[Optoacoustic imaging-Applications and advancements of innovative imaging techniques]. / Optoakustische Bildgebung ­ innovative Bildgebungsverfahren auf dem Vormarsch.

Optoacoustic imaging (OAB) has developed steadily in recent years. By means of partly pulsed light, in a wide variety of wavelengths, different colour carriers (chromophores) are excited to form sound waves. These in turn are detected by the newly developed systems and converted into three-dimensional images by means of various algorithms. The technique is characterised by a good ratio between contrast and penetration depth and can create macro-, meso- and microscopic images due to its scalability. Optoacoustic macroscopy broadly irradiates the area to be examined with laser light. This can produce images with a high penetration depth, but only with a moderate resolution. Clinically interesting fields of application are for example the results of sentinel lymph nodes (SLNs) examined ex vivo using macroscopic optoacoustics. Due to the ability of OAB to visualise melanin, the detection rate of metastases was superior to previous methods, but not to histology. The ability to visualise dermal and epidermal structures, especially vessels, with good resolution makes optoacoustic mesoscopy useful in the examination of inflammatory skin diseases and could contribute to the verification of the success of therapy, e.g., with biologics for psoriasis vulgaris or atopic eczema (AE), in the future. Optoacoustic microscopy, which has so far been limited mainly to preclinical in vivo research, could be used in the future to detect even finer vascular structures and their changes. The clinical possibilities of OAB seem to be of great benefit and continue to be the subject of intensive research.

Non-invasive imaging in dermatology and the unique potential of raster-scan optoacoustic mesoscopy.

In recent years, several non-invasive imaging methods have been introduced to facilitate diagnostics and therapy monitoring in dermatology. The microscopic imaging methods are restricted in their penetration depth, while the mesoscopic methods probe deeper but provide only morphological, not functional, information. 'Raster-scan optoacoustic mesoscopy' (RSOM), an emerging new imaging technique, combines deep penetration with contrast based on light absorption, which provides morphological, molecular and functional information. Here, we compare the capabilities and limitations of currently available dermatological imaging methods and highlight the principles and unique abilities of RSOM. We illustrate the clinical potential of RSOM, in particular for non-invasive diagnosis and monitoring of inflammatory and oncological skin diseases.

Uniqueness in multispectral constant-wave epi-illumination imaging.

Multispectral tissue imaging based on optical cameras and continuous-wave tissue illumination is commonly used in medicine and biology. Surprisingly, there is a characteristic absence of a critical look at the quantities that can be uniquely characterized from optically diffuse matter by multispectral imaging. Here, we investigate the fundamental question of uniqueness in epi-illumination measurements from turbid media obtained at multiple wavelengths. By utilizing an analytical model, tissue-mimicking phantoms, and an in vivo imaging experiment we show that independent of the bands employed, spectral measurements cannot uniquely retrieve absorption and scattering coefficients. We also establish that it is, nevertheless, possible to uniquely quantify oxygen saturation and the Mie scattering power-a previously undocumented uniqueness condition.

Optical innovations in surgery.

BACKGROUND: In the past decade, there has been a major drive towards clinical translation of optical and, in particular, fluorescence imaging in surgery. In surgical oncology, radical surgery is characterized by the absence of positive resection margins, a critical factor in improving prognosis. Fluorescence imaging provides the surgeon with reliable and real-time intraoperative feedback to identify surgical targets, including positive tumour margins. It also may enable decisions on the possibility of intraoperative adjuvant treatment, such as brachytherapy, chemotherapy or emerging targeted photodynamic therapy (photoimmunotherapy). METHODS: This article reviews the use of optical imaging for intraoperative guidance and decision-making. RESULTS: Image-guided cancer surgery has the potential to be a powerful tool in guiding future surgical care. Photoimmunotherapy is a theranostic concept (simultaneous diagnosis and treatment) on the verge of clinical translation, and is highlighted as an effective combination of image-guided surgery and intraoperative treatment of residual disease. Multispectral optoacoustic tomography, a technique complementary to optical image-guided surgery, is currently being tested in humans and is anticipated to have great potential for perioperative and postoperative application in surgery. CONCLUSION: Significant advances have been achieved in real-time optical imaging strategies for intraoperative tumour detection and margin assessment. Optical imaging holds promise in achieving the highest percentage of negative surgical margins and in early detection of micrometastastic disease over the next decade.

Three-dimensional imaging of whole mouse models: comparing nondestructive X-ray phase-contrast micro-CT with cryotome-based planar epi-illumination imaging.

In this study, we compare two evolving techniques for obtaining high-resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome-based planar epi-illumination imaging (cryo-imaging). On the other hand, we examine X-ray phase-contrast micro-computed tomography (micro-CT) using synchrotron radiation. Cryo-imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo-frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X-ray phase-contrast imaging is based on the minute refraction of X-rays inside the specimen and features higher soft-tissue contrast than conventional, attenuation-based micro-CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft-tissue contrast is strongly enhanced in the X-ray phase images compared to the attenuation-based images. This identifies phase-contrast micro-CT as a powerful tool for the study of small animal disease models.

Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an α(v)ß(3-)integrin targeted agent.

BACKGROUND: In ovarian cancer, optimal cytoreductive surgery is of the utmost importance for long-term survival. The ability to visualize minuscule tumor deposits is important to ensure complete resection of the tumor. The purpose of our study was to estimate the in vivo sensitivity, specificity and diagnostic accuracy of an intra-operative fluorescence imaging system combined with an α(v)ß(3)-integrin targeted near-infrared fluorescent probe. METHOD: Tumor bearing mice were injected intravenously with a fluorescent probe targeting α(v)ß(3) integrins. Fluorescent spots and non-fluorescent tissue were identified and resected. Standard histopathology and fluorescence microscopy were used as gold-standard for tumor detection. RESULTS: Fifty-eight samples excised with support of intra-operative image-guided surgery were analyzed. The mean target to background ratio was 2.2 (SD 0.5). The calculated sensitivity of the imaging system was 95%, and the specificity was 88% with a diagnostic accuracy of 96.5%. CONCLUSION: Near-infrared image-guided surgery in this model has a high diagnostic accuracy and a fair target to background ratio that supports the development towards clinical translation of α(v)ß(3)-integrin targeted imaging.

Raster-scanning optoacoustic mesoscopy biomarkers for atopic dermatitis skin lesions.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease worldwide. Its severity is assessed using scores that rely on visual observation of the affected body surface area, the morphology of the lesions and subjective symptoms, like pruritus or insomnia. Ideally, such scores should be complemented by objective and accurate measurements of disease severity to standardize disease scoring in routine care and clinical trials. Recently, it was shown that raster-scanning optoacoustic mesoscopy (RSOM) can provide detailed three-dimensional images of skin inflammation processes that capture the most relevant features of their pathology. Moreover, precise RSOM biomarkers of inflammation have been identified for psoriasis. However, the objectivity and validity of such biomarkers in repeated measurements have not yet been assessed for AD. Here, we report the results of a study on the repeatability of RSOM inflammation biomarkers in AD to estimate their precision. Optoacoustic imaging analysis revealed morphological inflammation biomarkers with precision well beyond standard clinical severity metrics. Our findings suggest that optoacoustic mesoscopy may be a good choice for quantitative evaluations of AD that are inaccessible by other methods. This could potentially enable the optimization of disease scoring and drug development.

Three-dimensional optoacoustic tomography at video rate.

Using optoacoustic excitation, a complete volumetric tomographic data sets from the imaged object can in principle be generated with a single interrogating laser pulse. Thus, optoacoustic imaging intrinsically has the potential for fast three-dimensional imaging. We have developed a system capable of acquiring volumetric optoacoustic data in real time and showcase in this work the undocumented capacity to generate high resolution three-dimensional optoacoustic images at a rate of 10 Hz, currently mainly limited by the pulse repetition rate of the excitation laser.

Multispectral optoacoustic tomography for in vivo detection of lymph node metastases in oral cancer patients using an EGFR-targeted contrast agent and intrinsic tissue contrast: A proof-of-concept study.

Oral cancer patients undergo diagnostic surgeries to detect occult lymph node metastases missed by preoperative structural imaging techniques. Reducing these invasive procedures that are associated with considerable morbidity, requires better preoperative detection. Multispectral optoacoustic tomography (MSOT) is a rapidly evolving imaging technique that may improve preoperative detection of (early-stage) lymph node metastases, enabling the identification of molecular changes that often precede structural changes in tumorigenesis. Here, we characterize the optoacoustic properties of cetuximab-800CW, a tumor-specific fluorescent tracer showing several photophysical properties that benefit optoacoustic signal generation. In this first clinical proof-of-concept study, we explore its use as optoacoustic to differentiate between malignant and benign lymph nodes. We characterize the appearance of malignant lymph nodes and show differences in the distribution of intrinsic chromophores compared to benign lymph nodes. In addition, we suggest several approaches to improve the efficiency of follow-up studies.

Intraoperative near-infrared fluorescence imaging for sentinel lymph node detection in vulvar cancer: first clinical results.

OBJECTIVE: Disadvantages of the combined sentinel lymph node (SLN) procedure with radiocolloid and blue dye in vulvar cancer are the preoperative injections of radioactive tracer in the vulva, posing a painful burden on the patient. Intraoperative transcutaneous imaging of a peritumorally injected fluorescent tracer may lead to a one-step procedure, while maintaining high sensitivity. Aim of this pilot study was to investigate the applicability of intraoperative fluorescence imaging for SLN detection and transcutaneous lymphatic mapping in vulvar cancer. METHODS: Ten patients with early stage squamous cell carcinoma of the vulva underwent the standard SLN procedure. Additionally, a mixture of 1 mL patent blue and 1 mL indocyanin green (ICG; 0.5 mg/mL) was injected immediately prior to surgery, with the patient under anesthesia. Color and fluorescence images and videos of lymph flow were acquired using a custom-made intraoperative fluorescence camera system. The distance between skin and femoral artery was determined on preoperative CT-scan as a measure for subcutaneous adipose tissue. RESULTS: In 10 patients, SLNs were detected in 16 groins (4 unilateral; 6 midline tumors). Transcutaneous lymphatic mapping was possible in five patients (5 of 16 groins), and was limited to lean patients, with a maximal distance between femoral artery and skin of 24 mm, as determined on CT. In total, 29 SLNs were detected by radiocolloid, of which 26 were also detected by fluorescence and 21 were blue. CONCLUSIONS: These first clinical results indicate that intraoperative transcutaneous lymphatic mapping using fluorescence is technically feasible in a subgroup of lean vulvar cancer patients.

Experimental demonstration of accurate Bragg peak localization with ionoacoustic tandem phase detection (iTPD).

Accurate knowledge of the exact stopping location of ions inside the patient would allow full exploitation of their ballistic properties for patient treatment. The localized energy deposition of a pulsed particle beam induces a rapid temperature increase of the irradiated volume and leads to the emission of ionoacoustic (IA) waves. Detecting the time-of-flight (ToF) of the IA wave allows inferring information on the Bragg peak location and can henceforth be used forin-vivorange verification. A challenge for IA is the poor signal-to-noise ratio at clinically relevant doses and viable machines. We present a frequency-based measurement technique, labeled as ionoacoustic tandem phase detection (iTPD) utilizing lock-in amplifiers. The phase shift of the IA signal to a reference signal is measured to derive theToF. Experimental IA measurements with a 3.5 MHz lead zirconate titanate (PZT) transducer and lock-in amplifiers were performed in water using 22 MeV proton bursts. A digital iTPD was performedin-silicoat clinical dose levels on experimental data obtained from a clinical facility and secondly, on simulations emulating a heterogeneous geometry. For the experimental setup using 22 MeV protons, a localization accuracy and precision obtained through iTPD deviates from a time-based reference analysis by less than 15µm. Several methodological aspects were investigated experimentally in systematic manner. Lastly, iTPD was evaluatedin-silicofor clinical beam energies indicating that iTPD is in reach of sub-mm accuracy for fractionated doses < 5 Gy. iTPD can be used to accurately measure theToFof IA signals online via its phase shift in frequency domain. An application of iTPD to the clinical scenario using a single pulsed beam is feasible but requires further development to reach <1 Gy detection capabilities.

Assessing nailfold microvascular structure with ultra-wideband raster-scan optoacoustic mesoscopy.

Nailfold capillaroscopy, based on bright-field microscopy, is widely used to diagnose systemic sclerosis (SSc). However it cannot reveal information about venules and arterioles lying deep under the nailfold, nor can it provide detailed data about surface microvasculature when the skin around the nail is thick. These limitations reflect the fact that capillaroscopy is based on microscopy methods whose penetration depth is restricted to about 200 µm. We investigated whether ultra-wideband raster-scan optoacoustic mesoscopy (UWB-RSOM) can resolve small capillaries of the nailfold in healthy volunteers and compared the optoacoustic data to conventional capillaroscopy examinations. We quantified UWB-RSOM-resolved capillary density and capillary diameter as features that relate to SSc biomarkers, and we obtained the first three-dimensional, in vivo images of the deeper arterioles and venules. These results establish the potential of UWB-RSOM for analyzing SSc-relevant markers.

Fluorescence molecular imaging of small animal tumor models.

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.

Unleashing Optics and Optoacoustics for Developmental Biology.

The past decade marked an optical revolution in biology: an unprecedented number of optical techniques were developed and adopted for biological exploration, demonstrating increasing interest in optical imaging and in vivo interrogations. Optical methods have become faster and have reached nanoscale resolution, and are now complemented by optoacoustic (photoacoustic) methods capable of imaging whole specimens in vivo. Never before were so many optical imaging barriers broken in such a short time-frame: with new approaches to optical microscopy and mesoscopy came an increased ability to image biology at unprecedented speed, resolution, and depth. This review covers the most relevant techniques for imaging in developmental biology, and offers an outlook on the next steps for these technologies and their applications.

Ionoacoustic characterization of the proton Bragg peak with submillimeter accuracy.

PURPOSE: Range verification in ion beam therapy relies to date on nuclear imaging techniques which require complex and costly detector systems. A different approach is the detection of thermoacoustic signals that are generated due to localized energy loss of ion beams in tissue (ionoacoustics). Aim of this work was to study experimentally the achievable position resolution of ionoacoustics under idealized conditions using high frequency ultrasonic transducers and a specifically selected probing beam. METHODS: A water phantom was irradiated by a pulsed 20 MeV proton beam with varying pulse intensity and length. The acoustic signal of single proton pulses was measured by different PZT-based ultrasound detectors (3.5 and 10 MHz central frequencies). The proton dose distribution in water was calculated by Geant4 and used as input for simulation of the generated acoustic wave by the matlab toolbox k-WAVE. RESULTS: In measurements from this study, a clear signal of the Bragg peak was observed for an energy deposition as low as 10(12) eV. The signal amplitude showed a linear increase with particle number per pulse and thus, dose. Bragg peak position measurements were reproducible within ±30 µm and agreed with Geant4 simulations to better than 100 µm. The ionoacoustic signal pattern allowed for a detailed analysis of the Bragg peak and could be well reproduced by k-WAVE simulations. CONCLUSIONS: The authors have studied the ionoacoustic signal of the Bragg peak in experiments using a 20 MeV proton beam with its correspondingly localized energy deposition, demonstrating submillimeter position resolution and providing a deep insight in the correlation between the acoustic signal and Bragg peak shape. These results, together with earlier experiments and new simulations (including the results in this study) at higher energies, suggest ionoacoustics as a technique for range verification in particle therapy at locations, where the tumor can be localized by ultrasound imaging. This acoustic range verification approach could offer the possibility of combining anatomical ultrasound and Bragg peak imaging, but further studies are required for translation of these findings to clinical application.

Differential diffuse optical tomography.

We formulate a perturbative solution for the heterogeneous diffusion equation which demonstrates how to use differential changes in diffuse light transmission to construct images of tissue absorption changes following contrast agent administration. The analysis exposes approximations leading to an intuitive and simplified inverse algorithm, shows explicitly why transmission geometries are less susceptible to error than the remission geometries, and why differential measurements are less susceptible to surface artifacts. These ideas about differential diffuse optical tomography are not only applicable to tumor detection and characterization using contrast agents, but also to functional activation studies with or without contrast agents and multiple-wavelength measurements.

Accuracy limits in the determination of absolute optical properties using time-resolved NIR spectroscopy.

We assess typical systematic experimental errors involved in a time-resolved measurement as applied to NIR diffuse optical spectroscopy and investigate their effect on the quantification accuracy of the absorption and the reduced scattering coefficient. We demonstrate that common systematic experimental uncertainties may lead to quantification errors of 10% or more, even when excellent signal to noise ratio conditions exist and accurate photon propagation models are employed. We further demonstrate that the accuracy of the calculation depends nonlinearly on the optical properties of the medium measured. High scattering and low absorbing media can be quantified more accurately than media with low scattering or high absorption using measurements of the same signal to noise ratio. We further discuss curve-shape fitting schemes that aid in improving the quantification accuracy in the presence of experimental errors. Finally, we identify uncertainties that set quantification accuracy limits and we find temporal resolution as the ultimate limiting factor in the quantification accuracy achieved. Our findings suggest that temporal resolution of the order of 10 ps is necessary for quantifying the absorption and reduced scattering coefficient of diffuse media with accuracy better than 5% using curve fitting methods. In that sense this analysis can be used in time-resolved system design and in predicting the expected errors given the technology selected for time-resolved measurements.

Oximetry based on diffuse photon density wave differentials.

The quantification of tissue optical properties for calculating blood saturation and hemoglobin concentration using measurements of diffuse photon density waves at some distance away from an intensity-modulated light source, generally requires the determination of the amplitude and phase of this light source. This determination may become a severe impediment for measurements performed in the clinical environment. In this work we extend a self-calibrating methodology developed for constant wave and modulation depth-phase measurements, to include amplitude and phase measurements of diffuse photon density waves. The method uses amplitude and phase changes of intensity modulated light, under the assumption of known index of refraction and invariant reduced scattering coefficient mu's, to quantify the absorption coefficient mu(a) without requiring initial amplitude and phase knowledge. Quantification of the mu(a) at selected time points during a measurement can then be employed to calibrate numerical solutions of the diffusion equation and compute the mu(a) for the remaining time points of the experiment. It is shown that the method is quite insensitive to the knowledge of the exact mu's value so that an assumption on the average mu's value for the tissue measured may be employed. The sensitivity of calculating blood saturation and hemoglobin concentration, as a function of the deviation of the mu's used in the calculation versus the real mu's value is investigated using simulated data. It is also demonstrated that the saturation calculation is especially insensitive to the mu's guess. The performance of the method to quantify blood oxygen saturation and the concentrations of oxy- and deoxy-hemoglobin is examined with experimental measurements at two wavelengths on specially constructed blood model phantoms. To validate the method the measurements are monitored by a time-resolved spectrometer. The method is shown to be accurate to within +/-5% in calculating blood saturation and to within +/-10% in calculating hemoglobin concentration compared to the results obtained with the time-resolved spectrometer and the expected theoretical values.

Three-dimensional diffuse optical tomography in the parallel plane transmission geometry: evaluation of a hybrid frequency domain/continuous wave clinical system for breast imaging.

Three-dimensional diffuse optical tomography (DOT) of breast requires large data sets for even modest resolution (1 cm). We present a hybrid DOT system that combines a limited number of frequency domain (FD) measurements with a large set of continuous wave (cw) measurements. The FD measurements are used to quantitatively determine tissue averaged absorption and scattering coefficients. The larger cw data sets (10(5) measurements) collected with a lens coupled CCD, permit 3D DOT reconstructions of a 1-liter tissue volume. To address the computational complexity of large data sets and 3D volumes we employ finite difference based reconstructions computed in parallel. Tissue phantom measurements evaluate imaging performance. The tests include the following: point spread function measures of resolution, characterization of the size and contrast of single objects, field of view measurements and spectral characterization of constituent concentrations. We also report in vivo measurements. Average tissue optical properties of a healthy breast are used to deduce oxy- and deoxy-hemoglobin concentrations. Differential imaging with a tumor simulating target adhered to the surface of a healthy breast evaluates the influence of physiologic fluctuations on image noise. This tomography system provides robust, quantitative, full 3D image reconstructions with the advantages of high data throughput, single detector-tissue coupling path, and large (1L) imaging domains. In addition, we find that point spread function measurements provide a useful and comprehensive representation of system performance.