherosclerosis imaging Nanowontons Wonton-shaped cobalt cores 30–90 nm 400–800 Phantoms and Dual mode PAI and MRI Bouchard and coated with layer of gold mouse model colleagues (61) Iron oxide-gold nanoshells Iron oxide cores with gold 30–40 nm 600–900 Phantoms Dual mode PAI and MRI Jin and colleagues (81) shells Superparamagnetic iron Iron oxide cores embedded in 80–150 nm 720 Rat model—ex vivo Photoacoustic intra- Grootendorst and oxide nanoparticles dextran coating operative lymph node colleagues (87) imaging with MRI imaging agent

acrResearch Cancer Magneto-plasmonic Liposomes encapsulating 100–200 nm 800 Phantoms Dual mode PAI and MRI Qu and colleagues (82) nanoparticles gold nanorods and iron oxide spheres

Abbreviation: EGFR, EGF receptor. aFDA approved. Published OnlineFirst February 10, 2014; DOI: 10.1158/0008-5472.CAN-13-2387

Translational Strategies for Photoacoustic Imaging

Figure 4. Illustration of several different types of PAI agents from small to large (left to right). See Table 1 for more detailed information on these imaging agents. The displayed imaging agents are nontargeted, however, they can all be functionalized by conjugation with specific targeting moieties. valid candidates for clinical use. As mentioned earlier, a few (46–57). Besides these regular shapes, some more complex biocompatible dyes have already been approved for clinical use shapes have been designed, such as nanostars, nanoroses, (ICG, Evans blue, and methylene blue). These dyes have been nanowontons, and nanoclusters (52–56, 58–62). The advantage studied in various animal models for PAI (3, 38–43), but not yet of these plasmonic nanoparticles is that their optical proper- in clinical trials. The downside of their small size is that it is less ties (absorption and scattering) are highly tunable over the NIR likely for the imaging agents to be delivered to or retained at the spectrum by changing their size or shape, which makes them target site during their short circulation time. In addition, very attractive for biomedical applications. An additional photostability is an important issue for these dyes as they suffer important advantage is that the surface characteristics of these from photobleaching (loss of optical absorption) after expo- nanoparticles are easy to chemically modify, which is impor- sure to prolonged laser irradiation. Circulation time and tant for in vivo use (e.g., to decrease cytotoxicity or to increase photostability can be improved by encapsulating more dye circulation time and stability) and for molecular targeting. molecules into a bigger nanoparticle, which was shown by Kim Functional groups such as polyethylene glycol (PEG) or integ- and colleagues who developed ICG-embedded nanoparticles rins can be attached to the surface in a straightforward way. based on PEBBLE (photonic explorers for biomedical use by The disadvantages of plasmonic nanoparticles are that they biologically localized embedding) technology (44). In PEBBLE, can deform after extended exposure to laser irradiation and high dye concentrations are encapsulated into biocompatible that their long-term safety is a concern, especially with the nanoparticles, for example, an ormosil matrix was used by Kim larger particles that do not fully clear the body as they are taken and colleagues for nanoparticle synthesis and each particle was up by the reticuloendothelial system (RES). The deformation of loaded with >20,000 ICG molecules. Gupta and colleagues also nanoparticles affects the photoacoustic signal and results in encapsulated many ICG molecules into bigger nanoparticles, inconsistent imaging results over time. A thin layer of silica but they made a construct composed of a protein shell, purified coating can increase stability of the nanoparticles and allow from the plant-infecting brome mosaic virus (BMV), to encap- them to maintain their original shape for a longer time (63). An sulate the ICG (45). extra advantage of the silica coating is that these hybrid nanoparticles can act as photoacoustic nanoamplifiers due Nanoparticles to more efficient heat dissipation to the tissue (64). Zhang and Nanoparticles are larger in size than small molecules. Their colleagues have demonstrated a new type of nanoparticle for size varies between a few to several hundred nm. On the basis of PAI. In addition to using conjugates of fluorescent nanodia- their mechanism, the nanoparticles can be divided into two monds and gold nanoparticles to enhance their otherwise low categories: plasmonic and nonplasmonic nanoparticles. The photoacoustic signal (65), they have shown the feasibility of plasmonic nanoparticles are made of a noble metal (gold or using nanodiamonds damaged by helium ion irradiation (66). silver) and use the surface plasmon resonance effect (SPR). These radiation-damaged nanoparticles gave a 70-fold higher This effect takes place when the electromagnetic field of photoacoustic signal as compared with gold nanoparticles of incoming light interacts with the conduction electrons on the similar dimensions, caused by increased optical absorption surface of the nanoparticle, resulting in mutual oscillation of due to vacancies in the diamond crystal lattice. these electrons at a resonance frequency relative to the lattice The nonplasmonic nanoparticles are strong light absorbers of positive ions. At this resonance frequency, the incoming light that do not rely on the SPR effect. Single-walled carbon is absorbed by the nanoparticle, with an optical absorption nanotubes (SWNT) are the most widely used example of that is five orders of magnitude greater than the absorption for nonplasmonic nanoparticles. They absorb light over a broad dyes (on a per particle basis; ref. 25). spectrum, including the NIR range. Their broad absorption The plasmonic nanoparticles exist in numerous sizes and spectrum even allows for "thermoacoustic imaging" using shapes (see Table 2). The most commonly used kinds in PAI are lower energy electromagnetic waves (microwaves instead of nanoshells, nanorods, and nanocages, usually made of gold NIR light; ref. 67). Like the plasmonic nanoparticles, the

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nonplasmonic nanoparticles can also be modified to improve oxide and gold-coupled core–shell nanoparticles, and lipo- their functionality. For example, several dyes can be attached somes containing iron oxide nanospheres and gold nanorods to the SWNTs to increase light absorption and targeting agents (58–62, 80–82). Kim and colleagues used multifunctional can be conjugated to the SWNTs for molecular imaging microbubbles and nanobubbles for concurrent photoacoustic purposes (Fig. 4; refs. 68–70). In addition, the SWNTs can be and ultrasound imaging in phantoms (83). Wilson and col- coated with a thin layer of gold, which enhances the photo- leagues developed photoacoustic nanodroplets, consisting of acoustic signal as with this layering the SPR effect is used (71). a droplet of liquid-phase shift perfluorocarbon with a bovine Another type of nonplasmonic nanoparticle is a structure in serum albumin shell in which optically absorbing nanoparti- which several dye molecules are grouped together, with stron- cles have been suspended (84). These nanodroplets act as dual- ger light absorption and longer circulation time than for the imaging agents for both photoacoustic and ultrasound imag- free dye. For example, the ICG-embedded PEBBLEs by Kim and ing through optically triggered vaporization. Gold nanorods colleagues are ormosil spheres containing many ICG dye have been labeled by 125I to allow for dual-mode SPECT and molecules, and it was shown that HER2-antibodies could be PAI after intra-articular injection in rat tails (85). For a triple- attached to the surface of these nanoparticles for cancer modality approach of MRI, PAI, and Raman imaging, our targeting (44). Quantum dots are semiconductor nanoparticles research group recently developed "MPR" nanoparticles by that are strongly fluorescent with sizes ranging from 2 to 10 modifying gold-based Surface Enhanced Raman Scattering nm. Shashkov and colleagues showed in their studies that these (SERS) nanoparticles with gadolinium (86). This novel molec- quantum dots can also be used as PAI agents (72). Copper ular imaging agent was then used to accurately help delineate sulfide nanoparticles, approximately 11 nm in diameter, also the margins of brain tumors in living mice (Fig. 5). Recently, have these semiconductor characteristics and produce photo- Grootendorst and colleagues proposed to use a commercially acoustic signal at longer wavelengths (1,064 nm) as shown by available MRI agent (Endorem; superparamagnetic iron oxide Ku and colleagues (73). nanoparticles), as a photoacoustic probe for intraoperative Limitations of SWNTs for human clinical applications com- imaging of lymph nodes and demonstrated the feasibility ex prise mainly the (long-term) toxicity concerns (74). Although vivo in rats (87). the first systematic toxicity evaluation of functionalized SWNTs following intravenous injection in living mice did not Genetically encoded probes show signs of acute or chronic toxicity, more evidence is Genetically encoded fluorescent proteins have been widely needed to determine the safety of these SWNTs for in vivo used for more than a decade in preclinical optical imaging medical applications (75). The nanoparticles encapsulating studies. The use of PAI for reporter gene imaging was first dyes have the problem of photobleaching, however, their demonstrated by Li and colleagues in 2008 (88). They used the stability can increase up to 500% as compared with the free lacZ gene, which encodes the enzyme b-galactosidase. After dye (76). In addition, the surface of the encapsulated dyes can injection of a colorless analog of lactose, "X-gal," cleavage of potentially be easier to chemically modify than just the free dye this substrate by b-galactosidase eventually leads to the for- by itself. Quantum dots are highly photostable, but their main mation of a blue product, which can then be detected by PAI drawback for biomedical applications is that their major due to its high optical absorption. In later studies, they showed components, cadmium and selenium, are toxic to cells and that they could reach imaging depths of approximately 5 cm organisms (77). with this technique, with spatial resolutions of approximately 1 Depending on their characteristics, the nanoparticles can mm, which is a major improvement over traditional optical also be loaded with drugs and used as delivery vehicles to allow (bioluminescence or fluorescence) reporter gene imaging, for simultaneous delivery of therapeutic and imaging agents in reaching depths of up to approximately 1 cm (89). Filonov vivo (78, 79), or their intrinsic properties can be used for and colleagues showed that a bacteriophytochrome-based NIR photothermal ablation therapy (80). This provides great oppor- fluorescent protein named iRFP could also serve as a probe for tunities for image-guided therapy/theranostics (combining PAI (90). It can produce stronger photoacoustic signal than therapy and diagnostics). blood in the NIR window, does not require injection of exogenous substrates, and can be delivered into the body by Multimodality imaging agents standard genetic manipulations of cells. Ha and colleagues Because imaging is becoming increasingly important in the then reported the potential of ferritin as a reporter gene diagnosis and treatment of disease, imaging agents that can be suitable for PAI in vitro (91). Recently, Qin and colleagues tracked by multiple imaging modalities can be very useful in demonstrated in vitro and in vivo in mice that the tyrosinase overcoming the limitations of a single imaging modality in the gene as a single reporter gene could be used for triple-modality different steps of the diagnosis/treatment process (e.g., using imaging by PAI, MRI, and PET, through catalyzing the synthesis different modalities for whole-body diagnostic imaging before of melanin (92). These genetically encoded proteins have vast surgery vs. real-time intraoperative imaging). Multimodality potential for whole-body animal imaging, however, the use of imaging agents that can enhance contrast for PAI in combi- genetically encoded probes remains challenging for human nation with other imaging modalities are under development. applications due to the requirement of introduction of a Nanoparticles that are both plasmonic and superparamagnetic reporter gene. Nevertheless, for many small animal model can be used for combined photoacoustic and MRI; examples applications or cell therapy applications in humans, reporter are nanostars, nanoroses, pearl necklaces, nanowontons, iron gene strategies provide a potentially powerful approach.

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Figure 5. Triple-modality detection of brain tumors in living mice with MPR nanoparticles. After orthotopic inoculation, tumor-bearing mice were injected intravenously with MPR nanoparticles. Photoacoustic, Raman, and MRI images of the brain were acquired before and 2, 3, and 4 hours postinjection, respectively. A, two-dimensional coronal MRI, photoacoustic, and Raman images. The postinjection images of all three modalities demonstrated clear tumor visualization. The photoacoustic and Raman images were coregistered with the MRI image, demonstrating good colocalization between the three modalities. B, 3D rendering of MRI image with the tumor segmented in red (top), overlay of MRI and 3D photoacoustic images (middle), and overlay of MRI, segmented tumor, and photoacoustic image (bottom) showing good colocalization of the photoacoustic signal with the tumor. C, quantiﬁcation of signal in the tumor shows signiﬁcant increase in MRI, photoacoustic, and Raman signals before versus after the injection (, P < 0.001; , P < 0.01). Error bars, SEM. Reprinted by permission from Macmillan Publishers Ltd: Nature Medicine, Kircher and colleagues (86), copyright 2012 Nat Med.

Clinical Applications and MRI. A combination of these methods usually yields Breast imaging relatively high sensitivity and speciﬁcity for cancer detection. The conventional techniques for breast imaging include Each of the techniques has some drawbacks. Mammography digital mammography, ultrasonography, scintimammography, is associated with ionizing radiation and has decreased

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ABC250 200 147 100 50 100 250 40 35 200 30 25 150

Y (mm) 20 15 10 100 5 50 5 10 15 20 25 30 35 40 45 X (mm)

Figure 6. PAI study of a suspicious lesion in the right breast of a 57-year-old woman that was conﬁrmed to be invasive ductal carcinoma by histopathology. A–C, the craniocaudal X-ray mammogram of this lesion (A); the ultrasound image of the lesion (B); and the craniocaudal photoacoustic maximum intensity projection of the same lesion (C); the higher intensity regions were attributed to tumor vascularization. Reprinted with permission from Manohar and colleagues (8). Copyright 2007 The Optical Society.

sensitivity for cancer detection in dense breasts. The results of technical reasons. These technical issues, such as the inability ultrasound greatly depend on the instrument performance and to image lesions located close to the chest wall, are challenging examiner's skill. MRI requires more time and cost and requires in the design of improved photoacoustic breast imaging sys- the use of imaging agents, to get dynamic information about tems. An example of a breast cancer imaged with mammog- neovascularization. New imaging technologies are needed that raphy, ultrasound, and PAI is shown in Fig. 6. can aid in early detection of breast cancer and provide com- Ermilov and colleagues investigated a laser-based optoa- prehensive information of the tumor properties potentially coustic system (LOIS) with a 64-element-annular array of important for treatment decision and especially in assessment rectangular transducer elements surrounding a hemicylindri- of response to therapy. Increased use of preoperative systemic cal volume in which the breast was suspended (31). A pulsed therapy and differentiation of therapy in general, depending on laser operating at 755 nm illuminated the breast and single the tumor properties, require more functional/molecular planes through the breast were captured one at a time. The tumor information that PAI may be able to provide. spatial resolution was reported to be 0.5 mm. Preliminary PAI is now being investigated in initial patient studies in clinical studies on 27 patients with suspicious breast lesions breast imaging. Five studies published so far have involved on mammogram and/or ultrasound were presented. The LOIS- patients (8, 9, 31, 93, 94). Two of the studies are continuing 64 was able to visualize 18 of 20 malignant lesions, as was clinical attempts with The Twente Photoacoustic Mammo- confirmed by breast biopsy performed after the PAI procedure. scope (PAM), developed at the University of Twente (Enschede, Kruger and colleagues recently modified a small animal the Netherlands). It consists of a bed on which the patient lies photoacoustic tomography (PAT) system to image human in the prone position with her breast pendant through an breasts (9). Two three-dimensional (3D) maximum intensity aperture. In the scanning compartment of the system, the point (MIP) projections of the left breast of a healthy breast is gently compressed between a glass plate and a flat volunteer were generated, depicting sub-millimeter vessels ultrasound detector matrix, with acoustic coupling gel applied to a depth of 40 mm without the use of a molecular imaging between the breast and the detector. The system was first agent (see Supplementary Data; http://www.optosonics. tested and evaluated in breast tissue phantoms (95, 96) and in com/breast-images.html). So far, no patients with breast 2007 2 of 5 clinical breast cancer cases were presented (8). In cancer have been imaged with this system, but neoplastic 2012, this group then presented 10 cases of breast malignancy breast lesions could potentially be visualized on the basis of and 2 cases of benign cysts (94). PAM imaging revealed higher their hemoglobin content. intensity regions based on increased optical absorption that Kitai and colleagues recently published an article in which 27 could be attributed to the cancer-associated vascular distri- tumors in 26 subjects were visualized using a prototype bution. This increased photoacoustic contrast was absent in photoacoustic mammography system (93). The patients were the cysts. However, the photoacoustic contrast seemed to be examined lying in the prone position with the breasts mildly independent of the mammographically estimated breast den- compressed in a craniocaudal direction between holding sity, a promising observation for imaging patients with dense plates. Pulsed laser beams irradiated the breast from both breast tissue (currently limited by decreased mammographic sides, and photoacoustic signals were detected on the caudal sensitivity). In these studies, 13 of 30 (first 8 of 13 and then 5 of side by a transducer array. The measurable area was 30 mm 17) measurements could not be included in the analysis due to 46 mm for one scan, which took 45 seconds. The lesions

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included 21 invasive breast cancers (IBC), five ductal carcino- colloid and a blue dye. The clinical procedure varies, but can ma in situ (DCIS), and one phyllodes tumor. Nine of 21 patients involve presurgical scintigraphic imaging, and pre/peri-oper- with IBC had received primary systemic therapy (PST). Eight of ative use of a gamma probe or Geiger counter to locate the SLN. 12 IBC without PST were visible. After visual confirmation, the SLN is surgically resected, and Detection was possible in all 5 cases with DCIS, whereas it the definitive presence of cancer is established with histologic was not in the 1 case with phyllodes tumor. Seven of nine IBCs methods. SLNB can help avoiding morbidities associated with PST were assigned as visible despite decreased tumor size with complete axillary nodal dissection, which was the prior after PST. The mean value of hemoglobin saturation in the standard. So far, radiocolloids and blue dyes are generally blood vessels of visible lesions was 78.6% (they reported values considered complementary in SLNB and should both be used of >92% for normal subcutaneous blood vessels) and the routinely. However, the procedure could be done without average total hemoglobin content in these lesions was 207 involving radioactivity if the signal from the blue dye alone mmol/L. These encouraging results show the potential to could be detected in real-time pre/perioperatively with PAI evaluate and quantify functional information, which may be and in combination with ultrasound for anatomic information. useful in diagnosing and assessing treatment response in Although to a lesser extent, morbidity has also been reported breast cancer. after SLNB (103, 104). Possibly, a PAI-guided approach could Other systems with potential applications for breast cancer avoid surgical intervention of the sentinel node and instead imaging have been developed, but not yet clinically tested. The guide a fine-needle aspiration. If PAI allows for noninvasive optoacoustic plus ultrasound system (OPUS), is a handheld identification of the SLN, the combination of fine-needle combination of a wavelength-tunable pulsed laser and a com- aspiration biopsy and real-time reverse transcription PCR to mercial ultrasound scanner (97). By using suitable image confirm lymph node status (malignant/benign) might be a reconstruction algorithms and quantification procedures, a minimally invasive alternative to SLNB. linear relation between local optical absorption and photo- Using PAI, Song and colleagues accurately mapped SLNs acoustic signal was found for absorbers at different depths and noninvasively in living mice and rats upon injection of Evans optical matrix properties. blue, a blue dye currently used in clinical SLNB (41). They were The Wang laboratory has developed an integrated PAT and also able to mimic PAI of "deeper" SLNs by layering additional thermoacoustic tomography (TAT) system that combines biologic tissues on top of the rats (>3 cm) and injecting either nonionizing radiofrequency waves and NIR laser light to obtain methylene blue (40), ICG (38), or gold nanocages (105). ICG- additional information from breast tissue, such as water/ion stained SNL photoacoustic imaging is shown in Fig. 7. In concentration, blood volume, and oxygenation level of hemo- addition, Kim and colleagues have reported in vivo photoa- globin (98). As opposed to breast compression from the sides, coustic and ultrasound mapping of ICG- or methylene blue– used in X-ray mammography, this PAT/TAT scanner uses front stained SLNs in rats using a clinical ultrasound array system, compression to give the breast a cylindrical shape and obtain a and they also demonstrated guidance of SLN fine-needle full 3D dataset. The front compression may possibly allow for aspiration biopsies using this technique (7, 106, 107). SNL imaging the regions close to the chest wall. Also, dry coupling is detection by PAI and ICG-enhanced SWNTs was evaluated in used instead of the usual coupling gel in conventional ultra- rats by Koo and colleagues (70). Akers and colleagues recently sound imaging. Good quality PAT and TAT images on tissue reported the use of SPECT/CT to validate PAT in SLN detection mimicking phantoms have so far been achieved (98, 99). (108). Although SLN imaging is a PAI application under inten- So far, out of all of the potential clinical applications, sive research in animal models in vivo, its feasibility remains to translation of PAI has reached the furthest in breast imaging. be shown in humans. Several groups are working on improved clinical systems with better depth penetration and higher spatial resolution (100, Urologic imaging 101), which will help move photoacoustic breast imaging Prostate imaging. Standard screening and diagnostic forward. methods for prostate cancer—such as blood screening for Sentinel lymph node detection in breast cancer. The prostate-specific antigen (PSA) and other biomarkers, digital presence of axillary lymph node metastases is the most impor- rectal examination (DRE), and transrectal ultrasound (TRUS)– tant factor in staging of early breast cancer and during the last guided prostate biopsy—have limited ability (sensitivity and decade, sentinel lymph node biopsy (SLNB) has taken a leading specificity) in the early diagnosis of prostate cancer (109, 110). role in the staging procedure. Although SLNB could also be Furthermore, the available techniques cannot fully differen- useful in other type of cancers, for example, melanoma or tiate between aggressive and more latent prostate cancers Merkel cell carcinoma (an aggressive form of skin cancer), it is with the consequence that there is no significant impact on most widely used in breast cancer and we will therefore focus survival. This leads to overdiagnosis and overtreatment on this application. Sentinel lymph nodes (SLN) are defined as (110). A combined transrectal probe with ultrasound and the first lymph nodes (usually a cluster of 3–4 nodes) in a tumor PAI for prostate imaging to combine anatomic, functional, bed that receive lymphatic drainage from the tumor tissues. and molecular imaging is a potentially useful approach to These nodes are thus most likely to harbor any metastasizing move prostate imaging and diagnosis forward. To date, there cancer cells along the path of lymph drainage of tumor tissues are no reports on PAI for prostate imaging in humans, yet (102). The SLNB technique usually involves injection at the several groups have conducted promising studies in vivo.In breast tumor site with the combination of a radiolabeled sulfur canine prostates, induced blood lesions have successfully

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ABC

Figure 7. Photoacoustic images of SLN in a rat in vivo after ICG injection. A, image at 668 nm 0.2 hour after ICG injection. B, image at 618 nm 2.2 hours after injection. C, image at 668 nm 2.8 hours after injection. D, graph shows comparison of spectroscopic photoacoustic (PA) signals within the SLN region over a period of time. E, graph shows comparison of D E spectroscopic photoacoustic signals within blood vessels 1.2 668 618 668 1.2 668 618 668 (BV) over a period of time. LV, lymphatic vessel. Numbers in colored bar at top of D and 0.8 1 E equal wavelength in nanometers. Reprinted with permission from The 0.4 0.8 Radiological Society of North America, Kim and colleagues Injection Injection signal at BV (AU) PA 0.6 (38). Copyright 2010 RSNA PA signal at SLN (AU) PA 0 0 123 0 123 Time (h) Time (h)

been imaged (Fig. 8; refs. 111–113);inaratmodel,prostate (118). Harrison and Zemp investigated PAI for brachyther- adenocarcinoma tumors have been visualized (32) and in a apy seed visualization in a tissue phantom, comparing the mouse window chamber model, prostate tumor progression received intensity to endogenous contrast. They found that has been imaged with 3D photoacoustic and pulse echo PAI at 1,064 nm could identify brachytherapy seeds uniquely imaging, both without the use of a molecular imaging agent at laser penetration depths of 5 cm in biologic tissue at the (114) and with the use of gold nanorods to enhance image ANSI limit for human exposure with a contrast-to-noise contrast (115). ICG embedded PEBBLEs conjugated with ratio of 26.5 dB (119). Kuo and colleagues recently showed HER-2 antibody showed high contrast and high efficiency imaging of brachytherapy seeds in an ex vivo dog prostate for binding to prostate cancer cells in initial in vitro char- phantom (120). acterization studies (44). In summary, to move the clinical translation of photoacous- Among the treatment options available for prostate can- tic prostate imaging forward, there is a need for the develop- cer, the use of brachytherapy seeds, metal implants that ment of a transrectal PAI/ultrasound device with sufficient deliver localized radiotherapy, is an important clinical depth penetration and spatial resolution. Also, better charac- approach (116, 117). Because visualization of the small seeds terization of biologic prostate tumor signatures (e.g., cell can be difficult, the TRUS-derived position of the needles surface receptors, degree of vascularization) is important to delivering the seeds to the tissues is often relied upon to identify potential targets for a more specific diagnosis of infer seed placement. Correct seed placement is important prostate cancer by PAI, with or without the use of extrinsic for dose planning management. Because metals have an molecular imaging agents. optical absorption that is orders of magnitude greater than Bladder imaging. Because urethrocystoscopy cannot that of soft tissue, PAI may be very valuable as an adjunct to determine the extent of invasive bladder cancer and is not TRUS in seed placement imaging. Su and colleagues exper- always able to discriminate between flat carcinoma in situ and imentally evaluated PAI in combination with TRUS for inflammation, PAI may be able to give additional diagnostic brachytherapy seed visualization in excised bovine prostate and/or prognostic information in these cases. In an initial tissue. They found that PAI improved imaging contrast from study by Xie and colleagues, the bladder microvasculature was 2.7 to 27.9 dB over TRUS alone (for seeds in the long-axis successfully visualized ex vivo using microscopic PAI on canine orientation) at imaging depths ranging from 4 to 13 mm and human bladder specimens (121).

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Figure 8. PAI study of ex vivo dog prostate. Photograph of the sliced prostate (A) shows an induced lesion, B shows the ultrasound image of this lesion in vivo, C and D show the in vivo CD photoacoustic images before and after the lesion induction. Prostate capsule (PC), urethra (U), needle insertion path (NIP), and lesion (L) can be distinguished. Reprinted with permission from Yaseen and colleagues (113). Copyright 2010 J Biomed Opt.

A different application is evaluation of vesicoureteral reflux noninvasive imaging techniques, including PAI, are under (VUR). VUR is abnormal leakage of urine from the bladder to investigation. Most melanomas contain melanin, which is a the upper urinary tract, which may be a source of infections highly light-absorbing pigment. Melanin provides great con- and eventually renal scarring, especially in children. Current trast for PAI in the "optical window" (600–1,000 nm; see Fig. imaging methods to diagnose and monitor VUR are X-ray or 2) without having to inject an imaging agent. PAM is most radionuclide based and the possibility to use a combination of frequently explored for melanoma detection and staging, using ultrasound and PAI instead, and hence avoid ionizing radiation various imaging systems such as dark-field PAM, with depth in children, would be a step forward. Kim and colleagues penetrations of up to 3 mm (17). The first proof-of-principle of explored noninvasive photoacoustic bladder imaging in vivo this technique in vivo was shown by Zhang and colleagues, by mapping rat bladders filled with methylene blue (122), imaging angiogenesis, melanoma, and SO2 of single vessels in whereas Koo and colleagues visualized the rat bladders using animals, and total hemoglobin concentration in humans (2). ICG-enhanced SWNTs (70). These methods may in the future This group illustrated that dual-wavelength imaging can sep- be useful for VUR monitoring in children. arate the contributions from two different pigments, in this Photoacoustic bladder imaging is certainly in its infancy and case hemoglobin and melanin, based on their absorption many technical issues need to be resolved before clinical spectra. In further studies, they measured the metabolic rate translation is feasible. In addition, further investigations of of oxygen in melanoma using PAM (124). In addition, they exogenous molecular imaging agents, especially about safety demonstrated that targeted melanoma imaging could enhance and suitability for this application, are warranted before explo- the photoacoustic contrast by approximately 300%, using ration of the technique in humans. bioconjugated gold nanocages targeted to the a-melanocyte–stimulating hormone receptors overexpressed on mela- Dermatologic imaging nomas (Fig. 9; ref. 53). Favazza and colleagues investigated Melanoma is the most aggressive form of cutaneous cancer, PAM in vivo in volunteers by imaging a melanocytic nevus and and the most difficult to diagnose and stage (123). Detection assessing the vascular and structural differences between palm typically relies on visual evaluation of the morphology of a and forearm skin (125). Recently, photoacoustic detection of lesion, followed by biopsy and histopathologic assessment of melanoma metastases based on their melanin content proved suspicious lesions. To improve melanoma detection, various to be possible in resected human lymph nodes (126). In

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summary, PAM can noninvasively (without the use of an human ovarian tissues were imaged successfully ex vivo, show- imaging agent) obtain functional information on tissue that ing the potential of the hybrid device over each modality alone may be helpful in the diagnosis and staging of melanoma. in ovarian tissue characterization. Kumavor and colleagues Biopsy is the standard method for histology and diagnosis of have recently developed a coregistered pulse-echo/photoa- melanoma, although it relies on biopsying a representative coustic transvaginal probe and in initial experiments human area of the often heterogeneous tumor. With the use of PAM, it ovaries ex vivo were imaged (136). The mentioned combined might be possible to "map" the lesion and guide what area to techniques for ovarian imaging show promise for clinical biopsy, potentially increasing the diagnostic accuracy and translation, but results remain to be validated in vivo and avoiding unnecessary biopsies. One of the challenges is that eventually tested for diagnostic accuracy compared with exist- a subgroup of melanomas does not contain melanin, and thus ing imaging techniques. PAI could potentially also play a role in require a different PAI approach, possibly involving the injec- other gynecologic diseases, such as cervical and endometrial tion of a molecular imaging agent. Another critical point in the cancer, but so far these areas have not been explored. development of more accurate diagnostic tools is the need for a high-resolution technique to decide whether the cancer cells Hematologic imaging have invaded through the basement membrane (123), enabling Circulating tumor cells (CTC) can often be detected in the them to spread to distant tissues. Further in vivo studies are blood of patients with early or advanced cancer by antibody- thus required to determine whether PAM can meet these based assays or molecular methods. CTC detection is an area of clinical needs. intense research, studying patients with different types of In addition to assessing malignant melanoma, PAI may also cancer, including breast, colorectal, prostate, and skin cancer. be valuable in the evaluation of other skin conditions, such as In several studies, CTC detection in early and/or metastatic burn wounds. Because clinical characterization of burn disease has been shown to correlate with unfavorable clinical wounds can be challenging, the assessment of tissue viability outcome (137). There are several different technologies for ex vivo by imaging intact dermal vasculature may help in determining CTC detection , but the two main approaches are the use an appropriate treatment plan. So far, PAM has been explored of monoclonal antibodies directed against histogenic proteins for burn wound assessment in rat and pig models with (e.g., cytokeratins and epithelial cell adhesion molecule) and fi promising results (127–130). Human studies have not been PCR-based molecular assays amplifying tissue-speci c tran- performed yet, but this seems to be within reach in the near scripts (138). The major drawback for both of these techniques future. is their limited sensitivity due to the analysis of only small blood volumes. To overcome this problem, flow cytometry Gynecologic imaging techniques could be used to assess larger blood volumes in vivo. Ovarian cancer has the highest mortality of all gynecologic The fluorescent labels commonly used in flow cytometry have cancers due to the late onset of symptoms in combination with certain limitations, such as their potential toxicity and immune a lack of effective methods for early detection (58). At present, response to the fluorescent tags. Photoacoustic flow cytometry there is no existing evidence that any screening test, including has the advantage that label-free detection of cells with high CA-125, ultrasound, or pelvic examination, reduces mortality absorption/scattering characteristics, such as the CTCs from from ovarian cancer (131). The currently used imaging tech- melanomas for instance, is possible. Malignant melanomas are niques, for instance ultrasound, have poor sensitivity and known to metastasize at early stages and the presence of CTCs specificity for the detection of early-stage cancers (132). seems to be a prognostic marker (139, 140). The intrinsic Because PAI can be easily integrated in a clinical ultrasound photoacoustic contrast, coming from the melanin in the cells, system, such a dual system could potentially enhance the makes photoacoustic flow cytometry an ideal candidate for the diagnostic accuracy of ovarian cancer detection. Aguirre and label-free detection of CTCs from melanoma, as has been colleagues developed a noninvasive transvaginal coregistered shown by several research groups in vitro (141–145) as well 3D ultrasound and PAI-system, and evaluated the optical as in vivo (146–148). Nedosekin and colleagues recently properties of normal porcine ovarian tissue with this system reported on in vivo ultrafast photoacoustic flow cytometry, in vivo (133). The results showed strong light absorption from showing the detection of circulating human melanoma cells in highly vascularized corpora lutea and low absorption from large blood vessels in mice by using the intrinsic photoacoustic follicles, demonstrating that PAI is capable of detecting vas- signal of melanin, and by targeting the tumor cells with cularized structures in the ovaries, otherwise not visible with magnetic nanoparticles (148). McCormack and colleagues ultrasound. These findings were further evaluated in 33 ex vivo used targeted gold nanoparticles to increase the signal from human ovaries (134). Quantification of the light absorption melanoma cells in a stationary in vitro system (144). The same levels in the ovary indicated that, in the postmenopausal group, group was able to use targeted gold nanoparticles in a breast malignant ovaries showed significantly higher light absorption cancer cell line and detect the nonpigmented CTCs with their than normal ones (P ¼ 0.0237) corresponding to a sensitivity of system. Although they did not specifically show the detection 83% and a specificity of 83%. The same group presented an of individual cells due to cell clumping, they claim that their integrated optical coherence tomography (OCT), ultrasound, system may be capable of this in clinical samples (149). Label- and PAI prototype endoscopy system, with a diameter suitable free or targeted photoacoustic CTC detection methods may be for insertion through a standard endoscopic laparoscopic port translated into human studies in the near future. Possible during minimally invasive surgery (5 mm; ref. 135). Porcine and clinical applications to be evaluated include blood screening

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[Nle4, D-Phe7]-α-MSH-AuNCs PEG-AuNCs A E

B F

Figure 9. In vivo noninvasive photoacoustic (PA) time-course coronal maximum amplitude projection (MAP) images of B16 melanomas using targeted [Nle4, D-Phe7]-a-melanocyte- stimulating hormone gold nanocages([Nle4, D-Phe7]- a-MSH-AuNCs) and nontargeted PEG gold nanocages (PEG-AuNCs). A and E, photographs of nude mice C G transplanted with B16 melanomas before injection of [Nle4,d-Phe7]-a-MSH-AuNCs (A) and PEG-AuNCs (E). Time- course photoacoustic images of the B16 melanomas after intravenous injection with 100 mL of 10 nmol/L [Nle4,d-Phe7]- a-MSH-AuNCs (B–D) and PEG- AuNCs (F–H) via tail vein. The background vasculature images were obtained at 570 nm (ultrasonic frequency ¼ 50 MHz), and the melanoma images were obtained at 778 nm (ultrasonic frequency ¼ 10 MHz). Red, blood vessels; yellow, the increase in D H photoacoustic amplitude. Reprinted with permission from Kim and colleagues (53). Copyright 2010 American Chemical Society.

2 mm

0 10 20 30 40 50 Increase in PA amplitude (%)

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for early CTCs before the development of metastases to predict Bone and joint imaging patient prognosis; testing for disease recurrence and individ- Peripheral joint imaging seems to be a highly accessible and ualized treatment monitoring through CTC counting; evalua- promising area for PAI, as demonstrated in several pilot studies tion of surgical margins for CTCs; and even the inhibition/ of human finger joints (166–169). In combination with con- prevention of the development of metastases through thera- ventional US, dual-modality imaging can potentially provide peutic elimination of CTCs (150). both morphologic and physiologic information on joints, such In addition to tumor cell detection in the circulation, PAI as blood flow, blood volume, hemoglobin oxygenation, and may also be very useful for visualization of the vessel wall itself, vascular density. It would be an advantage to be able to show as several reports on atherosclerotic plaque characterization that PAI early on can visualize the physiologic changes in the illustrate (37, 151, 152). Catheters that integrate intravascular articular tissue appearing before the late morphologic changes. ultrasound (IVUS) and intravascular photoacoustic (IVPA) This would help to diagnose and treat joint disease at an earlier imaging have been designed to approach tissues of interest stage before it has become more manifested. In addition, by through the blood vessels (153–159). In the future, these types visualizing these physiologic features, PAI may be able to play a of catheters could be used during intravascular procedures in a role in the diagnosis of primary bone cancer or bone metas- cardiovascular disease setting (e.g., coronary plaque charac- tases. A first, study by Hu and colleagues demonstrated in vivo terization and stent placement), but possibly also in an onco- PAI of osteosarcoma in a rat leg, monitoring tumor develop- logic setting (e.g., guidance of intravascular drug delivery ment and showing apparent changes in imaging characteris- directly to the tumor site). tics over the course of 2 weeks after cell injection (170). Although these initial results are encouraging, more research Ophthalmic imaging is needed to verify findings in human studies and to overcome Ultrasound, OCT, and fluorescent angiography play an certain critical challenges of PAI, such as the limited depth important role in diagnostic imaging of the eye. Because PAI penetration in bone (even more limited than in soft tissue). depicts optical absorption, which is independent of the tissue characteristics imaged by OCT or ultrasound, this technique Neonatal brain imaging can provide additional physiologic information in ocular imag- Ultrasound imaging is an established pediatric brain imaging. The eye is a very suitable application for optical imaging ing modality that is used before the fontanelles are closed. After due to its accessibility and optical properties. The major light the closure of the fontanelles, the image quality degrades absorbing molecules in the eye are melanin and hemoglobin. significantly because the skull severely attenuates and scatters Melanin is present in the uvea (iris, choroid, ciliary body) and in ultrasonic waves. Transcranial ultrasonic brain imaging of pigmented tumors, whereas hemoglobin is present in the adults is also limited by the inhomogeneous aberrating effect abundant microvasculature of the uveal tract, in superficial of the skull bone. Yang and Wang applied PAT to image the microvessels of the eye (retina, conjunctiva) and in pathologic brain cortex of a monkey through the intact scalp and skull ex neovascularization (including tumors). The physiologic infor- vivo. The reconstructed PAT image showed the major blood mation provided by PAI, for example, changes in microcircu- vessels on the monkey brain cortex (171). Because the human lation, hypoxia, and nevi formation, could prove highly valuable skull scatters light strongly, the same group added a photon in the diagnosis of ocular disease. Not only for the detection recycler to the PAT system, to reflect back-scattered light back and characterization of cancer of the eye (e.g., intraocular to the skull, which in turn increased light transmittance melanoma, lymphoma, retinoblastoma, and metastases), but through the skull (172). Recently Sussman and colleagues used also for other blinding diseases such as diabetic retinopathy, PAT in a neonatal rat model to demonstrate in vivo alterations age-related macular degeneration, and glaucoma. To date, in cerebral hemodynamics in a noninvasive and near real-time several efforts have been made to develop PAI methods suit- fashion (173). These results suggest that PAT may have the able for ophthalmologic applications, although so far only potential for human brain cortex imaging in infants or even tested in experimental settings. Our laboratories have devel- adults although further technical development is needed oped a photoacoustic ocular imaging device and demon- before proceeding to humans. strated its utility in imaging in living rabbits (the deeper layers of the eye including the retina, choroid, and optic Gastrointestinal imaging nerve; Fig. 10 (160). Hu and colleagues have shown label-free Endoscopic imaging is the main diagnostic tool in gastro- photoacoustic angiography of the eye in mice (161). This intestinal diseases. Mostly, endoscopic imaging relies on wide- is an alternative to the commonly used fluorescence angi- field white-light optical methods, which are limited by what the ography that requires injection of an imaging agent (fluo- human eye can see and therefore lack sensitivity to subsurface rescein or ICG), which may have side effects. Song and or physiologic changes. Attempts to overcome these limita- colleagues have built several PAI platforms for ophthalmic tions include techniques such as autofluorescence imaging, imaging in rodents, including hybrid systems that combine narrow-band imaging, and OCT (174, 175). Photoacoustic photoacoustic ophthalmoscopy with other techniques such endoscopic imaging may have the ability to visualize physio- as OCT and fluorescence imaging (162–165). Nevertheless, logic processes and to resolve molecular imaging agents that there is a need for further optimization and evaluation of target specific molecular changes in tissue. Subsurface imaging ocular PAI systems before they can be tested in humans and is very well possible with this technique because of the favor- evaluated against existing diagnostic techniques. able penetration depth (up to several centimeters). The

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Figure 10. A, photographic, horizontal photoacoustic (B), vertical ultrasound (C), and vertical photoacoustic (D) images of an eye of a living CD rabbit. The area of the eye imaged in B is outlined by the red boundary (dotted box) in A, and the depth is noted by the dashed red line in C. Reprinted with permission from de la Zerda and colleagues (160). Copyright 2010 The Optical Society.

0 100 Photoacoustic signal (AU)

combination of deeper tissue penetration, high resolution, and device that can acquire photoacoustic and ultrasonic data real-time imaging makes PAI a valuable add-on to endoscopy. simultaneously. This device was tested in vivo in rabbit esoph- It may allow for the detection of very small lesions (e.g., micro- agi and in rat colons. With this technique, the esophagus and metastases) or otherwise invisible "ﬂat" lesions, probably even surrounding organs could be visualized, including the adjacent more so with the use of targeted molecular imaging agents. vasculature that was only visible in photoacoustic mode. Also, Intraoperatively, PAI could help in determining the extent of the vasculature in the colon wall could be seen, and after the tumor before surgical removal, and the completeness of the injection of Evans blue dye, the adjacent lymph structures excision could be checked immediately. Also, this technique became visible. Moreover, differences in oxygen saturation could assist in guiding the anastomosis procedures. values could be indicated between the aorta and caudal Only a few studies have been published in this application vena cava in the rabbits (176). Yuan and colleagues and Sheaff area. Recently, Yang and colleagues presented their endoscopy and colleagues both describe a preclinical photoacoustic

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endoscope that they are developing (177, 178). The endoscope they point out the need for development of a handheld probe built by Yuan and colleagues was able to discriminate cancer- with increased depth resolution before clinical translation of ous human colorectal tissue from normal human colorectal the technique. Furthermore, to deal with the strong effect of tissue ex vivo, with contrast ratios of approximately 2 (177). No blood (from intraoperative bleeding) on PAI absorption, they clinical studies have been performed yet. suggest the use of targeted molecular imaging agents to provide enough contrast. In a recent review, Su and colleagues Thyroid imaging discuss potential future intraoperative imaging methods for Follicular thyroid carcinomas cannot be distinguished from malignant gliomas, a common type of brain tumor. Among adenomas based on their clinical, imaging, or cytologic assess- other techniques, they point out that PAI in combination with ment, but always require surgical biopsy for diagnosis. optical imaging agents could be promising (180). This appli- Because the majority of nodules (3/4) are benign, the need cation has been explored by our laboratory, with the develop- for new noninvasive detection methods to avoid unnecessary ment of a triple-modality MRI–PAI –Raman imaging nano- surgeries is clear, and PAI may be a valid candidate. To date, particle that can accurately help delineate the margins of brain there are no studies published on photoacoustic thyroid tumors in living mice both preoperatively and intraoperatively imaging, but this application is currently under exploration (see also Fig. 5; ref. 86). by our research group (36). We developed an enzyme-activa- Besides tumor delineation and assessment of its margins, table photoacoustic probe that was previously described in PAI could also play a role in the assessment of metastases, in this review (35). The presence and activity of two members particular the lymph node status. Previously, we discussed the of the MMP family, MMP-2 and MMP-9, suggested to differ- SLN procedure for breast cancer. The lymph node status for entiate between malignant and benign lesions, were studied in other types of cancer could also be explored by PAI during a thyroid tumor mouse model. In vivo PAI results showed tumor resection, for instance, in the case of an abdominal significantly higher signal in tumors injected with the enzyme- tumor, the surgeon would be able to make quick decisions on activatable probe than in tumors injected with the control the resection of suspicious (mesenteric) lymph nodes during (noncleavable) probe, indicating the probe's potential to laparotomy or laparoscopy. For this situation, most likely an distinguish between benign adenomas and MMP-rich follic- imaging agent would have to be injected to provide enough ular carcinomas. We believe that with the combination of high sensitivity and specificity. Grootendorst and colleagues have spatial resolution and signal specificity, molecular PAI holds shown in healthy rats that superparamagnetic iron oxide great promise as a noninvasive method for early diagnosis of nanoparticles (Endorem) can be used as a PAI agent for lymph follicular thyroid carcinomas. node visualization (87). More recently, they have shown promising results using the same nanoparticles in an ex vivo Intraoperative imaging intraoperative setting in a metastatic prostate cancer model Intraoperative is another area where PAI has great potential, in rats (181). particularly because it can help the surgeon to see immediately beyond the resection surface. A portable PAI could be brought Combination of diagnostics and therapy into the operating room and provide real-time images similar PAI (combined with other modalities) could also be used in a to intraoperative ultrasound. However, the optical contrast clinical setting to guide treatment and to combine diagnostic provides additional information over ultrasound images and information with therapy ("theranostics"). PAI has recently could for instance help assess oxygen saturation and perfusion been proposed to guide and/or monitor therapy based on high- status of an organ (viability), useful in, for example, bowel intensity focused ultrasound (HIFU; ref. 182). Prost and col- resections, organ transplants, and amputations. Surgical resec- leagues show a lesion obtained in vitro in chicken breast by tion also plays a critical role in the treatment of many cancers. focusing HIFU based on the photoacoustic signals emitted by It is of ultimate importance to reassure complete resection of an optical absorber placed in the tissue (183). Gold nanopar- the tumor mass to diminish the risk for local recurrence and ticle–mediated hyperthermia shows particular promise in improve survival rates. CT, MRI, or PET-CT imaging (depend- animal studies and is currently being investigated in early ing on tumor type) can give information on tumor extent clinical studies (184). The visualization and tracking of tem- before surgery, but during the operative procedure, the sur- porarily or permanently implanted metal devices, such as geon currently relies on visual appearance and palpation. biopsy needles and needles used for localized drug delivery, Several intraoperative imaging methods have been evaluated, have been studied with PAI (185). The results suggest that PAI, such as ultrasound and intraoperative MRI, but they have been combined with ultrasound imaging, is capable of real-time, found either too time consuming or lacking sufficient spatial high-contrast, and high-spatial resolution visualization of resolution. PAI could potentially provide a solution to this metal implants within anatomic landmarks of the background problem. The exploration of PAI in the intraoperative setting is tissue. still in its infancy. Xi and colleagues report a microelectrome- chanical system–based intraoperative PAT (iPAT) technique, and demonstrate its ability to accurately map tumors in 3D and Outlook to inspect the completeness of tumor resection during surgery PAI holds the promise of becoming a valuable tool in in a tumor-bearing mouse model (179). They propose potential multiple clinical areas in which there are unmet needs. This use of the iPAT technique in breast cancer surgery although technique has several important strengths over the currently

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available imaging techniques; in particular, its ability to obtain information on tissue. To enhance photoacoustic image con- functional and molecular information from tissue, even with- trast, exogenous molecular imaging agents are under devel- out the use of a molecular imaging agent, along with its low opment and will need to be approved for use in humans. cost, portability, lack of ionizing radiation, and feasibility of Several key concerns about these molecular imaging agents real-time imaging at high resolution and clinically relevant remain. These include (i) the ability to produce sufficient PAI depths. PAI can be used not only in a noninvasive way, but also signal with a relatively low dose of injected imaging agent. It in an intraoperative or endoscopic imaging setting. The com- may be the case that for some agents, insufficient amounts can bination of PAI with existing imaging techniques (multimod- be delivered to the target site(s) because of the limits in the ality imaging) or delivery of therapeutics will likely add even total mass that can be injected while still being nontoxic to the more to its potential. subject. (ii) Regulatory issues that have held up the translation Besides these major advantages, there are still important of optical imaging agents will likely slow down the clinical challenges to overcome. Clinicians will need to get familiar translation of PAI agents as well. (iii) The ability to show the with the PAI technique and become aware of its capabilities added value of using an imaging agent in addition to intrinsic and limitations. The most obvious limitation for clinical appli- PAI signal will have to be carefully studied. Nevertheless, once cations of PAI is its penetration depth, dependent on the relevant molecular imaging agents become available for clin- limited penetration of light in tissue, which will likely not ical PAI, this technique may play an even more significant role reach beyond several cm (7 cm; Table 1) in the NIR wave- in a variety of diseases with respect to their detection, staging, length range. Because spatial resolution is associated with treatment planning, and monitoring (see also Fig. 3). penetration depth in PAI, the detection of smaller lesions (a In conclusion, the recent surge in interest in PAI has few mm) located deeper in the tissue will need to be investi- provided us with a wealth of experience and knowledge from gated. As for the propagation of ultrasound waves in tissue, the preclinical studies. Promising results and technologic same limitations apply as in ultrasound imaging, that is, advances lay the foundation for clinical translation of this limited penetration through bone and air. technique. Although challenges remain, few of them are Although the PAI technology has already reached a level that beyond reach. Further developments in laser sources and can be used in clinical studies, there is still much room for detectors, image processing algorithms, and molecular imag- improvement. Fast and tunable systems that can provide mul- ing agents, will open up opportunities for PAI in disease tispectral images in real-time should be developed, and PAI detection, treatment surveillance, and targeted therapy in a systems need to become stable and user-friendly with regard to range of pathologies. Its ability for high-resolution molecular data acquisition and analysis; in particular, progress in quan- imaging in real-time, its nonionizing properties, and its com- titative data analysis is essential. The development of miniatur- patibility with existing imaging systems (ultrasound in par- ized probes is needed for endoscopic, intravesicular, and intra- ticular), will help strengthen PAI's position in the fast-growing vascular PAI. For intravascular clinical use, the photoacoustic imaging arena in the near future. probeshouldbescaleddownto<1mmindiameter(157). Image contrast and sensitivity of detection represent anoth- Disclosure of Potential Conflicts of Interest er area of needed improvement for PAI. Because one of the S.S. Gambhir has ownership interest (including patents) in Endra Inc. and is a main advantages of PAI is that endogenous chromophores can consultant/advisory board member of Visualsonics Inc. No potential conflicts of be imaged without the injection of an exogenous imaging interest were disclosed by the other authors. agent, this intrinsic contrast will most likely be the primary focus of initial clinical studies. Therefore, we will need to learn Authors' Contributions more about the distribution of intrinsic features, such as Conception and design: S. Zackrisson, S.M.W.Y. van de Ven, S.S. Gambhir neoangiogenesis and hypoxia/oxygen saturation, within and Writing, review, and/or revision of the manuscript: S. Zackrisson, S.M.W.Y. between the various cancer types and benign lesions. Tumors van de Ven, S.S. Gambhir that are less vascularized could be a caveat, but overall this Received August 19, 2013; revised September 9, 2013; accepted November 25, intrinsic contrast may provide us with substantial functional 2013; published OnlineFirst February 10, 2014.

References 1. Bell AG. On the production of sound by light. Am J Sci 1880;XX: 5. Maslov K, Zhang HF, Hu S, Wang LV. Optical-resolution photoa- 305–24. coustic microscopy for in vivo imaging of single capillaries. Opt Lett 2. Zhang HF, Maslov K, Stoica G, Wang LV. Functional photoacoustic 2008;33:929–31. microscopy for high-resolution and noninvasive in vivo imaging. Nat 6. Wang LV, Hu S. Photoacoustic tomography: in vivo imaging from Biotechnol 2006;24:848–51. organelles to organs. Science 2012;335:1458–62. 3. Ku G, Wang LV. Deeply penetrating photoacoustic tomography in 7. Kim C, Erpelding TN, Jankovic L, Pashley MD, Wang LV. Deeply biological tissues enhanced with an optical contrast agent. Opt Lett penetrating in vivo photoacoustic imaging using a clinical ultrasound 2005;30:507–9. array system. Biomed Opt Express 2010;1:278–84. 4. Emelianov SYA, Karpiouk SR, Mallidi AB, Park S, Sethuraman S, Shah 8. Manohar S, Vaartjes SE, van Hespen JC, Klaase JM, van den Engh S, et al. Synergy and applications of combined ultrasound, elasticity, FM, Steenbergen W, et al. Initial results of in vivo non-invasive cancer and photoacoustic imaging. In: Proceedings of the IEEE International imaging in the human breast using near-infrared photoacoustics. Opt Ultrasonics Symposium [Internet] 2006; Vancouver, Canada. Express 2007;15:12277–85.

www.aacrjournals.org Cancer Res; 74(4) February 15, 2014 999

Zackrisson et al.

9. KrugerRA,LamRB,ReineckeDR,DelRioSP,DoyleRP. 34. Yang Q, Cui H, Cai S, Yang X, Forrest ML. In vivo photoacoustic Photoacoustic angiography of the breast. Med Phys 2010;37: imaging of chemotherapy-induced apoptosis in squamous cell car- 6096–100. cinoma using a near-infrared caspase-9 probe. J Biomed Opt 2011; 10. Esenaliev RO, Karabutov AA, Oraevsky AA. Sensitivity of laser opto- 16:116026. acoustic imaging in detection of small deeply embedded tumors. 35. Levi J, Kothapalli SR, Ma TJ, Hartman K, Khuri-Yakub BT, Gambhir IEEE J Selected Top Quantum Electron 1999;5:981–8. SS. Design, synthesis, and imaging of an activatable photoacoustic 11. Wang LV. Multiscale photoacoustic microscopy and computed probe. J Am Chem Soc 2010;132:11264–9. tomography. Nat Photonics 2009;3:503–09. 36. Levi J, Kothapalli SR, Bohndiek S, Yoon JK, Dragulescu-Andrasi A, 12. Su JL, Wang B, Wilson KE, Bayer CL, Chen YS, Kim S, et al. Advances Nielsen C, et al. Molecular photoacoustic imaging of follicular thyroid in clinical and biomedical applications of photoacoustic imaging. carcinoma. Clin Cancer Res 2013;19:1494–502. Expert Opin Med Diagn 2010;4:497–510. 37. Razansky D, Harlaar NJ, Hillebrands JL, Taruttis A, Herzog E, Zeeb- 13. Kruger RA, Kiser WL Jr, Reinecke DR, Kruger GA. Thermoacoustic regts CJ, et al. Multispectral optoacoustic tomography of matrix computed tomography using a conventional linear transducer array. metalloproteinase activity in vulnerable human carotid plaques. Mol Med Phys 2003;30:856–60. Imaging Biol 2012;14:277–85. 14. Yin B, Xing D, Wang Y, Zeng Y, Tan Y, Chen Q. Fast photoacoustic 38. Kim C, Song KH, Gao F, Wang LV. Sentinel lymph nodes and imaging system based on 320-element linear transducer array. Phys lymphatic vessels: noninvasive dual-modality in vivo mapping by Med Biol 2004;49:1339–46. using indocyanine green in rats–volumetric spectroscopic photoa- 15. Kothapalli S, Ma T, Vaithilingam S, Oralkan O, Khuri-Yakub B, coustic imaging and planar fluorescence imaging. Radiology 2010; Gambhir S. Deep tissue photoacoustic imaging using a miniaturized 255:442–50. 2-D capacitive micromachined ultrasonic transducer array. IEEE 39. Wang X, Ku G, Wegiel MA, Bornhop DJ, Stoica G, Wang LV. Non- Trans Biomed Eng 2012;59:1199–204. invasive photoacoustic angiography of animal brains in vivo with 16. Zemp RJ, Bitton R, Li ML, Shung KK, Stoica G, Wang LV. Photo- near-infrared light and an optical contrast agent. Opt Lett 2004;29: acoustic imaging of the microvasculature with a high-frequency 730–2. ultrasound array transducer. J Biomed Opt 2007;12:010501. 40. Song KH, Stein EW, Margenthaler JA, Wang LV. Noninvasive photo- 17. Li C, Wang LV. Photoacoustic tomography and sensing in biomed- acoustic identification of sentinel lymph nodes containing methylene icine. Phys Med Biol 2009;54:R59–97. blue in vivo in a rat model. J Biomed Opt 2008;13:054033. 18. Gibson AP, Hebden JC, Arridge SR. Recent advances in diffuse 41. Song L, Kim C, Maslov K, Shung KK, Wang LV. High-speed dynamic optical imaging. Phys Med Biol 2005;50:R1–43. 3D photoacoustic imaging of sentinel lymph node in a murine model 19. Schulze R, Zangerl G, Holotta M, Meyer D, Handle F, Nuster R, et al. using an ultrasound array. Med Phys 2009;36:3724–9. On the use of frequency-domain reconstruction algorithms for photo- 42. Li C, Aguirre A, Gamelin J, Maurudis A, Zhu Q, Wang LV. Real-time acoustic imaging. J Biomed Opt 2011;16:086002. photoacoustic tomography of cortical hemodynamics in small ani- 20. Xia J, Guo Z, Maslov K, Aguirre A, Zhu Q, Percival C, et al. Three- mals. J Biomed Opt 2010;15:010509. dimensional photoacoustic tomography based on the focal-line 43. Li M, Oh JT, Xie X, Ku G, Wang W, Li C, et al. Simultaneous molecular concept. J Biomed Opt 2011;16:090505. and hypoxia imaging of brain tumors in vivo using spectroscopic 21. Yao L, Jiang H. Photoacoustic image reconstruction from few-detec- photoacoustic tomography. Proc IEEE 2008;96:481–9. tor and limited-angle data. Biomed Opt Express 2011;2:2649–54. 44. Kim G, Huang SW, Day KC, O'Donnell M, Agayan RR, Day MA, et al. 22. Zhu B, Sevick-Muraca EM. Reconstruction of sectional images in Indocyanine-green-embedded PEBBLEs as a contrast agent for frequency-domain based photoacoustic imaging. Opt Express 2011; photoacoustic imaging. J Biomed Opt 2007;12:044020. 19:23286–97. 45. Gupta S, Chatni MR, Rao AL, Vullev VI, Wang LV, Anvari B. Virus- 23. Sheu YL, Chou CY, Hsieh BY, Li PC. Image reconstruction in intra- mimicking nano-constructs as a contrast agent for near infrared vascular photoacoustic imaging. IEEE Trans Ultrason Ferroelectr photoacoustic imaging. Nanoscale 2013;5:1772–6. Freq Control 2011;58:2067–77. 46. Bayer CL, Chen YS, Kim S, Mallidi S, Sokolov K, Emelianov S. 24. Oraevsky A, Karabutov A. Optoacoustic tomography. In:Vo-Dinh T, Multiplex photoacoustic molecular imaging using targeted silica- editor. Biomedical photonics handbook: Boca Raton, FL: CRC Press; coated gold nanorods. Biomed Opt Express 2011;2:1828–35. 2003. 47. Xie H, Diagaradjane P, Deorukhkar AA, Goins B, Bao A, Phillips WT, 25. Wang LV, Wu HI. Biomedical optics: principles and imaging. Hobo- et al. Integrin alphavbeta3-targeted gold nanoshells augment tumor ken, NJ: Wiley; 2007. vasculature-specific imaging and therapy. Int J Nanomedicine 2011; 26. Wang LV. Prospects of photoacoustic tomography. Med Phys 6:259–69. 2008;35:5758–67. 48. Li ML, Wang JC, Schwartz JA, Gill-Sharp KL, Stoica G, Wang LV. In- 27. Xu MH, Wang LH. Photoacoustic imaging in biomedicine. Rev Sci vivo photoacoustic microscopy of nanoshell extravasation from solid Instrum 2006;77:041101. tumor vasculature. J Biomed Opt 2009;14:010507. 28. Emelianov SY, Li PC, O'Donnell M. Photoacoustics for molecular 49. Eghtedari M, Liopo AV, Copland JA, Oraevsky AA, Motamedi M. imaging and therapy. Phys Today 2009;62:34–9. Engineering of hetero-functional gold nanorods for the in vivo 29. Rice A, Quinn CM. Angiogenesis, thrombospondin, and ductal car- molecular targeting of breast cancer cells. Nano Lett 2009;9: cinoma in situ of the breast. J Clin Pathol 2002;55:569–74. 287–91. 30. Wang B, Su JL, Amirian J, Litovsky SH, Smalling R, Emelianov S. 50. Eghtedari M, Oraevsky A, Copland JA, Kotov NA, Conjusteau A, Detection of lipid in atherosclerotic vessels using ultrasound-guided Motamedi M. High sensitivity of in vivo detection of gold nanorods spectroscopic intravascular photoacoustic imaging. Opt Express using a laser optoacoustic imaging system. Nano Lett 2007;7: 2010;18:4889–97. 1914–8. 31. Ermilov SA, Khamapirad T, Conjusteau A, Leonard MH, Lacewell R, 51. Li PC, Wang CR, Shieh DB, Wei CW, Liao CK, Poe C, et al. In vivo Mehta K, et al. Laser optoacoustic imaging system for detection of photoacoustic molecular imaging with simultaneous multiple selec- breast cancer. J Biomed Opt 2009;14:024007. tive targeting using antibody-conjugated gold nanorods. Opt Express 32. Kumon RE, Deng CX, Wang X. Frequency-domain analysis of photo- 2008;16:18605–15. acoustic imaging data from prostate adenocarcinoma tumors in a 52. Copland JA, Eghtedari M, Popov VL, Kotov N, Mamedova N, Mota- murine model. Ultrasound Med Biol 2011;37:834–9. medi M, et al. Bioconjugated gold nanoparticles as a molecular based 33. Bhattacharyya S, Wang S, Reinecke D, Kiser W Jr, Kruger RA, contrast agent: implications for imaging of deep tumors using optoa- DeGrado TR. Synthesis and evaluation of near-infrared (NIR) dye- coustic tomography. Mol Imaging Biol 2004;6:341–9. herceptin conjugates as photoacoustic computed tomography (PCT) 53. Kim C, Cho EC, Chen J, Song KH, Au L, Favazza C, et al. In vivo probes for HER2 expression in breast cancer. Bioconjug Chem molecular photoacoustic tomography of melanomas targeted by 2008;19:1186–93. bioconjugated gold nanocages. ACS Nano 2010;4:4559–64.

1000 Cancer Res; 74(4) February 15, 2014 Cancer Research

Translational Strategies for Photoacoustic Imaging

54. Skrabalak SE, Chen J, Sun Y, Lu X, Au L, Cobley CM, et al. Gold 75. Schipper ML, Nakayama-Ratchford N, Davis CR, Kam NW, Chu P, Liu nanocages: synthesis, properties, and applications. Acc Chem Res Z, et al. A pilot toxicology study of single-walled carbon nanotubes in 2008;41:1587–95. a small sample of mice. Nat Nanotechnol 2008;3:216–21. 55. Yang X, Skrabalak SE, Li ZY, Xia Y, Wang LV. Photoacoustic tomog- 76. Altinoglu EI, Russin TJ, Kaiser JM, Barth BM, Eklund PC, Kester M, raphy of a rat cerebral cortex in vivo with au nanocages as an optical et al. Near-infrared emitting ﬂuorophore-doped calcium phosphate contrast agent. Nano Lett 2007;7:3798–802. nanoparticles for in vivo imaging of human breast cancer. ACS Nano 56. Jokerst JV, Thangaraj M, Kempen PJ, Sinclair R, Gambhir SS. 2008;2:2075–84. Photoacoustic imaging of mesenchymal stem cells in living mice via 77. Bottrill M, Green M. Some aspects of quantum dot toxicity. Chem silica-coated gold nanorods. ACS Nano 2012;6:5920–30. Commun 2011;47:7039–50. 57. Jokerst JV, Cole AJ, Van de Sompel D, Gambhir SS. Gold nanorods 78. Fernandez-Fernandez A, Manchanda R, McGoron AJ. Theranostic for ovarian cancer detection with photoacoustic imaging and resec- applications of nanomaterials in cancer: drug delivery, image-guided tion guidance via Raman imaging in living mice. ACS Nano 2012; therapy, and multifunctional platforms. Appl Biochem Biotechnol 6:10366–77. 2011;165:1628–51. 58. Kim C, Song HM, Cai X, Yao J, Wei A, Wang LV. In vivo photoacoustic 79. Yavuz MS, Cheng Y, Chen J, Cobley CM, Zhang Q, Rycenga M, et al. mapping of lymphatic systems with plasmon-resonant nanostars. Gold nanocages covered by smart polymers for controlled release J Mater Chem 2011;21:2841–4. with near-infrared light. Nat Mater 2009;8:935–9.

59. Ma LL, Feldman MD, Tam JM, Paranjape AS, Cheruku KK, Larson TA, 80. Wang C, Chen J, Talavage T, Irudayaraj J. Gold nanorod/Fe3O4 et al. Small multifunctional nanoclusters (nanoroses) for targeted nanoparticle "nano-pearl-necklaces" for simultaneous targeting, cellular imaging and therapy. ACS Nano 2009;3:2686–96. dual-mode imaging, and photothermal ablation of cancer cells. 60. Ma LL, Tam JO, Willsey BW, Rigdon D, Ramesh R, Sokolov K, et al. Angew Chem Int Ed Engl 2009;48:2759–63. Selective targeting of antibody conjugated multifunctional nanoclus- 81. Jin Y, Jia C, Huang SW, O'Donnell M, Gao X. Multifunctional nano- ters (nanoroses) to epidermal growth factor receptors in cancer cells. particles as coupled contrast agents. Nat Commun 2010;1:41. Langmuir 2011;27:7681–90. 82. Qu M, Mallidi S, Mehrmohammadi M, Truby R, Homan K, Joshi P, 61. Bouchard LS, Anwar MS, Liu GL, Hann B, Xie ZH, Gray JW, et al. et al. Magneto-photo-acoustic imaging. Biomed Opt Express Picomolar sensitivity MRI and photoacoustic imaging of cobalt 2011;2:385–96. nanoparticles. Proc Natl Acad Sci U S A 2009;106:4085–9. 83. Kim C, Qin R, Xu JS, Wang LV, Xu R. Multifunctional microbubbles 62. Yoon SJ, Mallidi S, Tam JM, Tam JO, Murthy A, Johnston KP, et al. and nanobubbles for photoacoustic and ultrasound imaging. Utility of biodegradable plasmonic nanoclusters in photoacoustic J Biomed Opt 2010;15:010510. imaging. Opt Lett 2010;35:3751–3. 84. Wilson K, Homan K, Emelianov S. Biomedical photoacoustics 63. Chen YS, Frey W, Kim S, Homan K, Kruizinga P, Sokolov K, et al. beyond thermal expansion using triggered nanodroplet vaporization Enhanced thermal stability of silica-coated gold nanorods for photo- for contrast-enhanced imaging. Nat Commun 2012;3:618. acoustic imaging and image-guided therapy. Opt Express 2010;18: 85. Agarwal A, Shao X, Rajian JR, Zhang H, Chamberland DL, Kotov NA, 8867–78. et al. Dual-mode imaging with radiolabeled gold nanorods. J Biomed 64. Chen YS, Frey W, Kim S, Kruizinga P, Homan K, Emelianov S. Silica- Opt 2011;16:051307. coated gold nanorods as photoacoustic signal nanoamplifiers. Nano 86. Kircher MF, De la Zerda A, Jokerst J, Zavaleta CL, Kempen P, Mittra E, Lett 2011;11:348–54. et al. A brain tumor molecular imaging strategy using a novel triple- 65. Zhang B, Fang CY, Chang CC, Peterson R, Maswadi S, Glickman RD, modality nanoparticle. Nat Med 2012;18:829–34. et al. Photoacoustic emission from fluorescent nanodiamonds 87. Grootendorst DJ, Jose J, Fratila RM, Visscher M, Velders AH, Ten enhanced with gold nanoparticles. Biomed Opt Express 2012;3: Haken B, et al. Evaluation of superparamagnetic iron oxide nano- 1662–29. particles (Endorem(R)) as a photoacoustic contrast agent for intra- 66. Zhang T, Cui H, Fang CY, Su LJ, Ren S, Chang HC, et al. Photo- operative nodal staging. Contrast Media Mol Imaging 2013;8:83–91. acoustic contrast imaging of biological tissues with nanodiamonds 88. Li L, Zhang HF, Zemp RJ, Maslov K, Wang L. Simultaneous imaging of fabricated for high near-infrared absorbance. J Biomed Opt 2013; a lacZ-marked tumor and microvasculature morphology in vivo by 18:26018. dual-wavelength photoacoustic microscopy. J Innov Opt Health Sci 67. Pramanik M, Swierczewska M, Green D, Sitharaman B, Wang LV. 2008;1:207–15. Single-walled carbon nanotubes as a multimodal-thermoacoustic 89. Cai X, Li L, Krumholz A, Guo Z, Erpelding TN, Zhang C, et al. Multi- and photoacoustic-contrast agent. J Biomed Opt 2009;14: scale molecular photoacoustic tomography of gene expression. 034018. PLoS ONE 2012;7:e43999. 68. De la Zerda A, Liu Z, Bodapati S, Teed R, Vaithilingam S, Khuri-Yakub 90. Filonov GS, Krumholz A, Xia J, Yao J, Wang LV, Verkhusha VV. Deep- BT, et al. Ultrahigh sensitivity carbon nanotube agents for photoa- tissue photoacoustic tomography of a genetically encoded near- coustic molecular imaging in living mice. Nano Lett 2010;10:2168–72. infrared fluorescent probe. Angew Chem Int Ed Engl 2012;51: 69. De la Zerda A, Zavaleta C, Keren S, Vaithilingam S, Bodapati S, Liu Z, 1448–51. et al. Carbon nanotubes as photoacoustic molecular imaging agents 91. Ha SH, Carson AR, Kim K. Ferritin as a novel reporter gene for in living mice. Nat Nanotechnol 2008;3:557–62. photoacoustic molecular imaging. Cytometry A 2012;81:910–5. 70. Koo J, Jeon M, Oh Y, Kang HW, Kim J, Kim C, et al. In vivo non- 92. Qin C, Cheng K, Chen K, Hu X, Liu Y, Lan X, et al. Tyrosinase as a ionizing photoacoustic mapping of sentinel lymph nodes and blad- multifunctional reporter gene for Photoacoustic/MRI/PET triple ders with ICG-enhanced carbon nanotubes. Phys Med Biol modality molecular imaging. Sci Rep 2013;3:1490. 2012;57:7853–62. 93. Kitai T, Torii M, Sugie T, Kanao S, Mikami Y, Shiina T, et al. Photo- 71. Kim JW, Galanzha EI, Shashkov EV, Moon HM, Zharov VP. Golden acoustic mammography: initial clinical results. Breast Cancer 2012 carbon nanotubes as multimodal photoacoustic and photothermal Apr 7. [Epub ahead of print]. high-contrast molecular agents. Nat Nanotechnol 2009;4:688–94. 94. Heijblom M, Piras D, Xia W, van Hespen JC, Klaase JM, van den Engh 72. Shashkov EV, Everts M, Galanzha EI, Zharov VP. Quantum dots as FM, et al. Visualizing breast cancer using the Twente photoacoustic multimodal photoacoustic and photothermal contrast agents. Nano mammoscope: what do we learn from twelve new patient Lett 2008;8:3953–8. measurements? Opt Express 2012;20:11582–97. 73. Ku G, Zhou M, Song S, Huang Q, Hazle J, Li C. Copper sulfide 95. Manohar S, Kharine A, van Hespen JC, Steenbergen W, van Leeuwen nanoparticles as a new class of photoacoustic contrast agent for TG. Photoacoustic mammography laboratory prototype: imaging of deep tissue imaging at 1064 nm. ACS Nano 2012;6:7489–96. breast tissue phantoms. J Biomed Opt 2004;9:1172–81. 74. Wick P, Clift MJ, Rosslein M, Rothen-Rutishauser B. A brief summary 96. Jose J, Manohar S, Kolkman RG, Steenbergen W, van Leeuwen TG. of carbon nanotubes science and technology: a health and safety Imaging of tumor vasculature using Twente photoacoustic systems. perspective. ChemSusChem 2011;4:905–11. J Biophotonics 2009;2:701–17.

www.aacrjournals.org Cancer Res; 74(4) February 15, 2014 1001

Zackrisson et al.

97. Haisch C, Eilert-Zell K, Vogel MM, Menzenbach P, Niessner R. 119. Harrison T, Zemp RJ. Coregistered photoacoustic-ultrasound imag- Combined optoacoustic/ultrasound system for tomographic absorp- ing applied to brachytherapy. J Biomed Opt 2011;16:080502. tion measurements: possibilities and limitations. Anal Bioanal Chem 120. Kuo N, Kang HJ, Song DY, Kang JU, Boctor EM. Real-time photo- 2010;397:1503–10. acoustic imaging of prostate brachytherapy seeds using a clinical 98. Ku G, Fornage BD, Jin X, Xu M, Hunt KK, Wang LV. Thermoacoustic ultrasound system. J Biomed Opt 2012;17:066005. and photoacoustic tomography of thick biological tissues toward 121. Xie Z, Roberts W, Carson P, Liu X, Tao C, Wang X. Evaluation of breast imaging. Technol Cancer Res Treat 2005;4:559–66. bladder microvasculature with high-resolution photoacoustic imag- 99. Pramanik M, Ku G, Li C, Wang LV. Design and evaluation of a novel ing. Opt Lett 2011;36:4815–7. breast cancer detection system combining both thermoacoustic (TA) 122. Kim C, Jeon M, Wang LV. Nonionizing photoacoustic cystography in and photoacoustic (PA) tomography. Med Phys 2008;35:2218–23. vivo. Opt Lett 2011;36:3599–601. 100. Xia W, Piras D, van Hespen JC, van Veldhoven S, Prins C, van 123. Smith L, Macneil S. State of the art in non-invasive imaging of Leeuwen TG, et al. An optimized ultrasound detector for photoa- cutaneous melanoma. Skin Res Technol 2011;17:257–69. coustic breast tomography. Med Phys 2013;40:032901. 124. Yao J, Maslov KI, Zhang Y, Xia Y, Wang LV. Label-free oxygen- 101. Montilla LG, Olafsson R, Bauer DR, Witte RS. Real-time photoacous- metabolic photoacoustic microscopy in vivo. J Biomed Opt 2011; tic and ultrasound imaging: a simple solution for clinical ultrasound 16:076003. systems with linear arrays. Phys Med Biol 2013;58:N1–12. 125. Favazza CP, Jassim O, Cornelius LA, Wang LV. In vivo photoacoustic 102. Cheng G, Kurita S, Torigian DA, Alavi A. Current status of sentinel microscopy of human cutaneous microvasculature and a nevus. lymph-node biopsy in patients with breast cancer. Eur J Nucl Med J Biomed Opt 2011;16:016015. Mol Imaging 2011;38:562–75. 126. Grootendorst DJ, Jose J, Wouters MW, van Boven H, Van der Hage J, 103. Purushotham AD, Upponi S, Klevesath MB, Bobrow L, Millar K, Myles Van Leeuwen TG, et al. First experiences of photoacoustic imaging JP, et al. Morbidity after sentinel lymph node biopsy in primary breast for detection of melanoma metastases in resected human lymph cancer: results from a randomized controlled trial. J Clin Oncol nodes. Lasers Surg Med 2012;44:541–9. 2005;23:4312–21. 127. Aizawa K, Sato S, Saitoh D, Ashida H, Obara M. Photoacoustic 104. Crane-Okada R, Wascher RA, Elashoff D, Giuliano AE. Long-term monitoring of burn healing process in rats. J Biomed Opt 2008;13: morbidity of sentinel node biopsy versus complete axillary dissection 064020. for unilateral breast cancer. Ann Surg Oncol 2008;15:1996–2005. 128. Hirao A, Sato S, Saitoh D, Shinomiya N, Ashida H, Obara M. In vivo 105. Song KH, Kim C, Cobley CM, Xia Y, Wang LV. Near-infrared gold photoacoustic monitoring of photosensitizer distribution in burned nanocages as a new class of tracers for photoacoustic sentinel lymph skin for antibacterial photodynamic therapy. Photochem Photobiol node mapping on a rat model. Nano Lett 2009;9:183–8. 2010;86:426–30. 106. Erpelding TN, Kim C, Pramanik M, Jankovic L, Maslov K, Guo Z, et al. 129. Yamazaki M, Sato S, Ashida H, Saito D, Okada Y, Obara M. Mea- Sentinel lymph nodes in the rat: noninvasive photoacoustic and US surement of burn depths in rats using multiwavelength photoacoustic imaging with a clinical US system. Radiology 2010;256:102–10. depth profiling. J Biomed Opt 2005;10:064011. 107. Kim C, Erpelding TN, Maslov K, Jankovic L, Akers WJ, Song L, et al. 130. Zhang HF, Maslov K, Stoica G, Wang LV. Imaging acute thermal Handheld array-based photoacoustic probe for guiding needle biop- burns by photoacoustic microscopy. J Biomed Opt 2006;11:054033. sy of sentinel lymph nodes. J Biomed Opt 2010;15:046010. 131. ACS. Cancer facts & figures 2009. American Cancer Society; 2009. 108. Akers WJ, Edwards WB, Kim C, Xu B, Erpelding TN, Wang LV, et al. 132. Shaaban A, Rezvani M. Ovarian cancer: detection and radiologic Multimodal sentinel lymph node mapping with single-photon emis- staging. Top Magn Reson Imaging 2010;21:247–59. sion computed tomography (SPECT)/computed tomography (CT) 133. Aguirre A, Guo P, Gamelin J, Yan S, Sanders MM, Brewer M, et al. and photoacoustic tomography. Transl Res 2012;159:175–81. Coregistered three-dimensional ultrasound and photoacoustic imag- 109. Heijmink SW, Futterer JJ, Strum SS, Oyen WJ, Frauscher F, Witjes JA, ing system for ovarian tissue characterization. J Biomed Opt et al. State-of-the-art uroradiologic imaging in the diagnosis of 2009;14:054014. prostate cancer. Acta Oncol 2011;50(Suppl 1):25–38. 134. Aguirre A, Ardeshirpour Y, Sanders MM, Brewer M, Zhu Q. Potential 110. Ilic D, Neuberger MM, Djulbegovic M, Dahm P. Screening for prostate role of coregistered photoacoustic and ultrasound imaging in ovarian cancer. Cochrane Database Syst Rev 2013;1:CD004720. cancer detection and characterization. Transl Oncol 2011;4:29–37. 111. Oraevsky AA, Ermilov S, Metha K, Miller T, Bell B, Orihuela E, et al. In 135. Yang Y, Li X, Wang T, Kumavor PD, Aguirre A, Shung KK, et al. vivo testing of laser optoacoustic system for image-guided biopsy of Integrated optical coherence tomography, ultrasound and photoa- prostate. Proc SPIE 2006;6086. coustic imaging for ovarian tissue characterization. Biomed Opt 112. Wang X, Roberts WW, Carson PL, Wood DP, Fowlkes JB. Photo- Express 2011;2:2551–61. acoustic tomography: a potential new tool for prostate cancer. 136. Kumavor PD, Alqasemi U, Tavakoli B, Li H, Yang Y, Sun X, et al. Co- Biomed Opt Express 2010;1:1117–26. registered pulse-echo/photoacoustic transvaginal probe for real time 113. Yaseen MA, Ermilov SA, Brecht HP, Su R, Conjusteau A, Fronheiser imaging of ovarian tissue. J Biophotonics 2013;6:475–84. M, et al. Optoacoustic imaging of the prostate: development toward 137. Paterlini-Brechot P. Organ-specific markers in circulating tumor cell image-guided biopsy. J Biomed Opt 2010;15:021310. screening: an early indicator of metastasis-capable malignancy. 114. Bauer DR, Olafsson R, Montilla LG, Witte RS. 3-D photoacoustic and Future Oncol 2011;7:849–71. pulse echo imaging of prostate tumor progression in the mouse 138. Mavroudis D. Circulating cancer cells. Ann Oncol 2010;21(Suppl 7): window chamber. J Biomed Opt 2011;16:026012. vii95–100. 115. Olafsson R, Bauer DR, Montilla LG, Witte RS. Real-time, contrast 139. Ireland A, Millward M, Pearce R, Lee M, Ziman M. Genetic factors in enhanced photoacoustic imaging of cancer in a mouse window metastatic progression of cutaneous melanoma: the future role of chamber. Opt Express 2010;18:18625–32. circulating melanoma cells in prognosis and management. Clin Exp 116. Davis BJ, Horwitz EM, Lee WR, Crook JM, Stock RG, Merrick GS, Metastasis 2011;28:327–36. et al. American Brachytherapy Society consensus guidelines for 140. Mocellin S, Hoon D, Ambrosi A, Nitti D, Rossi CR. The prognostic transrectal ultrasound-guided permanent prostate brachytherapy. value of circulating tumor cells in patients with melanoma: a system- Brachytherapy 2012;11:6–19. atic review and meta-analysis. Clin Cancer Res 2006;12:4605–13. 117. Yamada Y, Rogers L, Demanes DJ, Morton G, Prestidge BR, Pouliot 141. Weight RM, Viator JA, Dale PS, Caldwell CW, Lisle AE. Photoacoustic J, et al. American Brachytherapy Society consensus guidelines for detection of metastatic melanoma cells in the human circulatory high-dose-rate prostate brachytherapy. Brachytherapy 2012; system. Opt Lett 2006;31:2998–3000. 11:20–32. 142. Gutierrez-Juarez G, Gupta SK, Al-Shaer M, Polo-Parada L, Dale PS, 118. Su JL, Bouchard RR, Karpiouk AB, Hazle JD, Emelianov SY. Photo- Papageorgio C, et al. Detection of melanoma cells in vitro using an acoustic imaging of prostate brachytherapy seeds. Biomed Opt optical detector of photoacoustic waves. Lasers Surg Med 2010; Express 2011;2:2243–54. 42:274–81.

1002 Cancer Res; 74(4) February 15, 2014 Cancer Research

Translational Strategies for Photoacoustic Imaging

143. Gutierrez-Juarez G, Gupta SK, Weight RM, Polo-Parada L, Papa- 165. Jiao S, Jiang M, Hu J, Fawzi A, Zhou Q, Shung KK, et al. Photo- giorgio C, Bunch JD, et al. Optical photoacoustic detection of circu- acoustic ophthalmoscopy for in vivo retinal imaging. Opt Express lating melanoma cells in vitro. Int J Thermophys 2010;31:784–92. 2010;18:3967–72. 144. McCormack DR, Bhattacharyya K, Kannan R, Katti K, Viator JA. 166. Sun Y, Sobel ES, Jiang H. First assessment of three-dimensional Enhanced photoacoustic detection of melanoma cells using gold quantitative photoacoustic tomography for in vivo detection of oste- nanoparticles. Lasers Surg Med 2011;43:333–8. oarthritis in the finger joints. Med Phys 2011;38:4009–17. 145. Weight RM, Dale PS, Viator JA. Detection of circulating melanoma 167. Wang X, Chamberland DL, Jamadar DA. Noninvasive photoacoustic cells in human blood using photoacoustic flowmetry. Conf Proc IEEE tomography of human peripheral joints toward diagnosis of inflam- Eng Med Biol Soc 2009;2009:106–9. matory arthritis. Opt Lett 2007;32:3002–4. 146. Zharov VP, Galanzha EI, Shashkov EV, Kim JW, Khlebtsov NG, 168. Xiao J, Yao L, Sun Y, Sobel ES, He J, Jiang H. Quantitative two- Tuchin VV. Photoacoustic flow cytometry: principle and application dimensional photoacoustic tomography of osteoarthritis in the finger for real-time detection of circulating single nanoparticles, pathogens, joints. Opt Express 2010;18:14359–65. and contrast dyes in vivo. J Biomed Opt 2007;12:051503. 169. Xu G, Rajian JR, Girish G, Kaplan MJ, Fowlkes JB, Carson PL, et al. 147. Galanzha EI, Shashkov EV, Kelly T, Kim JW, Yang L, Zharov VP. In vivo Photoacoustic and ultrasound dual-modality imaging of human magnetic enrichment and multiplex photoacoustic detection of cir- peripheral joints. J Biomed Opt 2013;18:10502. culating tumour cells. Nat Nanotechnol 2009;4:855–60. 170. Hu J, Yu M, Ye F, Xing D. In vivo photoacoustic imaging of osteo- 148. Nedosekin DA, Sarimollaoglu M, Ye JH, Galanzha EI, Zharov VP. In sarcoma in a rat model. J Biomed Opt 2011;16:020503. vivo ultra-fast photoacoustic flow cytometry of circulating human 171. Yang X, Wang LV. Monkey brain cortex imaging by photoacoustic melanoma cells using near-infrared high-pulse rate lasers. Cytometry tomography. J Biomed Opt 2008;13:044009. A 2011;79:825–33. 172. Nie L, Cai X, Maslov K, Garcia-Uribe A, Anastasio MA, Wang LV. 149. Bhattacharyya K, Goldschmidt BS, Hannink M, Alexander S, Jurkevic Photoacoustic tomography through a whole adult human skull with a A, Viator JA. Gold nanoparticle-mediated detection of circulating photon recycler. J Biomed Opt 2012;17:110506. cancer cells. Clin Lab Med 2012;32:89–101. 173. Sussman CB, Rossignol C, Zhang Q, Jiang H, Zheng T, Steindler D, 150. Galanzha EI, Shashkov EV, Spring PM, Suen JY, Zharov VP. In vivo, et al. Photoacoustic tomography can detect cerebral hemodynamic noninvasive, label-free detection and eradication of circulating met- alterations in a neonatal rodent model of hypoxia-ischemia. Acta astatic melanoma cells using two-color photoacoustic flow cytome- Neurobiol Exp 2012;72:253–63. try with a diode laser. Cancer Res 2009;69:7926–34. 174. Kiesslich R, Goetz M, Hoffman A, Galle PR. New imaging techniques 151. Allen TJ, Hall A, Dhillon AP, Owen JS, Beard PC. Spectroscopic and opportunities in endoscopy. Nat Rev Gastroenterol Hepatol photoacoustic imaging of lipid-rich plaques in the human aorta in the 2011;8:547–53. 740 to 1400 nm wavelength range. J Biomed Opt 2012;17:061209. 175. Zhang JG, Liu HF. Functional imaging and endoscopy. World 152. Graf IM, Kim S, Wang B, Smalling R, Emelianov S. Noninvasive J Gastroenterol 2011;17:4277–82. detection of intimal xanthoma using combined ultrasound, strain 176. Yang JM, Favazza C, Chen R, Yao J, Cai X, Maslov K, et al. Simul- rate and photoacoustic imaging. Ultrasonics 2012;52:435–41. taneous functional photoacoustic and ultrasonic endoscopy of inter- 153. Karpiouk AB, Wang B, Emelianov SY. Development of a catheter for nal organs in vivo. Nat Med 2012;18:1297–1302. combined intravascular ultrasound and photoacoustic imaging. Rev 177. Yuan Y, Yang S, Xing D. Preclinical photoacoustic imaging endo- Sci Instrum 2010;81:014901. scope based on acousto-optic coaxial system using ring transducer 154. Wei W, Li X, Zhou Q, Shung KK, Chen Z. Integrated ultrasound array. Opt Lett 2010;35:2266–8. and photoacoustic probe for co-registered intravascular imaging. 178. Sheaff C, Lau N, Patel H, Huang SW, Ashkenazi S. Photoacoustic J Biomed Opt 2011;16:106001. imaging endoscope. Conf Proc IEEE Eng Med Biol Soc 2009;2009: 155. Hsieh BY, Chen SL, Ling T, Guo LJ, Li PC. Integrated intravascular 1983–6. ultrasound and photoacoustic imaging scan head. Opt Lett 179. Xi L, Grobmyer SR, Wu L, Chen R, Zhou G, Gutwein LG, et al. 2010;35:2892–4. Evaluation of breast tumor margins in vivo with intraoperative photo- 156. Emelianov S, Wang B, Su J, Karpiouk A, Yantsen E, Sokolov K, et al. acoustic imaging. Opt Express 2012;20:8726–31. Intravascular ultrasound and photoacoustic imaging. Conf Proc IEEE 180. Su X, Cheng K, Wang C, Xing L, Wu H, Cheng Z. Image-guided Eng Med Biol Soc 2008;2008:2–5. resection of malignant gliomas using fl uorescent nanoparticles. 157. Jansen K, van der Steen AF, van Beusekom HM, Oosterhuis JW, van Wiley Interdiscip Rev Nanomed Nanobiotechnol 2013;5:219–32. Soest G. Intravascular photoacoustic imaging of human coronary 181. Grootendorst DJ, Fratila RM, Visscher M, Haken BT, van Wezel RJ, atherosclerosis. Opt Lett 2011;36:597–9. Rottenberg S, et al. Intra-operative ex vivo photoacoustic nodal 158. Sethuraman S, Aglyamov SR, Amirian JH, Smalling RW, Emelianov staging in a rat model using a clinical superparamagnetic iron oxide SY. Intravascular photoacoustic imaging using an IVUS imaging nanoparticle dispersion. J Biophotonics 2013;6:493–504. catheter. IEEE Trans Ultrason Ferroelectr Freq Control 2007;54: 182. Cui H, Yang X. In vivo imaging and treatment of solid tumor using 978–86. integrated photoacoustic imaging and high intensity focused ultra- 159. Sethuraman S, Amirian JH, Litovsky SH, Smalling RW, Emelianov SY. sound system. Med Phys 2010;37:4777–81. Ex vivo characterization of atherosclerosis using intravascular photo- 183. Prost A, Funke A, Tanter M, Aubry JF, Bossy E. Photoacoustic- acoustic imaging. Opt Express 2007;15:16657–66. guided ultrasound therapy with a dual-mode ultrasound array. 160. de la Zerda A, Paulus YM, Teed R, Bodapati S, Dollberg Y, Khuri- J Biomed Opt 2012;17:061205. Yakub BT, et al. Photoacoustic ocular imaging. Opt Lett 2010;35: 184. Kennedy LC, Bickford LR, Lewinski NA, Coughlin AJ, Hu Y, Day ES, 270–2. et al. A new era for cancer treatment: gold-nanoparticle-mediated 161. Hu S, Rao B, Maslov K, Wang LV. Label-free photoacoustic oph- thermal therapies. Small 2011;7:169–83. thalmic angiography. Opt Lett 2010;35:1–3. 185. Su J, Karpiouk A, Wang B, Emelianov S. Photoacoustic imaging of 162. Song W, Wei Q, Feng L, Sarthy V, Jiao S, Liu X, et al. Multimodal clinical metal needles in tissue. J Biomed Opt 2010;15:021309. photoacoustic ophthalmoscopy in mouse. J Biophotonics 2013;6: 186. Kim C, Erpelding TN, Jankovic L, Wang LV. Performance benchmarks 505–12. of an array-based hand-held photoacoustic probe adapted from a 163. Song W, Wei Q, Jiao S, Zhang HF. Integrated photoacoustic oph- clinical ultrasound system for non-invasive sentinel lymph node thalmoscopy and spectral-domain optical coherence tomography. J imaging. Philos Trans A Math Phys Eng Sci 2011;369:4644–50. Vis Exp 2013:e4390. 187. Yao J, Maslov K, Hu S, Wang LV. Evans blue dye-enhanced capillary- 164. Song W, Wei Q, Liu T, Kuai D, Burke JM, Jiao S, et al. Integrating resolution photoacoustic microscopy in vivo. J Biomed Opt 2009; photoacoustic ophthalmoscopy with scanning laser ophthalmosco- 14:054049. py, optical coherence tomography, and fluorescein angiography for a 188. Xiang L, Yuan Y, Xing D, Ou Z, Yang S, Zhou F. Photoacoustic multimodal retinal imaging platform. J Biomed Opt 2012;17:061206. molecular imaging with antibody-functionalized single-walled carbon

www.aacrjournals.org Cancer Res; 74(4) February 15, 2014 1003

Zackrisson et al.

nanotubes for early diagnosis of tumor. J Biomed Opt 2009;14: 193. Lu W, Melancon MP, Xiong C, Huang Q, Elliott A, Song S, et al. Effects 021008. of photoacoustic imaging and photothermal ablation therapy medi- 189. de la Zerda A, Bodapati S, Teed R, May SY, Tabakman SM, Liu Z, ated by targeted hollow gold nanospheres in an orthotopic mouse et al. Family of enhanced photoacoustic imaging agents for high xenograft model of glioma. Cancer Res 2011;71:6116–21. sensitivity and multiplexing studies in living mice. ACS Nano 194. Lu W, Xiong C, Zhang G, Huang Q, Zhang R, Zhang JZ, et al. Targeted 2012;6:4694–701. photothermal ablation of murine melanomas with melanocyte-stim- 190. Akers WJ, Kim C, Berezin M, Guo K, Fuhrhop R, Lanza GM, et al. ulating hormone analog-conjugated hollow gold nanospheres. Clin Noninvasive photoacoustic and fluorescence sentinel lymph node Cancer Res 2009;15:876–86. identification using dye-loaded perfluorocarbon nanoparticles. ACS 195. Pan D, Pramanik M, Senpan A, Allen JS, Zhang H, Wickline SA, et al. Nano 2011;5:173–82. Molecular photoacoustic imaging of angiogenesis with integrin-tar- 191. Zhang Q, Iwakuma N, Sharma P, Moudgil BM, Wu C, McNeill J, geted gold nanobeacons. FASEB J 2011;25:875–82. et al. Gold nanoparticles as a contrast agent for in vivo tumor 196. Pan D, Pramanik M, Senpan A, Ghosh S, Wickline SA, Wang LV, et al. imaging with photoacoustic tomography. Nanotechnology Near infrared photoacoustic detection of sentinel lymph nodes with 2009;20:395102. gold nanobeacons. Biomaterials 2010;31:4088–93. 192. Lu W, Huang Q, Ku G, Wen X, Zhou M, Guzatov D, et al. Photo- 197. Homan KA, Souza M, Truby R, Luke GP, Green C, Vreeland E, et al. acoustic imaging of living mouse brain vasculature using hollow gold Silver nanoplate contrast agents for in vivo molecular photoacoustic nanospheres. Biomaterials 2010;31:2617–26. imaging. ACS Nano 2012;6:641–50.

1004 Cancer Res; 74(4) February 15, 2014 Cancer Research

Light In and Sound Out: Emerging Translational Strategies for Photoacoustic Imaging

S. Zackrisson, S.M.W.Y. van de Ven and S.S. Gambhir

Cancer Res 2014;74:979-1004. Published OnlineFirst February 10, 2014.

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