Optoacoustic Imaging: An Emerging Modality for the Gastrointestinal Tract

Abstract

Imaging of the gastrointestinal (GI) tract largely relies on the inspection of the wall lining using optical endoscopy procedures or virtual endoscopic methods based on image analysis of radiologic approaches such as computed tomography. Other endoscopic methods of imaging have been also considered, including ultrasonography and optical tissue-sectioning microscopy methods. Whole-body methods are useful in obtaining a volumetric impression of the GI tract and investigate for disease spread, but they cannot match optical endoscopy resolution and sensitivity.1Hoeffel C. Mulé S. Romaniuk B. et al.Advances in radiological imaging of gastrointestinal tumours.Crit Rev Oncol Hematol. 2009; 69: 153-167Crossref PubMed Scopus (16) Google Scholar Current application of virtual computed tomography endoscopy using barium administration remains important in the investigation of the GI tract. Magnetic resonance imaging is also employed, although less frequently, owing to using nonionizing radiation and providing good soft tissue contrast that can be further enhanced by the use of gadolinium. Nuclear imaging methods are useful in cancer staging applications and treatment monitoring, but their role in the primary diagnosis of GI tract disease has not been well defined as of yet. Used more extensively compared with radiology methods, endoscopic optical imaging remains a major diagnostic method in GI tract diseases. Wide-field white-light endoscopy allows the observation of anatomic features and discolorations indicative of disease and is utilized to guide tissue biopsies. Despite its wide clinical acceptance, the method is limited by human vision, namely, the lack of sensitivity to subsurface activity or to particular physiologic or molecular disease features. In response, several methods have been investigated to alleviate those issues.2Goetz M. Wang T.D. Molecular imaging in gastrointestinal endoscopy.Gastroenterology. 2010; 138: 828-833Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Using adapted white-light endoscopy to detect fluorescence, autofluorescence imaging has been employed to investigate disease related intrinsic tissue fluorochromes modification. Similarly, narrow-band imaging uses the spectral properties of hemoglobin present in the mucosal patterns and vessels to highlight angiogenesis as a biomarker for early neoplastic changes. Autofluorescence imaging and narrow-band imaging are applied under research protocols but have not yet been diagnostically confirmed for large-scale clinical adoption. Consequently, attention has turned to the endoscopic application of tissue sectioning microscopy methods. Confocal endomicroscopy can achieve penetration depths of ∼200 μm and ×1,000 magnification, attaining submicrometer resolution, whereby 2-photon microscopy endoscopes can penetrate deeper (∼600 μm) albeit sacrificing some of the resolution achieved by confocal. Endoscopic tissue-sectioning microscopy is generally limited by small fields of view (∼500 × 500 μm), but can reveal tissue morphology at the detail level of histopathology; offering valuable information in vivo. A current limitation is the ability to interrogate only small parts of the GI wall lining by bringing small fiber bundles in contact with tissue. Methods that can visualize deeper in the tract wall have also been investigated. High-frequency ultrasonic endoscopic imaging can penetrate for several millimeters to centimeters in tissue; however, the contrast achieved is generally poor and interpretation of data remains difficult. The optical equivalent of ultrasonography, that is, the optical coherence tomography, allows for higher resolution compared with ultrasound and achieves a penetration depth of a few millimeters. By providing good anatomic contrast, optical coherence tomography is also investigated as an alternative to tissue sectioning microscopy methods and ultrasonography. A common challenge is the limitation of diagnostic potential when intrinsic tissue contrast or nonspecific optical agents are visualized. However, similar to progress in other imaging applications, endoscopic imaging can significantly benefit from the administration of optical agents that can target disease biomarkers. Indeed, similarly to the recent propagation of a fluorescence probe targeting the folate receptor in surgical imaging,3Van Dam et al.Nat Med. 2011; 17: 1315-1319Crossref PubMed Scopus (1244) Google Scholar agents with specificity to GI biomarkers can be employed for enhancing the performance and diagnostic ability of optical endoscopic approaches. Under this light, optoacoustic imaging (OI) and tomography are emerging as a potent optical imaging modalities that combine high-resolution volumetric imaging over several millimeters to centimeters of tissue depth with the ability to resolve agents with molecular specificity, vasculature, and physiologic parameters. These unique features provide the rationale for GI OI. Of particular importance for the detection of intrinsic or extrinsic compounds is accurate detection granted by a multiwavelength approach. Herein we have outlined the basics of the technology and discuss operational characteristics and possible application areas in endoscopic GI imaging. Imaging based on optoacoustics is a truly hybrid modality, because it utilizes light as the source but acoustic detectors for data collection. The method emits narrow (typically nanosecond range) light pulses that, as they are absorbed by tissue photoabsorbing compounds, generate a transient temperature increase. This transient temperature rise results in a local thermoelastic expansion that generates ultrasonic waves, with frequencies ranging from several KHz to tens of MHZ (Figure 1). Tomographic images of the biodistribution of light absorbers in tissues can then be retrieved by capturing the emitted ultrasonic waves using detector scanning arrangements or detector arrays and mathematical inversion procedures.4Ntziachristos V. Going deeper than microscopy: the optical imaging frontier in biology.Nat Meth. 2010; 7: 603-614Crossref PubMed Scopus (1341) Google Scholar Reconstructed optoacoustic measurements obtained at a single wavelength reveal an image of light absorption by tissue absorbers, such as hemoglobin or melanin. The administration of contrast agents (chromophores, fluorochromes and photoabsorbing nanoparticles) can further enhance anatomic contrast or reveal physiologic parameters. Because multispectral optoacoustic tomography (MSOT) illuminates tissue with light at multiple wavelengths, techniques that can resolve the different spectral contributions of the compounds of interest can be employed (spectral unmixing).5Ntziachristos V. Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT).Chem Rev. 2010; 110: 2783-2794Crossref PubMed Scopus (619) Google Scholar Therefore, from a set of images at multiple wavelengths, MSOT produces images of the biodistribution of various molecules in tissue, including images of oxy- and deoxygenated hemoglobin, external photoabsorbing agents or labeled optical probes with molecular specificity. Spectral unmixing does not require baseline measurements; therefore, the clinical application of MSOT is straightforward, even when baseline measurements are not available. This is particularly important in applications such as GI imaging because it is impractical to obtain a baseline measurement. The operational characteristics of endoscopic MSOT are similar to endoscopic ultrasonography, with the exception that optical contrast is resolved. MSOT can image GI wall lining cross-sections and beyond, with resolutions of 20–40 microns or better. Although the resolution offered is not as high as in optical microscopy methods, larger fields of view can be inspected with molecular specificity. This can reveal the presence of particular biomarkers (eg, hypoxia, permeability, protein/receptor up-regulation) based on the presence of a targeted probe, not on anatomic features obtained with high-resolution as in tissue sectioning microscopy. In addition, OI can penetrate much deeper than optical microscopy methods; imaging through ≥6 mm has been achieved with 38-micron resolution and penetration depth can be exchanged for resolution5Ntziachristos V. Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT).Chem Rev. 2010; 110: 2783-2794Crossref PubMed Scopus (619) Google Scholar (Figure 1). By focusing a light beam, diffraction-limited optical resolution images can also be derived although at more superficial depths that are comparable with tissue-sectioning optical microscopy methods.6Hu S. Maslov K. Wang L.V. Noninvasive label-free imaging of microhemodynamics by optical-resolution photoacoustic microscopy.Opt Express. 2009; 17: 7688-7693Crossref PubMed Scopus (102) Google Scholar Similar to sonography, optoacoustic detection requires direct coupling of the detector with tissue, or the use of media with acoustic propagation properties that match those of tissue. Current technology allows for the implementation of different geometries for imaging or scanning hollow organs such as the GI tract; following after ultrasound approaches. An optoacoustic probe inserted through an endoscope and guided by imaging can be brought in contact with tissue for cross-sectional imaging of the lining wall and beyond.7Yang J.-M. Maslov K. Yang H.C. et al.Photoacoustic endoscopy.Opt Lett. 2009; 34: 1591-1593Crossref PubMed Scopus (202) Google Scholar Axial imaging through water-filled balloons, using 360° viewing arrangements can also be achieved for obtaining larger fields of view compared with direct contact scopes. Finally, it would be possible to achieve transabdominal imaging as well; in this case, however, limitations may arise from the light depth penetration into tissue beyond 2–4 cm. Currently, this area is under investigation. To achieve accurate decomposition of the spectral contributions of contrast sources imaged, it is important to obtain accurate image reconstruction performance. In particular, light and sound attenuation in the tissue needs to be considered—the attenuation effects being stronger in the visible (for light) and when increasing the imaging resolution using higher frequency ultrasonic components (for sound). In response, matrix-based inversion methods that can model experimental parameters, including the frequency response and size of the detector and light or sound attenuation and reflection effects, can greatly improve image quality. Optoacoustic imaging can be employed to investigate a range of pathologies using intrinsic or extrinsic contrast (Table 1), leading to high-resolution anatomic, functional and molecular imaging5Ntziachristos V. Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT).Chem Rev. 2010; 110: 2783-2794Crossref PubMed Scopus (619) Google Scholar, 8Kim C. Favazza C. Wang L.V. In vivo photoacoustic tomography of chemicals: high-resolution functional and molecular optical imaging at new depths.Chem Rev. 2010; 110: 2756-2782Crossref PubMed Scopus (655) Google Scholar using a single modality. Few methods currently have this ability with a single scan.Table 1Characteristics of Various Contrast Sources Available for Optoacoustic Imaging and Their Foreseeable DevelopmentContrast sourceExampleStrengthsWeaknessesForeseen developmentIntrinsic absorbing agentsBloodStability, access to specific informationLimited choice, low contrastOxygen mapping of organs, 3D tumor viability studiesUnbound fluorophoresIndocyanine greenHigh contrast, large available libraryRapid metabolization, photobleachingBlood flow mapping, metabolism quantificationFunctionalized fluorophoreIntegriSense (ViSen)Possibility of targeting, increased circulation time compared with unbound counterpartsPhotobleachingTumour imaging, metabolism imagingGold nanoparticlesGold nanorodsHigh contrast, high circulation time, targeting possibility, potential drug carriers, photodynamic therapyToxicity unassayedCancer applications (diagnosis, theragnosis)Carbon nanotubesSingle-walled carbon nanotubesHighly absorbing, targetable, potential drug carriersNo specific absorption peak, toxicity unassayedTumour and lymph node imaging, monitored drug releaseActivatable agentsMMPsense (ViSen; MMP activation)High signal-to-noise ratio, biomarker specificComplexity, detection of activation can be a challengeFunctional MSOT imaging of various pathology biomarkers (cancer, inflammation) Open table in a new tab Single wavelength optoacoustic measurements yield images of tissue absorption distribution, typically revealing vascular structures, with hemoglobin being a prominent light absorber. Depending on the wavelength selected different contrast enhancement can be achieved (Figure 2A): In the visible, blood vessels can be detected with high contrast over a few millimeters depth owing to the strong absorption of light by hemoglobin, whereas deeper seated vascular structures require a red or near infra-red wavelength shift (Figure 3A, B shows high-resolution imaging of blood vessels in the mouse ear and mouse colon).9Hu S. Wang L.V. Photoacoustic imaging and characterization of the microvasculature.J Biomed Opt. 2010; 15: 011101Crossref PubMed Scopus (327) Google Scholar Other absorbers and structures can potentially contribute to optoacoustic signal, such as melanin and other tissue chromophores, and sound reflectors such as calcifications involved in leiomyoma, leiomyosarcoma, or mucinous adenocarcinoma.Figure 3Current performance of different modalities of OI. Structural image of a mouse ear microvasculature in vivo (A(i)) using optoacoustic tomography and oxygen saturation mapping of the same area (A(ii)).6Hu S. Maslov K. Wang L.V. Noninvasive label-free imaging of microhemodynamics by optical-resolution photoacoustic microscopy.Opt Express. 2009; 17: 7688-7693Crossref PubMed Scopus (102) Google Scholar Large intestinal tract of a rat (B) imaged by endoscopic OI to locate blood vessels (BV) using a moving laser beam (LB) and showing a wall thickness of approximately 1 mm.7Yang J.-M. Maslov K. Yang H.C. et al.Photoacoustic endoscopy.Opt Lett. 2009; 34: 1591-1593Crossref PubMed Scopus (202) Google Scholar Perfusion of a mouse kidney with indocyanide green followed by in vivo multispectral optoacoustic tomography at 800 nm (C(i)) and using image difference to highlight indocyanine green distribution (C(ii)).10Buehler A. Herzog E. Razansky D. et al.Video rate optoacoustic tomography of mouse kidney perfusion.Opt Lett. 2010; 35: 2475-2477Crossref PubMed Scopus (164) Google Scholar Mouse tumor images obtained by multispectral optoacoustic tomography using gold nanorods as a contrast agent; anatomic image acquired at 800 nm (D(i)), AuNP detected using multispectral acquisition overlaid on the anatomic image (D(ii)).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Multispectral methods such as MSOT can provide differentiation of physiologic conditions based on differentiation of absorbers by spectral unmixing (oxy- from deoxyhemoglobin, leading to hypoxia characterization; melanin or lipofuscin can be differentiated from hemoglobin and their contributions characterized in GI tract discoloration). Such characterization can also be achieved with conventional multispectral optical imaging; however, in this case the contributions of scattering (photon diffusion) and depth perplex the quantification. In contrast, MSOT is insensitive to photon scattering and can more accurately reveal blood and pigment contributions compared with diffusive optical measurements. Moreover, it can resolve the contrast as a function of depth and retrieve the distribution of tissue chromophores within the lining wall and beyond. Optoacoustic methods can be also implemented for real-time imaging,10Buehler A. Herzog E. Razansky D. et al.Video rate optoacoustic tomography of mouse kidney perfusion.Opt Lett. 2010; 35: 2475-2477Crossref PubMed Scopus (164) Google Scholar achieving repetition rates of up to several KHz frequencies, allowing the observation of hemodynamic changes and video-rate scanning procedures during endoscopy. In addition to imaging intrinsic tissue chromophores, MSOT can detect extrinsically administered photoabsorbing agents with molecular sensitivity.11Razansky D. Distel M. Claudio Vinegoni C. et al.Multispectral optoacoustic tomography of deep-seated fluorescent proteins in vivo.Nat Photonics. 2009; 3: 412-417Crossref Scopus (575) Google Scholar The use of agents targeted toward biomarkers involved in disease progression can amplify the capabilities of endoscopic imaging over visual inspection by offering disease differentiation, leading to earlier and more accurate and sensitive detection. Various dyes, including fluorochromes and gold nanorods present distinct absorption spectral signatures and can be differentiated over background absorption, typically hemoglobin, offering additional diagnostic information (Figure 2B). Conjugation of such photoabsorbers with targeting moieties such as antibodies or peptides can lead to agents with the ability to differentiate for example cancerous or inflammatory changes before anatomic or discoloration differences from healthy tissue become apparent. US Food and Drug Administration–approved molecules are particularly interesting. For example, cetuximab (targeting the epidermal growth factor receptor) can be potentially labeled by an imaging agent. Although labeling of an approved molecule is treated as a new molecule for regulatory processes, it may retain memory of toxicity or targeting efficacy and biodistribution after expert labeling, compared with a new active molecule with unknown toxicity. In this case, clinical acceptance could be accelerated, once safety is established for a known molecule labeled with a known, nontoxic optical agent. Regardless, current developments of novel molecules developed as dedicated imaging agents can further enhance the application potential of the method. Whereas fluorescent agents in particular can be detected by wide-field fluorescence imaging or endoscopic tissue-sectioning microscopy methods, MSOT allows the ability to employ a larger number of contrast generating agents and offer detection that is insensitive to photon scattering, allowing high-resolution volumetric imaging of these agents deeper than that currently allowed by purely optical methods. The ability to detect fluorochromes brings MSOT closer to functional and molecular imaging applications, because they present characteristic absorption spectra that can be differentiated over other light absorption contributions by tissue. With quantum yields of single percentage digits, a large part of the absorbed energy on many near-infrared cyanine dyes, including indocyanine green is not converted to fluorescence re-emission and is available for generating optoacoustic contrast. Figure 3C shows MSOT images obtained at different time points of mouse kidney perfusion, showing the ability for dynamic, high-resolution fluorochrome imaging through several millimeters of tissue. In the particular implementation shown in Figure 3C, the field of view shown was ∼15 mm and the resolution was approximately 150 microns; although resolutions of <40 microns have been demonstrated as well.11Razansky D. Distel M. Claudio Vinegoni C. et al.Multispectral optoacoustic tomography of deep-seated fluorescent proteins in vivo.Nat Photonics. 2009; 3: 412-417Crossref Scopus (575) Google Scholar The typical sensitivity of detecting organic fluorochromes with the current state of the art MSOT methods is of the order of 100 picomoles. In addition to common fluorochromes, other nonfluorescent dyes can also be detected: Of interest in this case are dyes with high absorption cross-section to lead to more sensitive detection in vivo. Photoabsorbing nanoparticles produce strong optoacoustic responses and have been considered for OI.5Ntziachristos V. Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT).Chem Rev. 2010; 110: 2783-2794Crossref PubMed Scopus (619) Google Scholar Carbon nanotubes and gold nanoparticles (AuNP) have been used in optoacoustic demonstrations, but other particles such as quantum dots could also offer optoacoustic contrast. Compared with organic dyes, a number of nanoparticles typically generates significantly stronger optoacoustic signal compared with the same amount of dye molecules. However their size is also bigger; therefore the in vivo detection sensitivity will be eventually determined by the amount of agent that can reach or be internalized into tissue. Although higher sensitivity is expected regardless from nanoparticles, their human use and toxicity is not as well established yet as in cyanine dyes, which may slow down their clinical propagation. AuNP come in different shapes: Spheres, rods, pyramids, and even hollow cages that can be used for drug transportation in vivo, and in various sizes (usually in the 30–100 nm range), directly correlated to their absorption properties. Of interest for increased detection sensitivity and deep-tissue penetration are gold nanorods with a length/width ratio of around 3.5, presenting a peak absorption wavelength located in the near infra-red region of the spectra. Another interesting property of AuNP is their theragnostic application in photothermal therapy, where detection can be coupled with thermic destruction of the targeted tissue after specific laser excitation. Coating AuNP using peptides or hydrophilic polymers such as polyethylene glycol typically prolongs the circulation times, for optimizing delivery through highly permeable membranes or availability for binding onto intended targets. Single-walled carbon nanotubes (1–2 nm in diameter, 50–300 nm in length) have been tested for OI in vivo for both passive tumor targeting and lymph node mapping. Although their absorption spectrum shows no specific absorption peak, they absorb a large amount of light and thus provide strong optoacoustic signals, with a possible detection threshold of 50 nmol/L. Polyethylene glycol grafting has been investigated to increase the blood circulation time with good results, as was RGD-peptide grafting to target integrins. Displaying both fluorescence properties and good optoacoustic profile, quantum dots are photostable, semiconducting crystals that can also be functionalized to target specific tissues in a way similar to other nanoparticles, with which they also share their toxicity and excretion profile drawbacks. A common complication when employing targeted optical or optoacoustic agents is that nonbound agent can also generate contrast. In this case, long times after injection are required for the nonbound probe to be cleared from tissue. An alternative mechanism to allow separation of a molecular target of interest is by employing photoabsorbing agents that undergo a detectable conformation change when they interact with their intended target. Termed “smart probes” or “molecular beacons,” fluorescent agents that are dark in their base state but fluoresce after target interaction, is a well-established example of activatable contrast agents. It was recently noted that the absorption spectrum may also change during the conformational change of these agents, which can be detected by MSOT, leading in 1 case to the detection of inflammation in excised human carotids.12Razansky D. Harlaar N.J. Hillebrands J.L. et al.Multispectral optoacoustic tomography of matrix metalloproteinase activity in vulnerable human carotid plaques.Mol Imaging Biol. 2011 Jul 1; ([Epub ahead of print])Google Scholar Activatable agents can be also engineered based on AuNP. The ability to elongate the apparent surface of gold particles shifts the absorption spectrum, achieving performance that is similar to elongated gold nanotubes. However, the same property can be exploited by bringing spherical nanoparticles close together, for example, when multiple particles targeting cellular receptors come into close proximity on a cellular surface.13Aaron J. Travis K. Harrison N. et al.Dynamic imaging of molecular assemblies in live cells based on nanoparticles plasmon resonance coupling.Nano Lett. 2009; 9: 3612-3618Crossref PubMed Scopus (150) Google Scholar MSOT imaging offers a promising add-on to GI tract endoscopic imaging. Guided by conventional color imaging or potentially fluorescence imaging of agents with cancer or inflammation sensitivity, OI can interrogate a wealth of information on and under the wall surface with resolutions that can match those of optical microscopy or with penetration depths that match those of endoscopic ultrasound. Aided by the ability to interrogate anatomic, functional, and molecular contrast, different screening, diagnostic, or interventional strategies can be devised to enhance the information content that is now available to the gastroenterologist. Although OI is at the early stages of clinical application, strategies can already be proposed as to its endoscopic use.7Yang J.-M. Maslov K. Yang H.C. et al.Photoacoustic endoscopy.Opt Lett. 2009; 34: 1591-1593Crossref PubMed Scopus (202) Google Scholar This specific use of the method depends mainly on hardware limitations inherent to the dual nature of OI. This technique (using light and sound) requires the design of a small-sized probe that holds both a light source able to illuminate tissue at a wide angle and an ultrasound transducer array. Although both will have to be miniaturized (small optical fibers or mirrors for light illumination, miniaturized arrays for ultrasound detection), optoacoustic endoscopy can inspire itself from currently available combined ultracompact video-ultrasound endoscopic convex probes for its future probe design. Capitalizing on its ability to visualize vascularization and hypoxia, and overall quantify color changes with depth, MSOT can be employed to examine suspicious lesions and entire masses within the wall lining and beyond. Although the surface detection of the primary tumor is required and, mostly, already achievable by other methods, the unparalleled combination of high resolution, penetration depth, and real-time imaging can allow for the localization of much smaller malignant lesions such as micrometastases. Pinpointing the stage of progression of pathologies, mapping their spread with high precision, and eventually assaying the efficiency of administered drugs can be an important milestone in gastroenterology. In interventional procedures, the method can be further utilized to guide and examine the success of anastomosis procedures. It can also be a unique tool in helping surgeons to evaluate the depth of a cancerous tumor in real time before and during its excision, as well as controlling the complete success of the procedure. When employing targeted contrast, the ability to further visualize molecular markers can also lead to interesting applications. In that respect, it is also clear that the method will largely benefit from every development that occurs in the field of contrast agent research, sharing interest with both fluorescence imaging and nanoparticle research, to widen its use in a large number of pathologies. Currently, advances in the hardware, image formation, and spectral unmixing methods contribute to a continuous improvement in resolution, quantification, and sensitivity achieved, bringing out the full potential of the method. Compared with ultrasound images, optoacoustic in general and more particularly MSOT lead to images of high contrast, which are free of speckle owing to the absence of a sound source producing echoes. The methods can be engineered to conform to ultrasonic probe designs and user operation, but seems to be able to significantly expand the applications allowed when it comes to endoscopic approaches owing to the rich contrast produced. Transabdominal and interventional implementation may also be beneficial when allowed by the depth reached by the various light sources employed, and within permissible light energy deposition limits allowed for safe biomedical imaging.