Broadband mesoscopic optoacoustic tomography reveals skin layers: publisher's note.

This publisher's note contains corrections to Opt. Lett.39, 6297 (2014)OPLEDP0146-959210.1364/OL.39.006297.

Optical features of human skin revealed by optoacoustic mesoscopy in the visible and short-wave infrared regions.

Detailed assessment of skin conditions or the efficacy of skin treatments could greatly benefit from noninvasively assessing the distribution of cutaneous and subcutaneous structures and biomolecules. We considered ultrawideband raster scan optoacoustic mesoscopy with an extended wavelength range from visible to short-wave infrared and observed previously unseen high-resolution images of lipids colocalized with water, melanin, and hemoglobin distribution in human skin. Based on this contrast, the technique resolves subcutaneous fat, the pilosebaceous unit with complete hair strand and bulb, dermal microvasculature, and epidermal structures. We further visualize melanoidins that form via the Maillard reaction in the ultrathin stratum corneum layer, analyze their absorption spectrum, and separate them from the melanin layer. The suggested method may allow novel interrogation of skin conditions, possibly impacting diagnostics and medical and cosmetic treatments.

Electrolytic conductivity-related radiofrequency heating of aqueous suspensions of nanoparticles for biomedicine.

The development of suitable contrast agents can significantly enhance the efficiency of modern imaging and treatment techniques, such as thermoacoustic (TA) tomography and radio-frequency (RF) hyperthermia of cancer. Here, we examine the heating of aqueous suspensions of silicon (Si) and gold (Au) nanoparticles (NPs) under RF irradiation in the MHz frequency range. The heating rate of aqueous suspensions of Si NPs exhibited non-monotonic dependency on the electrical conductivity of the suspension. The experimental results were explained by the mathematical model considering oscillating solvated ions as the main source of Joule heating. These ions could be the product of the dissolution of Si NPs or organic coating of Au NPs. Thus, the ions governed the conductivity of the suspensions, which in turn governs both the heating rate and the near-field RF TA response. The model predicted the contrast in different tissues taking into account both Joule heating and dielectric losses.

Hybrid multiphoton and optoacoustic microscope.

We present a hybrid microscope combining multiphoton microscopy incorporating second-harmonic generation contrast and optical-resolution optoacoustic (photoacoustic) microscopy. We study the relative performance of the two systems and investigate the complementarity of contrast by demonstrating the label-free imaging capabilities of the hybrid microscope on zebrafish larvae ex vivo, concurrently visualizing the fish musculature and melanocytes. This implementation can prove useful in multiparametric microscopy studies, enabling broader information to be collected from biological specimens.

Ultrawideband reflection-mode optoacoustic mesoscopy.

We developed a reflection-mode optoacoustic mesoscopy system, based on raster-scanning of a custom designed spherically focused ultrasound detector, enabling seamless epi-illumination of the volume imaged. We study the performance of acoustic-resolution mesoscopy operating at an ultrawideband bandwidth of 20-180 MHz. i.e., a frequency band spreading over virtually an order of magnitude. Using tomographic reconstruction we showcase previously unreported, to our knowledge, axial resolutions of 4 µm and transverse resolutions of 18 µm reaching depths of up to 5 mm. We further investigate the frequency-dependence of features seen on the images to understand the implications of ultrawideband measurements. We show the overall imaging performance and the frequency ranges that contribute to observable resolution improvements from phantoms and animals.

Broadband mesoscopic optoacoustic tomography reveals skin layers.

We have imaged for the first time to our knowledge human skin in vivo with a raster-scan optoacoustic mesoscopy system based on a spherically focused transducer with a central frequency of 102.8 MHz and large bandwidth (relative bandwidth 105%). Using tissue phantoms we have studied the ability of the system to image vessels of sizes within the anatomically significant range from the key anatomical vasculature sites. The reconstructed images from experiments in vivo show several structures from the capillary loops at the dermal papillae, the horizontal plexus, and the difference between the dermis and the epidermis layers.

Raster-scan optoacoustic mesoscopy in the 25-125 MHz range.

We developed a raster-scan acoustic resolution broadband optoacoustic mesoscopy system and investigated the imaging performance using ultrasonic frequencies up to 125 MHz. The developed system achieves 7 µm axial resolution and transverse resolution of 30 µm reaching depths of at least 5 mm. This unprecedented performance is achieved by operating at out-of-focus ultrasonic detection and tomographic reconstruction. We demonstrate the limits reached due to the width of the laser pulse employed and showcase the technique on drosophila fly and drosophila pupae ex vivo.

Opening a window to skin biomarkers for diabetes stage with optoacoustic mesoscopy.

Being the largest and most accessible organ of the human body, the skin could offer a window to diabetes-related complications on the microvasculature. However, skin microvasculature is typically assessed by histological analysis, which is not suited for applications to large populations or longitudinal studies. We introduce ultra-wideband raster-scan optoacoustic mesoscopy (RSOM) for precise, non-invasive assessment of diabetes-related changes in the dermal microvasculature and skin micro-anatomy, resolved with unprecedented sensitivity and detail without the need for contrast agents. Providing unique imaging contrast, we explored a possible role for RSOM as an investigational tool in diabetes healthcare and offer the first comprehensive study investigating the relationship between different diabetes complications and microvascular features in vivo. We applied RSOM to scan the pretibial area of 95 participants with diabetes mellitus and 48 age-matched volunteers without diabetes, grouped according to disease complications, and extracted six label-free optoacoustic biomarkers of human skin, including dermal microvasculature density and epidermal parameters, based on a novel image-processing pipeline. We then correlated these biomarkers to disease severity and found statistically significant effects on microvasculature parameters as a function of diabetes complications. We discuss how label-free RSOM biomarkers can lead to a quantitative assessment of the systemic effects of diabetes and its complications, complementing the qualitative assessment allowed by current clinical metrics, possibly leading to a precise scoring system that captures the gradual evolution of the disease.

Dermal features derived from optoacoustic tomograms via machine learning correlate microangiopathy phenotypes with diabetes stage.

Skin microangiopathy has been associated with diabetes. Here we show that skin-microangiopathy phenotypes in humans can be correlated with diabetes stage via morphophysiological cutaneous features extracted from raster-scan optoacoustic mesoscopy (RSOM) images of skin on the leg. We obtained 199 RSOM images from 115 participants (40 healthy and 75 with diabetes), and used machine learning to segment skin layers and microvasculature to identify clinically explainable features pertaining to different depths and scales of detail that provided the highest predictive power. Features in the dermal layer at the scale of detail of 0.1-1 mm (such as the number of junction-to-junction branches) were highly sensitive to diabetes stage. A 'microangiopathy score' compiling the 32 most-relevant features predicted the presence of diabetes with an area under the receiver operating characteristic curve of 0.84. The analysis of morphophysiological cutaneous features via RSOM may allow for the discovery of diabetes biomarkers in the skin and for the monitoring of diabetes status.

Near-field thermoacoustic imaging with transmission line pulsers.

PURPOSE: Near-field radiofrequency thermoacoustic (NRT) tomography has been recently introduced for imaging electromagnetic (EM) properties of tissues using ultrawideband, high-energy impulses, which induce thermoacoustic responses. Operation in the near-field allows for more effective energy coupling into tissue, compared to using radiating sources, which in turn enables the use of shorter excitation pulses and leads to higher image resolution. This work aimed at investigating transmission lines as a method to generate excitation pulses to improve the NRT resolution over previous implementations without compromising the energy coupled into tissue. METHODS: The authors implemented a number of custom-made transmission lines to overcome the challenges of the broadband nature of the impulse excitation required in NRT. The authors further constructed phantoms and investigated the performance of the lines in regard to the pulse duration, energy coupling and the resulting resolution, and image quality achieved. Finally, the authors employed mice in order to investigate the performance of the approach in tissue imaging. RESULTS: The authors found that the use of transmission lines resulted in the generation of RF impulses in the range of tens of nanoseconds and shorter. This performance resulted to resolution improvements over previous thermoacoustic imaging implementations, reaching 45 µm resolution, while retaining several tens to hundreds of milli-Joules of energy per pulse. This performance further allowed the visualization and clear differentiation of different mouse structures such as the heart, lung, or spinal cord. CONCLUSIONS: The use of transmission lines significantly improved the NRT performance leading to high thermoacoustic tomography imaging quality by coupling adequate amounts of energy within short times at a relatively low cost.

In Vivo Three-Dimensional Raster Scan Optoacoustic Mesoscopy Using Frequency Domain Inversion.

Optoacoustic signals are typically reconstructed into images using inversion algorithms applied in the time-domain. However, time-domain reconstructions can be computationally intensive and therefore slow when large amounts of raw data are collected from an optoacoustic scan. Here we considered a fast weighted ω-k (FWOK) algorithm operating in the frequency domain to accelerate the inversion in raster-scan optoacoustic mesoscopy (RSOM), while seamlessly incorporating impulse response correction with minimum computational burden. We investigated the FWOK performance with RSOM measurements from phantoms and mice in vivo and obtained 360-fold speed improvement over inversions based on the back-projection algorithm in the time-domain. This previously unexplored inversion of in vivo optoacoustic data with impulse response correction in frequency domain reconstructions points to a promising strategy of accelerating optoacoustic imaging computations, toward video-rate tomography.

Noninvasive visualization of electrical conductivity in tissues at the micrometer scale.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 µm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.

Spatial heterogeneity of oxygenation and haemodynamics in breast cancer resolved in vivo by conical multispectral optoacoustic mesoscopy.

The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 µm throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.

Longitudinal imaging of T cell-based immunotherapy with multi-spectral, multi-scale optoacoustic tomography.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

High-resolution optoacoustic imaging of tissue responses to vascular-targeted therapies.

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy.

Optoacoustic mesoscopy for biomedicine.

Fuelled by innovation, optical microscopy plays a critical role in the life sciences and medicine, from basic discovery to clinical diagnostics. However, optical microscopy is limited by typical penetration depths of a few hundred micrometres for in vivo interrogations in the visible spectrum. Optoacoustic microscopy complements optical microscopy by imaging the absorption of light, but it is similarly limited by penetration depth. In this Review, we summarize progress in the development and applicability of optoacoustic mesoscopy (OPAM); that is, optoacoustic imaging with acoustic resolution and wide-bandwidth ultrasound detection. OPAM extends the capabilities of optical imaging beyond the depths accessible to optical and optoacoustic microscopy, and thus enables new applications. We explain the operational principles of OPAM, its placement as a bridge between optoacoustic microscopy and optoacoustic macroscopy, and its performance in the label-free visualization of tissue pathophysiology, such as inflammation, oxygenation, vascularization and angiogenesis. We also review emerging applications of OPAM in clinical and biological imaging.

Fully automated identification of skin morphology in raster-scan optoacoustic mesoscopy using artificial intelligence.

PURPOSE: Identification of morphological characteristics of skin lesions is of vital importance in diagnosing diseases with dermatological manifestations. This task is often performed manually or in an automated way based on intensity level. Recently, ultra-broadband raster-scan optoacoustic mesoscopy (UWB-RSOM) was developed to offer unique cross-sectional optical imaging of the skin. A machine learning (ML) approach is proposed here to enable, for the first time, automated identification of skin layers in UWB-RSOM data. MATERIALS AND METHODS: The proposed method, termed SkinSeg, was applied to coronal UWB-RSOM images obtained from 12 human participants. SkinSeg is a multi-step methodology that integrates data processing and transformation, feature extraction, feature selection, and classification. Various image features and learning models were tested for their suitability at discriminating skin layers including traditional machine learning along with more advanced deep learning algorithms. An support vector machines-based postprocessing approach was finally applied to further improve the classification outputs. RESULTS: Random forest proved to be the most effective technique, achieving mean classification accuracy of 86.89% evaluated based on a repeated leave-one-out strategy. Insights about the features extracted and their effect on classification accuracy are provided. The highest accuracy was achieved using a small group of four features and remained at the same level or was even slightly decreased when more features were included. Convolutional neural networks provided also promising results at a level of approximately 85%. The application of the proposed postprocessing technique was proved to be effective in terms of both testing accuracy and three-dimensional visualization of classification maps. CONCLUSIONS: SkinSeg demonstrated unique potential in identifying skin layers. The proposed method may facilitate clinical evaluation, monitoring, and diagnosis of diseases linked to skin inflammation, diabetes, and skin cancer.

Motion Quantification and Automated Correction in Clinical RSOM.

Raster-scan optoacoustic mesoscopy (RSOM) offers high-resolution non-invasive insights into skin pathophysiology, which holds promise for disease diagnosis and monitoring in dermatology and other fields. However, RSOM is quite vulnerable to vertical motion of the skin, which can depend on the part of the body being imaged. Motion correction algorithms have already been proposed, but they are not fully automated, they depend on anatomical segmentation pre-processing steps that might not be performed successfully, and they are not site- specific. Here, we determined for the first time the magnitude of the micrometric vertical skin displacements at different sites on the body that affect RSOM. The quantifi- cation of motion allowed us to develop a site-specific correction algorithm. The algorithm is fully automated and does not need prior anatomical information. We found that the magnitude of the vertical motion depends strongly on the site of imaging and is caused by breathing, heart beating, and arterial pulsation. The developed algorithm resulted in more than 2-fold improvement in the signal-to-noise ratio of the reconstructed images at every site tested. Proposing an effective automated motion correction algorithm paves the way for realizing the full clinical potential of RSOM.

Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

Optoacoustic microscopy at multiple discrete frequencies.

Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.