Development of large-scale multi-wavelength optoacoustic and ultrasound mesoscopy for biomedical applications

Optoacoustic (OA) imaging is an emerging technology capturing volumetric representations of absorbing structures in biological tissues at depths and resolutions not attainable with other imaging approaches due to strong light scattering. Technical implementations can be generally categorized into acoustic resolution (AR) and optical resolution (OR) systems. In AR mode, absorbers are localized based on the emitted ultrasound (US) wave taking multiple sensing positions into account. Tomographic implementations employ arrays of US sensors for parallel acquisition of volumetric data thus allowing for fast imaging frame rates (real-time), spatial resolutions in the 100 µm range, and penetration depth of several centimeters. In OR mode, the excitation beam is focused onto the sample’s surface resulting in micron-scale lateral resolutions down to the optical diffraction limit in superficial tissue layers, rapidly diminishing after a few hundred micrometers depth due to strong light scattering. OA mesoscopy - also known as AR-OAM - bridges the resolution gap between OR-OAM and OA tomography by sensing high frequency US waves with spherically focused, single element US detectors. Nevertheless, the technique requires mechanical scanning of light source and detector over the sample, resulting in slow scan times, small field-of-views, and poor usability. Furthermore, image reconstruction commonly relies on delay-and-sum approaches, which are know to introduce major artifacts and inaccuracies into the acoustic inversion making the images difficult to interpret and quantify. The goal of this thesis was to advance OA mesoscopy towards a more practically usable tool in biology and medicine. The developed system thus should allow for fast acquisition of a large field-of-view while minimizing motion related image artifacts and increasing usability for a broad application range. For functional imaging, multi-wavelength image scans are accomplished in a single mechanical overfly scan. Furthermore, image reconstruction techniques were advanced to allow for an accurate and quantitative reconstruction of the initial pressure distribution based on the measured sensor signals. The new OA mesoscope was built based on a custom-made PVDF transducer allowing the detection of waveforms over a broad acoustic spectrum spanning 1 MHz - 90 MHz frequency band. The US detector allows through hole illumination of the sample for inherent alignment of acoustic and optical axes thus overcoming the inherent disadvantages of commonly employed illumination configurations. Fiber and transducer are rapidly moved over the sample with images from large areas spanning up to 50 mm by 50 mm rendered in a matter of minutes. Three light sources are coupled into the same multimode fiber and a burst mode trigger scheme allows acquisition of multiwavelength images within a single mechanical overfly scan. To reconstruct the acquired datasets, a new model-based (MB) reconstruction approach was presented for OA mesoscopy relying on the translational symmetry of the transducer sensitivity field along the scan plane: by iteratively inverting the acoustic problem, resolution, signal-to-noise ratio, and contrast-to-noise ratio could be improved beyond existing delay-and-sum techniques representing an important step towards quantitative OA imaging. The developed system was used to image the vasculature in human skin at depths beyond reach for the existing implementations spanning areas as large as 20 mm by 20 mm. Vessels at 3.8 mm below the skin surface could be resolved while maintaining 28 µm lateral resolution in superficial tissue layers. Three wavelengths ranging from 532 nm to 1064 nm were acquired within a single mechanical overfly scan. The system was furthermore used to image skin vasculature in rodents showcasing its potential to image beyond the scattering limiting imposed on optical microscopy by light diffusion. The full vascular tree was visualized accurately resolving differently sized vessels due to the broadband PVDF detector sensitivity. The developed MB reconstruction approach showcased its performance by accurately reconstructing intensities from different depths. In an alternative application, the system was used as a screening platform to accurately extract absorption properties of proteins expressed by bacterial colonies. With its high-throughput capabilities, the setup can be used for screening large protein libraries to optimized their light absorption properties for OA imaging. The developed OA mesoscope showcased its usability in different application examples and could outperform other technical implementations presented in the past decades. Overall, the presented hardware and software developments represent an important step for OA mesoscopy to become a standard tool in medicine and biology. Hybrid US and OA microscopy can be incorporated into the same scan head to additionally acquire information about the skin structure including sebaceous glands and hair follicles. Additional developments are envisioned as essential future work, such as incorporation of low cost diodes to further decrease the cost footprint of the system