Acoustic-resolution optoacoustic microscopy (AR-OAM) visualizes internal tissue structures at millimeter to centimeter scale depths with high spatial resolution. The imaging performance mainly depends on the geometry and detection characteristics of the ultrasound transducer. Reconstruction methods incorporating transducer effects are essential to optimize achievable resolution, contrast and overall image quality. Model-based (MB) reconstruction has been shown to provide excellent imaging performance in several optoacoustic embodiments, due to its capacity to accurately model the transducer. However, the applicability of MB reconstruction methods in AR-OAM has been hampered by the high computational cost. Here, we propose an efficient MB reconstruction framework for largescale AR-OAM by considering scanning symmetries, which enabled capitalizing the computational power of a graphics processing unit. The suggested MB reconstruction method is shown to significantly improve the imaging performance of AR-OAM compared to synthetic aperture focusing technique, as validated in in vivo mouse skin experiment.
We present a fast, multispectral acoustic resolution optoacoustic microscope using a new burst-mode triggering scheme. Three pulsed laser sources are combined to retrieve spectral images across large field-of-views extending over 25mm by 25mm at 28μm lateral and 14μm axial resolution with an overfly scan of a few minutes. Highly sensitive PVDF transducer allows detection of structures 3.8mm below the human skin surface with per pulse energies of only 25μJ. The newly developed system overcomes limitations of previously reported scanning optoacoustic microscopy and mesoscopy implementations, offering a major leap forward in terms of clinical usability, laser safety, effective penetration depth and spectral unmixing capabilities.
Optoacoustic imaging is a highly scalable and versatile method that can be used for optical resolution (OR) microscopy applications at superficial depth yet can be adapted for tomographic imaging with ultrasonic resolution at centimeter penetration scales. However, imaging speed of the commonly employed scanning-based microscopy methods is slow as far as concerned with acquisition of volumetric data. Herein, we propose a new approach using multifocal structured illumination in conjunction with a spherical matrix ultrasonic array detection to achieve fast volumetric optoacoustic imaging in both optical and acoustic resolution modes. In our approach, the laser beam is raster scanned by an acousto-optic deflector running at hundred hertz scanning rate with the beam then split into hundreds of mini-beams by a beamsplitting grating, which are subsequently focused by a condensing lens to generate multifocal structured illumination. Phantom experimental results show that 10 x 10 x 5 cm3 volumetric imaging can be accomplished with spatial resolution around 29 μm. We believe by further speeding up the data acquisition in the further, the system will be operated in full power, making it possible to study functional, kinetic and metabolic processes across multiple penetration scales.
Fluorescence imaging is widely employed in biological discovery due to its excellent molecular sensitivity and contrast. However, due to light scattering wide-field fluorescence images are blurred resulting in very low spatial resolution and low image contrast. The existing scanning optical microscopy techniques are commonly restricted to sub-millimeter field-of-view or otherwise slow imaging speeds, limiting their applicability for imaging of fast biological dynamics occurring on larger spatial scales. Herein, we developed a rapid scanning wide-field multifocal structured illumination microscopy method based on a beam-splitting grating and an acousto-optic deflector synchronized with a high speed camera. The multi-beam pattern is focused by a condensing lens and a macroscopic objective to generate multifocal structured illumination profile on the imaged sample that is rapidly scanned at kHz rates. Experimental results show that the proposed method can achieve real-time fluorescence microscopy over a centimeter-scale field of view. Owing to the low numerical aperture of the diffracted beams, the illumination has a large depth of focus and hence is generally not affected by the sample’s curvature, which allowed here imaging of perfusion in the entire mouse cerebral cortex noninvasively. The new approach can be readily incorporated into traditional wide-field microscopes to attain optimal tradeoff between spatial resolution and field of view. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus anticipated to find broad applicability in a variety of applications looking at large-scale fluorescent-based biodynamics.
Neuronal activity occurs simultaneously and in a highly coordinated fashion in many different areas across the brain. Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with the existing neuroimaging methods that are limited in their ability to image large tissue volumes at high speeds. Genetically encoded calcium indicators (GECIs) that modulate their fluorescence intensity as a function of intracellular calcium concentrations are powerful tools for the observation of large neuronal networks. Optoacoustic imaging has been shown capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep in mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as lack of activity labels excitable in the near-infrared window. We developed and validated an isolated whole mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions and exhibit functional activity for several hours to several days. An optoacoustic imaging system coupled to a superfusion system was further devised and used for rapid volumetric monitoring of calcium dynamics in the brain evoked using an epilepsy-inducing drug. The new technique captures calcium fluxes as true 3D information across the entire brain with temporal resolution of 10ms and spatial resolution of 150µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. The system could be readily adapted to work with future generations of far-red- and near-infrared GECIs.
KEYWORDS: Brain, Skull, In vivo imaging, Ionizing radiation, Ultrasonography, Photoacoustic spectroscopy, Radiotherapy, Tumors, 3D image processing, Data acquisition
Radiation-induced brain damage could arise as a side effect in radiotherapy of brain tumors. We present evidence of radiation-induced damage to the skull and brain vasculature of mice as revealed by transcranial optoacoustic and ultrasound bio-microscopy in-vivo. The three-dimensional nature of the acquired optoacoustic images combined with a clear anatomical reference of the pulse-echo ultrasound data allowed clear differentiation of the skull vasculature from the superficial brain vasculature. The irradiation was selectively applied to one brain hemisphere and the effects of the ionizing radiation were evident without introduction of extrinsic labeling, owing to the strong haemoglobin contrast of optoacoustics.
Electrosurgery, i.e. the application of radiofrequency current for tissue ablation, is a frequently used treatment for many cardiac arrhythmias. Electrophysiological and anatomic mapping, as well as careful radiofrequency power control typically guide the radiofrequency ablation procedure. Despite its widespread application, accurate monitoring of the lesion formation with sufficient spatio-temporal resolution remains challenging with the existing imaging techniques. We present a novel integrated catheter for simultaneous radiofrequency ablation and optoacoustic monitoring of the lesion formation in real time and 3D. The design combines the delivery of both electric current and optoacoustic excitation beam in a single catheter consisting of copper-coated multimode light-guides and its manufacturing is described in detail. The electrical current causes coagulation and desiccation while the excitation light is locally absorbed, generating OA responses from the entire treated volume. The combined ablation-monitoring capabilities were verified using ex-vivo bovine tissue. The formed ablation lesions showed a homogenous coagulation while the ablation was monitored in realtime with a volumetric frame rate of 10 Hz over 150 seconds.
We present an optoacoustic microscope, combining structural and functional opticalresolution optoacoustic and ultrasound pulse-echo imaging. The system was applied to image Zebrafish larvae and complex vascular networks in the murine brain and ear.
Optoacoustic imaging is a rapidly developing area of biomedical imaging due its combination of rich optical
contrast and ultrasound depth penetration. Just like conventional pulse-echo ultrasound imaging, optoacoustic
tomography relies on the use of ultrasound detector arrays with a large number of elements. The precise
knowledge of the transducer’s sensitivity is crucial for the prediction of its performance for a given imaging
task. Sensitivity characteristics such as the central frequency and bandwidth are routinely characterized.
However, this characterization is typically performed solely under normal incidence since the measurement of
the angle and frequency depended sensitivity (directivity) is difficult and time consuming with existing
ultrasound characterization methods. We present a simple and fast characterization method for broadband
directivity measurements of the angular transducer sensitivity based on the optoacoustic effect. The method
utilizes a thin absorbing suture in order to generate omnidirectional and broadband optoacoustic signals,
which are calibrated using a needle hydrophone. We applied this method to characterize and compare the
directivity of a conventional piezoelectric (PZT) transducer to the directivity of a capacitive micromachined
ultrasonic (cMUT) transducer. Both technologies showed a similar broadband response at normal incidence
and the PZT transducer displayed a more than two times larger signal to noise ratio at normal incidence.
However, the cMUT transducer’s sensitivity was significantly less angle-depended and outperformed the
PZT’s sensitivity for angles larger than 20°.
Model organisms such as zebrafish play an important role for developmental biologists and experimental geneticists. Still, as they grow into their post-embryonic stage of development it becomes more and more difficult to image them because of high light scattering inside biological tissue. Optoacoustic mesoscopy based on spherically focused, high frequency, ultrasound detectors offers an alternative, where it relies on the focusing capabilities of the ultrasound detectors in generating the image rather than on the focusing of light. Nonetheless, because of the limited numerical aperture the resolution is not isotropic, and many structures, especially elongated ones, such as blood vessels and other organs, are either invisible, or not clearly identifiable on the final image. Herein, based on high frequency ultrasound detectors at 100 MHz and 50 MHz we introduce multi orientation (view) optoacoustic mesoscopy. We collect a rich amount of signals from multiple directions and combine them using a weighted sum in the Fourier domain and a Wiener deconvolution into a single high resolution three-dimensional image. The new system achieves isotropic resolutions on the order of 10 μm in-plane, 40 μm axially, and SNR enhancement of 15 dB compared to the single orientation case. To showcase the system we imaged a juvenile zebrafish ex vivo, which is too large to image using optical microscopic techniques, the reconstructed images show unprecedented performance in terms of SNR, resolution, and clarity of the observed structures. Using the system we see the inner organs of the zebrafish, the pigmentation, and the vessels with unprecedented clarity.
Many neurological disorders are linked to abnormal activation or pathological alterations of the vasculature in the affected brain region. Obtaining simultaneous morphological and physiological information of neurovasculature is very challenging due to the acoustic distortions and intense light scattering by the skull and brain. In addition, the size of cerebral vasculature in murine brains spans an extended range from just a few microns up to about a millimeter, all to be recorded in 3D and over an area of several dozens of mm2. Numerous imaging techniques exist that excel at characterizing certain aspects of this complex network but are only capable of providing information on a limited spatiotemporal scale. We present a hybrid ultrasound and dual-wavelength optoacoustic microscope, capable of rapid imaging of murine neurovasculature in-vivo, with high spatial resolution down to 12 μm over a large field of view exceeding 50mm2. The dual wavelength imaging capability allows for the visualization of functional blood parameters through an intact skull while pulse-echo ultrasound biomicroscopy images are captured simultaneously by the same scan head. The flexible hybrid design in combination with fast high-resolution imaging in 3D holds promise for generating better insights into the architecture and function of the neurovascular system.
The skull bone, a curved solid multilayered plate protecting the brain, constitutes a big challenge for the use of ultrasound-mediated techniques in neuroscience. Ultrasound waves incident from water or soft biological tissue are mostly reflected when impinging on the skull. To this end, skull properties have been characterized for both high-intensity focused ultrasound (HIFU) operating in the narrowband far-field regime and optoacoustic imaging applications. Yet, no study has been conducted to characterize the near-field of water immersed skulls. We used the thermoelastic effect with a 532 nm pulsed laser to trigger a wide range of broad-band ultrasound modes in a mouse skull. In order to capture the waves propagating in the near-field, a thin hydrophone was scanned in close proximity to the skull's surface. While Leaky pseudo-Lamb waves and grazing-angle bulk water waves are clearly visible in the spatio-temporal data, we were only able to identify skull-guided acoustic waves after dispersion analysis in the wavenumber-frequency space. The experimental data was found to be in a reasonable agreement with a flat multilayered plate model.
Frequency characteristics of ultrasound detectors used in optoacoustic tomography have a major impact on imaging performance. It is common practice to select transducers based on their sensitivity at the central frequency and under normal incidence. However, the bandwidth and angular sensitivity play an equally important role in establishing the quality and accuracy of the reconstructed images. Here, we developed a calibrated optoacoustic characterization method specifically tailored for broadband measurements of the angular transducer sensitivity (directivity). Ultrawideband omnidirectional optoacoustic responses were generated by uniformly illuminating thin absorbing sutures with nanosecond laser pulses and characterized with a needle hydrophone. This calibrated optoacoustic source was used to characterize the frequency dependence of the angular response by a conventional piezoelectric transducer (PZT) and a capacitive micromachined ultrasonic transducer (cMUT) with similar size and central frequency. Furthermore, both transducers had no preamplification electronics directly attached to the detection elements. While the PZT presented a 7.8 dB sensitivity advantage at normal incidence, it was able to provide detectable signal-to-noise levels only at incidence angles of up to 20 deg whereas the cMUT maintained reasonable sensitivity levels and broadband response at incidence angles of 40 deg and beyond. We further experimentally showcase a reduction in the limited-view image artifacts resulting from the broader acceptance angle of the cMUT.
In photoacoustic imaging, the angular reception performance of ultrasonic transducers is a critical parameter to be considered for system designers. The quantitative comparison between cMUT and PZT emphasizes the difference between the transducer requirements and specifications between conventional ultrasound and photoacoustic imaging. In this present work, we show significant benefits of cMUT based array transducers over conventional PZT arrays for the improvement of quality in photoacoustic imaging systems.
The acoustically-mismatched skull bone poses significant challenges for the application of ultrasonic and optical techniques in neuroimaging, still typically requiring invasive approaches using craniotomy or skull thinning. Optoacoustic imaging partially circumvents the acoustic distortions due to the skull because the induced wave is transmitted only once as opposed to the round trip in pulse-echo ultrasonography. To this end, the mouse brain has been successfully imaged transcranially by optoacoustic scanning microscopy. Yet, the skull may adversely affect the lateral and axial resolution of transcranial brain images. In order to accurately characterize the complex behavior of the optoacoustic signal as it traverses through the skull, one needs to consider the ultrawideband nature of the optoacoustic signals.
Here the insertion loss of murine skull has been measured by means of a hybrid optoacoustic-ultrasound scanning microscope having a spherically focused PVDF transducer and pulsed laser excitation at 532 nm of a 20 μm diameter absorbing microsphere acting as an optoacoustic point source. Accurate modeling of the acoustic transmission through the skull is further performed using a Fourier-domain expansion of a solid-plate model, based on the simultaneously acquired pulse-echo ultrasound image providing precise information about the skull's position and its orientation relative to the optoacoustic source. Good qualitative agreement has been found between the a solid-plate model and experimental measurements.
The presented strategy might pave the way for modeling skull effects and deriving efficient correction schemes to account for acoustic distortions introduced by an adult murine skull, thus improving the spatial resolution, effective penetration depth and overall image quality of transcranial optoacoustic brain microscopy.
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