[Show abstract][Hide abstract] ABSTRACT: Light propagating in tissue attains a spectrum that varies with location due
to wavelength-dependent fluence attenuation by tissue optical properties, an
effect that causes spectral corruption. Predictions of the spectral variations
of light fluence in tissue are challenging since the spatial distribution of
optical properties in tissue cannot be resolved in high resolution or with high
accuracy by current methods. Spectral corruption has fundamentally limited the
quantification accuracy of optical and optoacoustic methods and impeded the
long sought-after goal of imaging blood oxygen saturation (sO2) deep in
tissues; a critical but still unattainable target for the assessment of
oxygenation in physiological processes and disease. We discover a new principle
underlying light fluence in tissues, which describes the wavelength dependence
of light fluence as an affine function of a few reference base spectra,
independently of the specific distribution of tissue optical properties. This
finding enables the introduction of a previously undocumented concept termed
eigenspectra Multispectral Optoacoustic Tomography (eMSOT) that can effectively
account for wavelength dependent light attenuation without explicit knowledge
of the tissue optical properties. We validate eMSOT in more than 2000
simulations and with phantom and animal measurements. We find that eMSOT can
quantitatively image tissue sO2 reaching in many occasions a better than
10-fold improved accuracy over conventional spectral optoacoustic methods.
Then, we show that eMSOT can spatially resolve sO2 in muscle and tumor;
revealing so far unattainable tissue physiology patterns. Last, we related
eMSOT readings to cancer hypoxia and found congruence between eMSOT tumor sO2
images and tissue perfusion and hypoxia maps obtained by correlative
[Show abstract][Hide abstract] ABSTRACT: Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three-dimensional detection of pathophysiological parameters within skin. It was recently shown that single-wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three-dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15-125 MHz. We employ a per-pulse tunable laser to inherently co-register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label-free visualization of physiological biomarkers in skin in vivo. Cross-sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50-90%. All scale bars: 250 µm.
Journal of Biophotonics 11/2015; DOI:10.1002/jbio.201500247 · 4.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High fidelity optoacoustic (photoacoustic) tomography requires dense spatial sampling of optoacoustic signals using point acoustic detectors. However, in practice, spatial resolution of the images is often limited by limited sampling either due to coarse multi-element arrays or time in raster scan measurements. Herein, we investigate a method that integrates information from multiple optoacoustic images acquired at sub-diffraction steps into one high resolution image by means of an iterative registration algorithm. Experimental validations performed in target phantoms and ex-vivo tissue samples confirm that the suggested approach renders significant improvements in terms of optoacoustic image resolution and quality without introducing significant alterations into the signal acquisition hardware or inversion algorithms.
IEEE Transactions on Medical Imaging 10/2015; DOI:10.1109/TMI.2015.2497159 · 3.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo. When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.
[Show abstract][Hide abstract] ABSTRACT: The optoacoustic (photoacoustic) technique has been shown to resolve anatomical, functional, and molecular features at depths that go beyond the reach of epi-illumination optical microscopy, offering new opportunities for endoscopic imaging. In this Letter, we investigate the merits of optoacoustic endoscopy implemented by translating a sound detector in linear or curved geometries. The linear and curved detection geometries are achieved by employing an intravascular ultrasound transducer within a plastic guide shaped to a line or a curve. This concept could be used together with optical endoscopes to yield hybrid optical and optoacoustic imaging.
[Show abstract][Hide abstract] ABSTRACT: The concept of sparsity is extensively exploited in the fields of data acquisition and image processing, contributing to better signal-to-noise and spatio-temporal performance of the various imaging methods. In the field of optoacoustic tomography, the image reconstruction problem is often characterized by computationally extensive inversion of very large datasets, for instance when acquiring volumetric multispectral data with high temporal resolution. In this article we seek to accelerate accurate model-based optoacoustic inversions by identifying various sources of sparsity in the forward and inverse models as well as in the single- and multi-frame representation of the projection data. These sources of sparsity are revealed through appropriate transformations in the signal, model and image domains and are subsequently exploited for expediting image reconstruction. The sparsity-based inversion scheme was tested with experimental data, offering reconstruction speed enhancement by a factor of 40 to 700 times as compared with the conventional iterative model-based inversions while preserving similar image quality. The demonstrated results pave the way for achieving real-time performance of model-based reconstruction in multi-dimensional optoacoustic imaging.
[Show abstract][Hide abstract] ABSTRACT: Discerning the accurate distribution of chromophores and biomarkers by means of optoacoustic imaging is commonly challenged by the highly heterogeneous excitation light patterns resulting from strong spatial variations of tissue scattering and absorption. Here we used the light-fluence dependent switching kinetics of reversibly switchable fluorescent proteins (RSFPs), in combination with real-time acquisition of volumetric multi-spectral optoacoustic data to correct for the light fluence distribution deep in scattering media. The new approach allows for dynamic fluence correction in time-resolved imaging, e.g., of moving organs, and can be extended to work with a large palette of available synthetic and genetically encoded photochromic substances for multiplexed wavelength-specific fluence normalization.
[Show abstract][Hide abstract] ABSTRACT: 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.
Trends in Biotechnology 10/2015; 33(11). DOI:10.1016/j.tibtech.2015.08.002 · 11.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unveiling mechanisms driving specification, recruitment and regeneration of melanophores is the key to understanding melanin-related disorders. This study reports on the applicability of a hybrid focus opto-acoustic microscope (HFOAM) for volumetric tracking of migratory melanophores in developing zebrafish. The excellent contrast from highly-absorbing melanin provided by the method is shown to be ideal for label-free dynamic visualization of melanophores in their unperturbed living environment. We established safe laser energy levels that enable high-contrast longitudinal tracking of the cells over an extended period of developmental time without causing cell toxicity or pigment bleaching. Owing to its hybrid optical and acoustic resolution, the new imaging technique can be seamlessly applied for non-invasive studies of both optically-transparent larval as well as adult stages of the zebrafish model organism, which is not possible using other optical microscopy methods.
Mechanisms of development 09/2015; DOI:10.1016/j.mod.2015.09.001 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intravital imaging within heterogenic solid tumours is important for understanding blood perfusion profiles responsible for establishment of multiple parameters within the tumour mass, such as hypoxic and nutrition gradients, cell viability, proliferation and drug response potentials.
Herein, we developed a method based on a volumetric multispectral optoacoustic tomography (vMSOT) for cancer imaging in preclinical models and explored its capacity for three-dimensional imaging of anatomic, vascular and functional tumour profiles in real time.
In contrast to methods based on cross-sectional (2D) image acquisition as a basis for 3D rendering, vMSOT has attained concurrent observations from the entire tumour volume at 10 volumetric frames per second. This truly four dimensional imaging performance has enabled here the simultaneous assessment of blood oxygenation gradients and vascularization in solid breast tumours and revealed different types of blood perfusion profiles in-vivo.
The newly introduced capacity for high-resolution three-dimensional tracking of fast tumour perfusion suggests vMSOT as a powerful method in preclinical cancer research and theranostics. As the imaging setup can be equally operated in both stationary and handheld mode, the solution is readily translatable for perfusion monitoring in a clinical setting.
• vMSOT visualizes 3D anatomic, vascular and functional tumour profiles in real time. • Three types of blood perfusion profiles are revealed in breast tumour model. • The method is readily adaptable to operate in a handheld clinical mode.
European Radiology 09/2015; DOI:10.1007/s00330-015-3980-0 · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
With recent advancement in hardware of optoacoustic imaging systems, highly detailed cross-sectional images may be acquired at a single laser shot, thus eliminating motion artifacts. Nonetheless, other sources of artifacts remain due to signal distortion or out-of-plane signals. The purpose of image reconstruction algorithms is to obtain the most accurate images from noisy, distorted projection data.
In this paper, the authors use the model-based approach for acoustic inversion, combined with a sparsity-based inversion procedure. Specifically, a cost function is used that includes the L1 norm of the image in sparse representation and a total variation (TV) term. The optimization problem is solved by a numerically efficient implementation of a nonlinear gradient descent algorithm. TV-L1 model-based inversion is tested in the cross section geometry for numerically generated data as well as for in vivo experimental data from an adult mouse.
In all cases, model-based TV-L1 inversion showed a better performance over the conventional Tikhonov regularization, TV inversion, and L1 inversion. In the numerical examples, the images reconstructed with TV-L1 inversion were quantitatively more similar to the originating images. In the experimental examples, TV-L1 inversion yielded sharper images and weaker streak artifact.
The results herein show that TV-L1 inversion is capable of improving the quality of highly detailed, multiscale optoacoustic images obtained in vivo using cross-sectional imaging systems. As a result of its high fidelity, model-based TV-L1 inversion may be considered as the new standard for image reconstruction in cross-sectional imaging.
Medical Physics 09/2015; 42(9):5444. DOI:10.1118/1.4928596 · 2.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cellular localisation and binding specificity of two NMDAR-targeted near-IR imaging probes has been examined by microscopy, followed by exemplification of MSOT to monitor simulated glutamate bursts in cellulo and a preliminary study in mice observing the signal in the brain.
Chemical Communications 09/2015; DOI:10.1039/c5cc06277b · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The imaging performance of fluorescence molecular tomography (FMT) improves when information from the underlying anatomy is incorporated into the inversion scheme, in the form of priors. The requirement for incorporation of priors has recently driven the development of hybrid FMT systems coupled to other modalities, such as X-ray CT and MRI. A critical methodological aspect in this case relates to the particular method selected to incorporate prior information obtained from the anatomical imaging modality into the FMT inversion. We propose herein a new approach for utilizing prior information, which preferentially minimizes residual errors associated with measurements that better describe the anatomical segments considered. This preferential minimization was realized using a weighted least square (WLS) approach, where the weights were optimized using a Mamdani-type fuzzy inference system. The method of priors introduced herein was deployed as a two-step structured regularization approach and was verified with experimental measurements from phantoms as well as ex vivo and in vivo animal studies. The results demonstrate accurate performance and minimization of reconstruction bias, without requiring user input for setting the regularization parameters. As such, the proposed method offers significant progress in incorporation of anatomical priors in FMT and, as a result, in realization of the full potential of hybrid FMT.
[Show abstract][Hide abstract] ABSTRACT: Oxidative-based diseases including diabetes, chronic renal failure, cardiovascular diseases and neurological disorders are accompanied by accumulation of advanced glycation endproducts (AGE). Therefore, AGE-associated changes in tissue optical properties could yield a viable pathological indicator for disease diagnostics and monitoring. We investigated whether skin glycation could be detected based on absorption changes associated with AGE accumulation using spectral optoacoustic measurements and interrogated the optimal spectral band for skin glycation determination. Glycated and non-glycated skin was optoacoustically measured at multiple wavelengths in the visible region. The detected signals were spectrally processed and compared to measurements of skin auto-fluorescence and to second harmonic generation multiphoton microscopy images. Optoacoustic measurements are shown to be capable of detecting skin glycolysis based on AGE detection. A linear dependence was observed between optoacoustic intensity and the progression of skin glycation. The findings where corroborated by autofluorescence observations. Detection sensitivity is enhanced by observing normalised tissue spectra. This result points to a ratiometric method for skin glycation detection, specifically at 540 nm and 620 nm. We demonstrate that optoacoustic spectroscopy could be employed to detect AGE accumulation, and possibly can be employed as a non-invasive quick method for monitoring tissue glycation.
[Show abstract][Hide abstract] ABSTRACT: Intravital imaging of large specimens is intrinsically challenging for postembryonic studies. Selective plane illumination microscopy (SPIM) has been introduced to volumetrically visualize organisms used in developmental biology and experimental genetics. Ideally suited for imaging transparent samples, SPIM can offer high frame rate imaging with optical microscopy resolutions and low phototoxicity. However, its performance quickly deteriorates when applied to opaque tissues. To overcome this limitation, SPIM optics were merged with optical and optoacoustic (photoacoustic) readouts. The performance of this hybrid imaging system was characterized using various phantoms and by imaging a highly scattering ex vivo juvenile zebrafish. The results revealed the system’s enhanced capability over that of conventional SPIM for high-resolution imaging over extended depths of scattering content. The approach described here may enable future visualization of organisms throughout their entire development, encompassing regimes in which the tissue may become opaque.
[Show abstract][Hide abstract] ABSTRACT: Biology requires observations at multiple geometrical scales, a feature that is not typically offered by a single imaging modality. We developed a hybrid optical system that not only provides different contrast modes but also offers imaging at different geometrical scales, achieving uniquely broad resolution and a 1000-fold volume sampling increase compared to volumes scanned by optical microscopy. The system combines optoacoustic mesoscopy, optoacoustic microscopy and two-photon microscopy, the latter integrating second and third harmonic generation modes. Label-free imaging of a mouse ear and zebrafish larva ex-vivo demonstrates the contrast and scale complementarity provided by the hybrid system. We showcase the superior anatomical orientation offered by the label-free capacity and hybrid operation, over fluorescence microscopy, and the dynamic selection between field of view and resolution achieved, leading to new possibilities in biological visualization.
[Show abstract][Hide abstract] ABSTRACT: The inversion accuracy in optoacoustic tomography depends on a number of parameters, including the number of detectors employed, discrete sampling issues or imperfectness of the forward model. These parameters result in ambiguities on the reconstructed image. A common ambiguity is the appearance of negative values, which have no physical meaning since optical absorption can only be higher or equal than zero. We investigate herein algorithms that impose non-negative constraints in model-based optoacoustic inversion. Several state-of-the-art non-negative constrained algorithms are analyzed. Furthermore, an algorithm based on the conjugate gradient method is introduced in this work. We are particularly interested in investigating whether positive restrictions lead to accurate solutions or drive the appearance of errors and artifacts. It is shown that the computational performance of non-negative constrained inversion is higher for the introduced algorithm than for the other algorithms, while yielding equivalent results. The experimental performance of this inversion procedure is then tested in phantoms and small animals, showing an improvement in image quality and quantitativeness with respect to the unconstrained approach. The study performed validates the use of non-negative constraints for improving image accuracy compared to unconstrained methods, while maintaining computational efficiency.
Physics in Medicine and Biology 08/2015; 60(17):6733-6750. DOI:10.1088/0031-9155/60/17/6733 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Optical mesoscopy extends the capabilities of biological visualization beyond the limited penetration depth achieved by microscopy. However, imaging of opaque organisms or tissues larger than a few hundred micrometers requires invasive tissue sectioning or chemical treatment of the specimen for clearing photon scattering, an invasive process that is regardless limited with depth. We developed previously unreported broadband optoacoustic mesoscopy as a tomographic modality to enable imaging of optical contrast through several millimeters of tissue, without the need for chemical treatment of tissues. We show that the unique combination of three-dimensional projections over a broad 500 kHz–40 MHz frequency range combined with multi-wavelength illumination is necessary to render broadband multispectral optoacoustic mesoscopy (2B-MSOM) superior to previous optical or optoacoustic mesoscopy implementations.