Angiogenesis is a central cancer hallmark, necessary for supporting tumor growth and metastasis. In vivo imaging of angiogenesis is commonly applied, to understand dynamic processes in cancer development and treatment strategies. However, most radiological modalities today assess angiogenesis based on indirect mechanisms, such as the rate of contrast enhancement after contrast agent administration. We studied the performance of raster-scan optoacoustic mesoscopy (RSOM), to directly reveal the vascular network supporting melanoma growth in vivo, at 50 MHz and 100 MHz, through several millimeters of tumor depth. After comparing the performance at each frequency, we recorded, for the first time, high-resolution images of melanin tumor vasculature development in vivo, over a period of several days. Image validation was provided by means of cryo-slice sections of the same tumor after sacrificing the mice. We show how optoacoustic (photoacoustic) mesoscopy reveals a potentially powerful look into tumor angiogenesis, with properties and features that are markedly different than other radiological modalities. This will facilitate a better understanding of tumor’s angiogenesis, and the evaluation of treatment strategies.

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.

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.

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.

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

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.

Optoacoustic (photoacoustic) dermoscopy offers two principal advantages over conventional optical imaging applied in dermatology. First, it yields high-resolution cross-sectional images of the skin at depths not accessible to other non-invasive optical imaging methods. Second, by resolving absorption spectra at multiple wavelengths, it enables label-free 3D visualization of morphological and functional features. However, the relation of pulse energy to generated bandwidth and imaging depth remains poorly defined. In this paper, we apply computer models to investigate the optoacoustic frequency response generated by simulated skin. We relate our simulation results to experimental measurements of the detection bandwidth as a function of optical excitation energy in phantoms and human skin. Using raster-scan optoacoustic mesoscopy, we further compare the performance of two broadband ultrasonic detectors (a bandwidth of 20-180 and 10-90MHz) in acquiring optoacoustic readouts. Based on the findings of this paper, we propose energy ranges required for skin imaging with considerations of laser safety standards.

Optical and optoacoustic (photoacoustic) microscopy have been recently joined in hybrid implementations that resolve extended tissue contrast compared to each modality alone. Nevertheless, the application of the hybrid technique is limited by the requirement to combine an optical objective with ultrasound detection collecting signal from the same micro-volume. We present an all-optical optoacoustic microscope based on a pi-phase-shifted fiber Bragg grating (π-FBG) with coherence-restored pulsed interferometry (CRPI) used as the interrogation method. The sensor offers an ultra-small footprint and achieved higher sensitivity over piezoelectric transducers of similar size. We characterize the spectral bandwidth of the ultrasound detector and interrogate the imaging performance on phantoms and tissues. We show the first optoacoustic images of biological specimen recorded with π-FBG sensors. We discuss the potential uses of π-FBG sensors based on CRPI.