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.
