Periodic Reporting for period 1 - MSIOAM (Multifocal structured illumination optoacoustic microscopy) Reporting period: 2018-02-01 to 2020-01-31 Summary of the context and overall objectives of the project Optoacoustic (photoacoustic) imaging is a powerful bio-imaging modality associated with the intrinsic combination of ultrasound and light-related advantages and thus can provide a unique set of capabilities for biomedical applications, such as high spatio-temporal resolution, deep penetration, spectrally enriched imaging contrast and resolution scalability. It can achieve optical resolution optoacoustic microscopy (OR-OAM) at superficial depths and can be adapted for optoacoustic tomography (OAT) with ultrasonic resolution. For conventional OR-OAM, a single focused laser spot in conjunction with a single ultrasound detection element is employed for optoacoustic signal generation and detection, resulting in a slow imaging speed as far as concerned with acquisition of volumetric (3D) data, which greatly restricts its usage in applications involving dynamic biological processes. In this MSCA project, we aim to break the bottleneck of OR-OAM by developing a new approach using multifocal structured illumination in conjunction with a spherical ultrasonic array detection to achieve a highly scalable high-speed optoacoustic imaging scheme to enable imaging samples at multiple penetration scales by gradually exchanging microscopic optical resolution in superficial tissues with ultrasonic resolution at diffuse (macroscopic) depths. The overall objective is to achieve real-time volumetric optoacoustic imaging in both optical and acoustic resolution modes. For this, several objectives need to be accomplished. Firstly, a multifocal structured illumination system with a beamsplitting gratings will be designed and fabricated. Secondly, optoacoustic signal unmixing and reconstruction methods for the spherical ultrasonic array detection geometry will be investigated. Thirdly, calibration methods for the proposed system will be investigated. Last but not least, high-speed volumetric optoacoustic imaging with the proposed method in living tissues will be demonstrated. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far For multifocal structured illumination generation, different phase-only transmission grating phase patterns were calculated and simulated, out of which a grating with random phase pattern was fabricated with HOLOEYE Photonics AG to split the beam into 15x15 mini-beams with identical inter-beam angle of 0.57deg and off-axis uniformity error < ±10% at the designed wavelength of 532nm. For laser beam scanning, a customized ultrafast scanning module based on a 2D acousto-optic deflector (AOD) was developed. Although AODs have much smaller deflection angle range (typically <0.05 rad) than mirror based deflectors, they can achieve much higher deflection velocity (up to 8x104 rad/s) with much higher angular resolution (< 0.1 μrad). In this project, the deflection angle range corresponded to the inter-angles of the grating, i.e. 0.57 degree which is within the deflection angle range of the 2D AOD. The MSIOAM system was subsequently developed (Fig. 1a). Basically, a nanosecond-duration laser beam used to excite OA signals was shaped to a grid of spots at the sample surface by means of the beamsplitting grating and a condensing lens. The spot size was measured to ~15 μm along the horizontal and vertical axis with a depth of focus (DOF) of more than 1 mm within spot size of 20 μm along the light path, as characterized with a beam profiler. For optoacoustic signal unmixting and image reconstruction, firstly filtered back projection (BP) reconstruction was performed for each scanning frame. Secondly, regional maxima were searched and localized within the reconstructed 3D volume. Thirdly, virtual pinholes were applied to the selected signal spots to zero out surrounding pixels. Finally, the filtered signals were superimposed to form the complete image. Thereby, the light spot size at the sample surface determines the achievable lateral resolution, while the axial resolution is associated with the ability to allocate the recorded optoacoustic responses to the individual spots. For system characterization, a phantom containing 7 μm diameter carbon fibers was imaged to estimate the effective spatial resolution. The maximum intensity projections (MIPs) of the rendered 3D images of OAT and MSIOAM are shown in xy and xz views (Figs. 1b, c) with the zoomed-in area of the green boxed region shown in Fig. 1d. The superior resolution and visibility achieved with MSIOAM is clearly evinced and single fibers with 7µm diameter from a cluster can clearly be distinguished. The full-width-at-half-maximum (FWHM) values of the green line profiles through the 3D image were 28.2 µm and 45.2 μm (Figs. 1e, f). For in vivo real-time imaging demonstration, three athymic nude-Fox1nu mice were used. Mouse ear and the perfusion dynamics of a contrast agent were imaged. All procedures involving mice conformed to the national guidelines of the Swiss Federal Act on animal protection and were approved by the Cantonal Veterinary Office Zurich. As expected, the relatively low detection bandwidth of the array sensing elements hindered visualization of microvascular structures with OAT (Fig. 1g). In contrast, the MSIOAM image (Fig. 1h) presented more details with great image contrast, reconstructed from 50x50 scanning positions over the mouse ear with all the 512 transducer channels. The size of the smallest resolvable vessels was ~50µm for the MSIOAM image versus ~200µm in the regular OAT image as depicted in the zoomed-in image (Fig. 1i) and further elaborated by the signal profiles comparison (Fig. 1j). Image acquisition was subsequently accelerated by reducing the scanning pattern to 10x10 positions and increasing the PRF to 1 kHz and reducing the number of actively recorded channels to random 128 channels resulting in 10 Hz frame rate (Fig. 1k). In vivo imaging of mouse ear perfusion dynamics was showcased with this configuration. Alexa Fluor 555 fluorescent dye (100 ul, 1 mg/ml) was administrated by tail vein injection and the time-lapse images post dye injection were color coded and displayed (Fig. 1l). Signal profiles from three regions of interests (ROIs) were shown in Fig. 1m. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The imaging speed of OAM has been the bottle neck of this technique for many years although tremendous efforts have been proposed. With the proposed method, real-time OR-OAM has been accomplished by integrating a multi-order beamsplitting grating, ultrafast 2D AOD and a hemisphere array detector. Thanks to the unique design of the hemisphere array detector, the proposed method also brings huge advantages for 3D volumetric imaging without additional axial scan, which helps to attain optical resolution in the superficial tissue along with ultrasonic resolution at diffuse depths. It is believed that these unique characteristics will make it possible to study dynamic functional, kinetic and metabolism parameters, e.g. for diagnostic purposes of peripheral vasculature disease, skin lesions, characterization of lymph nodes and other indications. When combining multi-wavelength illumination, MSIOAM will enable functional and molecular imaging with various exogenous contrast agents (e.g. nanoparticles, organic dyes and fluorescent proteins), thus open new possibilities for imaging of multi-scale dynamics in pre-clinical and clinical applications, e.g. for in vivo cell tracking, targeted molecular imaging, visualization of tumor neovasculature, functional brain imaging.