Application of Photoacoustic Methods and Confocal Microscopy for Monitoring of Therapeutic Response in Plaque Psoriasis.

Psoriasis is prone to relapses and requires long-term therapy that may induce a range of adverse effects; therefore, an efficient and early detection of relapses is desirable. In this study, photoacoustic imaging and confocal laser scanning microscopic (CLSM) methods were investigated for their suitability in psoriasis follow-up examinations. Using a high-resolution photoacoustic system, the vascular structures of 11 psoriatic patients and 6 healthy volunteers were investigated. No differences were detected with respect to the average vessel diameter and vasculature per unit volume in the tissue of healthy volunteers and non-lesional and lesional skin areas of psoriatic patients. By means of CLSM, the diameters of the dermal papillae of 6 volunteers and 6 psoriatic patients were determined. The diameters of the dermal papillae of the healthy volunteers (0.074 ± 0.006 mm) revealed no significant difference when compared to non-lesional skin areas of psoriatic patients (0.079 ± 0.005 mm). The results obtained for the lesions in psoriatic patients showed a significant difference (Wilcoxon test, p = 0.028) between the diameters of the dermal papillae of the lesional skin areas (0.114 ± 0.012 mm) and the non-lesional skin areas (0.079 ± 0.005 mm). Thus, CLSM can be applied for monitoring psoriasis follow-up examinations.

Comparison of the optoacoustic signal generation efficiency of different nanoparticular contrast agents.

Optoacoustic imaging represents a new modality that allows noninvasive in vivo molecular imaging with optical contrast and acoustical resolution. Whereas structural or functional imaging applications such as imaging of vasculature do not require contrast enhancing agents, nanoprobes with defined biochemical binding behavior are needed for molecular imaging tasks. Since the contrast of this modality is based on the local optical absorption coefficient, all particle or molecule types that show significant absorption cross sections in the spectral range of the laser wavelength used for signal generation are suitable contrast agents. Currently, several particle types such as gold nanospheres, nanoshells, nanorods, or polymer particles are used as optoacoustic contrast agents. These particles have specific advantages with respect to their absorption properties, or in terms of biologically relevant features (biodegradability, binding to molecular markers). In the present study, a comparative analysis of the signal generation efficiency of gold nanorods, polymeric particles, and magnetite particles using a 1064 nm Nd:YAG laser for signal generation is described.

Antitumor necrosis factor-α antibody-coupled gold nanorods as nanoprobes for molecular optoacoustic imaging in arthritis.

Optoacoustic molecular imaging can provide spatially resolved information about the presence of molecular markers in vivo. We synthesized elongated gold nanorods having an absorption maximum in the range of 1064 nm modified with the antibodies infliximab and certolizumab for targeting TNF-α to detect inflammation in arthritic mouse knees. We showed an differential enhancement of optoacoustic signal amplitudes after the injection of infliximab-, but not certolizumab-modified and PEGylated control particles on arthritic and healthy control mice by using a fast-scanning optoacoustic imaging platform based on a pulsed Nd:YAG laser and a single focused ultrasound transducer. The excellent photoacoustic properties of the gold nanorods confirmed the overexpression of TNF-α in arthritic knees. Due to the uncomplicated coupling chemistry and the scalability of ultrasound-based imaging approaches, these results potentially allow a transfer to various preclinical and clinical applications. From the Clinical Editor: Gold nanorods were modified with TNF-α targeting antibodies and used to detect inflammation in arthritic mouse knees via optoaoustic imaging. A fast-scanning optoacoustic imaging platform based on a pulsed Nd:YAG laser and a single focused ultrasound transducer was utilized for imaging. The excellent photoacoustic properties of these gold nanorods confirmed the overexpression of TNF-α, paving the way towards further preclinical and future clinical applications.

Preparation and biological evaluation of multifunctional PLGA-nanoparticles designed for photoacoustic imaging.

Nanoparticulate contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. Here we introduce polymer-based multifunctional nanoparticles exhibiting a near-infrared absorption in the range of the Nd:YAG laser wavelength of 1064 nm as a novel resorbable photoacoustic (PA) contrast system and report about their biological evaluation. Submicron-sized spherical nanoparticles with a high encapsulation efficiency (>87%) were created by incorporation of near-infrared dyes (IR5/IR26) in poly[(rac-lactide)-co-glycolide] (PLGA) with 50 mol% glycolide content via a specific spray-drying process in good yield (>75%). Subsequent application of a centrifugation protocol produced two different size fractions with diameters in the ranges 445-540 nm and 253-305 nm; these were further used for investigation of PA properties and cytotoxic effects. The prepared PLGA nanoparticles exhibited PA properties using a Nd:YAG laser-based system. After exposure of particle concentrations up to 10 µg·ml(-1) for 2 days no effects on viability, mitochondrial activity and proliferation, and cell death of human hepatocarcinoma cells and monkey kidney cells were observed. The excellent PA properties in combination with the positive biological results qualify the dye-loaded PLGA particles as promising candidates for a resorbable PA contrast system. FROM THE CLINICAL EDITOR: Photoacoustics (PA), a new modality, in which laser light is shined into tissue and absorbed by inherent proteins or synthetic particles is reflected back and received as ultrasound. This technique was shown to be effective with an erodible polymer particle containing near infrared dyes. In vitro, the PA properties of the PLGA particles persisted for 2 days in cell culture.

Enhancement of Acoustic Microscopy Lateral Resolution: A Comparison Between Deep Learning and Two Deconvolution Methods.

Scanning acoustic microscopy (SAM) provides high-resolution images of biological tissues. Since higher transducer frequencies limit penetration depth, image resolution enhancement techniques could help in maintaining sufficient lateral resolution without sacrificing penetration depth. Compared with existing SAM research, this work introduces two novelties. First, deep learning (DL) is used to improve lateral resolution of 180-MHz SAM images, comparing it with two deconvolution-based approaches. Second, 316-MHz images are used as ground truth in order to quantitatively evaluate image resolution enhancement. The samples used were mouse and rat brain sections. The results demonstrate that DL can closely approximate ground truth (NRMSE = 0.056 and PSNR = 28.4 dB) even with a relatively limited training set (four images, each smaller than 1 mm ×1 mm). This study suggests the high potential of using DL as a single image superresolution method in SAM.

Wave front analysis for enhanced time-domain beamforming of point-like targets in optoacoustic imaging using a linear array.

Using linear array transducers in combination with state-of-the-art multichannel electronics allows to perform optoacoustic imaging with frame rates only limited by the laser pulse repetition frequency and the acoustic time of flight. However, characteristic image artefacts resulting from the limited view and a lower SNR when compared to systems based on single-element focused transducers represent a burden for the clinical acceptance of the technology. In this paper, we present a new method for the improvement of image quality based on the analysis of the signal amplitudes along summation curves during the delay-and-sum beamforming process (DAS). The algorithm compares amplitude distributions along wave fronts with theoretical patterns from optoacoustic point sources. The method was validated on simulated and experimental phantom as well as in-vivo data. An improvement of the lateral resolution by more than a factor of two when comparing conventional DAS and our approach could be shown (numeric and experimental phantom data). For instance, on experimental data from a wire phantom, a PSF in the range of 0.18-0.22 mm was obtained with our approach against 0.48 mm for standard DAS. Furthermore, the SNR of a subcutaneous vessel 2.5 mm below the skin surface was improved by about 30 dB when compared to standard DAS.

Hybrid Photoacoustic/Ultrasound Tomograph for Real-Time Finger Imaging.

We report a target-enclosing, hybrid tomograph with a total of 768 elements based on capacitive micromachined ultrasound transducer technology and providing fast, high-resolution 2-D/3-D photoacoustic and ultrasound tomography tailored to finger imaging. A freely programmable ultrasound beamforming platform sampling data at 80 MHz was developed to realize plane wave transmission under multiple angles. A multiplexing unit enables the connection and control of a large number of elements. Fast image reconstruction is provided by GPU processing. The tomograph is composed of four independent and fully automated movable arc-shaped transducers, allowing imaging of all three finger joints. The system benefits from photoacoustics, yielding high optical contrast and enabling visualization of finger vascularization, and ultrasound provides morphologic information on joints and surrounding tissue. A diode-pumped, Q-switched Nd:YAG laser and an optical parametric oscillator are used to broaden the spectrum of emitted wavelengths to provide multispectral imaging. Custom-made optical fiber bundles enable illumination of the region of interest in the plane of acoustic detection. Precision in positioning of the probe in motion is ensured by use of a motor-driven guide slide. The current position of the probe is encoded by the stage and used to relate ultrasound and photoacoustic signals to the corresponding region of interest of the suspicious finger joint. The system is characterized in phantoms and a healthy human finger in vivo. The results obtained promise to provide new opportunities in finger diagnostics and establish photoacoustic/ultrasound-tomography in medical routine.

Calibrated Linear Array-Driven Photoacoustic/Ultrasound Tomography.

The anisotropic resolution of linear arrays, tools that are widely used in diagnostics, can be overcome by compounding approaches. We investigated the ability of a recently developed calibration and a novel algorithm to determine the actual radial transducer array distance and its misalignment (tilt) with respect to the center of rotation in a 2-D and 3-D tomographic setup. By increasing the time-of-flight accuracy, we force in-phase summation during the reconstruction. Our setup is composed of a linear transducer and a rotation and translation axis enabling multidimensional imaging in ultrasound and photoacoustic mode. Our approach is validated on phantoms and young mice ex vivo. The results indicate that application of the proposed analytical calibration algorithms prevents image artifacts. The spatial resolution achieved was 160 and 250 µm in photoacoustic mode of 2-D and 3-D tomography, respectively.

Optoacoustic imaging of subcutaneous microvasculature with a class one laser.

We developed a combined imaging platform allowing optoacoustic and ultrasound imaging based on a low energy laser and a handheld probe. The device is based on a sensitive single element 35-MHz focused transducer, a 2-D piezoscanner and a dual-wavelength switchable Nd:YAG laser. Acoustical detection and optical illumination are confocal for optimization of optoacoustic signal-to-noise ratio. The system allows to scan over a range up to 12 mm ×12 mm in xy-direction with an isotropic lateral resolution of about 90 µm. Although the device is a class 1 laser product having pulse energies in the range, in vivo images of subcutaneous microvasculature could be obtained from human skin with signal-to-noise levels as good as 20 dB.

Near-infrared dye-loaded PLGA nanoparticles prepared by spray drying for photoacoustic applications.

INTRODUCTION: Nanoparticulate contrast agents are of great interest for diagnostic applications with high resolution in medicine. Here we present polymer-based degradable nanoparticles exhibiting a near infrared (NIR) absorption suitable for photoacoustic imaging. METHODS: The nanoparticles were prepared by incorporation of indocyanine green (ICG) as NIR dye in poly[(rac-lactide)-co-glycolide] (PLGA) via an optimized spray drying process. By application of a multi-step centrifugation protocol, two different size fractions were achieved. The biocompatibitilty of the nanoparticles was tested in 2D cell cultures (human hepatocarcinoma cells and monkey kidney cells) using WST-1, BrdU and LDH assay. RESULTS: Spherical particles were obtained with a good yield (>81%), showing a high NIR-dye encapsulation efficiency (>98%). By multi-step centrifugation, two different size fractions with a mean diameter of 640 nm and 390 nm were obtained. Cytotoxicity studies of the synthesized ICG-loaded PLGA particles were performed. No cytotoxic effect on metabolic activity, proliferation, or membrane integrity was observed. CONCLUSION: The high optical absorption at the relevant NIR-wavelength around 800 nm in combination with absence of cytotoxicity qualifies the ICG-loaded PLGA particles as promising candidates for degradable photoacoustic contrast agents.

High frequency optoacoustic microscopy.

Photoacoustic imaging--also called optoacoustic imaging--is a new hybrid modality of high tissue contrast which is based on the varying optical properties of tissue. The acoustic signal generated by pulsed laser absorption reports tissue-specific information with high spatial resolution. To increase the intrinsic contrast in tissue, absorbing particles are of great interest for optical imaging because of their considerable capacity to absorb and scatter light at visible and near-infrared wavelengths. The aim of the work presented here is to establish a scalable photoacoustic technology for volume imaging of biological samples down to diffraction limited microscopy. For this purpose a versatile photoacoustic microscopy platform has been developed with unmatched spatial resolution consisting of a microchip laser and a measurement cell with different transducers attached allowing generation and detection of laser-induced ultrasound signals in a frequency range up to 400 MHz. The performance of a versatile photoacoustic microscopy platform was evaluated via 2D optoacoustic images of light absorbing microparticles (5 microm Fe(3)0(4) and 1 micromblack toner particles) embedded in a polystyrene matrix. High frequency signals in the frequency range of 400 MHz generated by a single 1 microm particle could be recorded with a high signal to noise ratio (SNR) of 34 dB.