Ultrasound-aided Multi-parametric Photoacoustic Microscopy of the Mouse Brain

Abstract

High-resolution quantitative imaging of cerebral oxygen metabolism in mice is crucial for understanding brain functions and formulating new strategies to treat neurological disorders, but remains a challenge. Here, we report on our newly developed ultrasound-aided multi-parametric photoacoustic microscopy (PAM), which enables simultaneous quantification of the total concentration of hemoglobin (C Hb ), the oxygen saturation of hemoglobin (sO 2 ), and cerebral blood flow (CBF) at the microscopic level and through the intact mouse skull. The three-dimensional skull and vascular anatomies delineated by the dual-contrast (i.e., ultrasonic and photoacoustic) system provide important guidance for dynamically focused contour scan and vessel orientation-dependent correction of CBF, respectively. Moreover, bi-directional raster scan allows determining the direction of blood flow in individual vessels. Capable of imaging all three hemodynamic parameters at the same spatiotemporal scale, our ultrasound-aided PAM fills a critical gap in preclinical neuroimaging and lays the foundation for high-resolution mapping of the cerebral metabolic rate of oxygen (CMRO 2 )-a quantitative index of cerebral oxygen metabolism. This technical innovation is expected to shed new light on the mechanism and treatment of a broad spectrum of neurological disorders, including Alzheimer's disease and ischemic stroke. The brain accounts for more than 20% of our oxygen consumption at the resting state 1 . Disruptions in cerebral oxygen metabolism play a key role in the initiation and progression of multiple life-threatening brain disorders, in particular Alzheimer's disease and ischemic stroke 2,3 . High-resolution imaging of the cerebral metabolic rate of oxygen (CMRO 2 ) in mice-a species with abundant disease models and genetic manipulations available-is crucial for understanding elusive pathogenic mechanisms and formulating new therapeutic strategies. However, existing techniques have yet to achieve this goal. Positron emission tomography (PET) allows quantifying CMRO 2 in absolute values, but lacks the spatial resolution to image the mouse brain 4,5 . Combining optical intrinsic signal and laser speckle imaging allows measuring CMRO 2 at the mesoscopic level, but rather qualitative 6,7 . Functional ultrasound enables high resolution imaging of the cerebral blood flow across the entire rodent brain 8,9 , but does not have access to the functional information of blood oxygenation.Photoacoustic microscopy (PAM) 10-12 holds great potential to address this long-standing challenge. Capitalizing on the optical absorption of hemoglobin-the primary carrier of oxygen in the circulation, PAM allows in vivo characterization of vascular anatomy 13,14 , hemodynamics 15 , and vasoactivity [16][17][18] . By measuring the total concentration of hemoglobin (C Hb ), the oxygen saturation of hemoglobin (sO 2 ), and blood flow at selected locations in feeding arteries and draining veins, Yao et al. previously demonstrated PAM of the total metabolic rate of oxygen ...

Multiparametric photoacoustic microscopy of the mouse brain with 300-kHz A-line rate

“…The multiparametric PAM provides a solution; 4 however, the correlation-based CBF quantification limits its B-scan rate to ∼1 mm∕s. To boost the speed of PAM while maintaining the slow B-scan rate for the CBF measurement, we adopted the synchronized one-dimensional optical and two-dimensional (2-D) mechanical hybrid scan.…”

“…4 In parallel, C Hb can be derived from the absolute value by analyzing the Brownian motion-induced statistical fluctuation in the amplitudes of the same 49 A-lines, which is known to depend on the number of RBCs within the detection volume of PAM but not on the flow speed. 15 As shown in Fig.…”

“…Thus, 20 dual-wavelength B-scans can be simultaneously acquired, leading to a 20-fold increase in the imaging speed over our previously reported multiparametric PAM, which is based on pure mechanical scanning. 4 Simultaneous mapping of CBF, C Hb , and sO 2 at the microscopic level is realized by correlation, statistical, and spectral analyses of individual B-scans. 4 Specifically, PAM is insensitive to sO 2 at 532 nm, a near-isosbestic point of hemoglobin.…”

“…4 Simultaneous mapping of CBF, C Hb , and sO 2 at the microscopic level is realized by correlation, statistical, and spectral analyses of individual B-scans. 4 Specifically, PAM is insensitive to sO 2 at 532 nm, a near-isosbestic point of hemoglobin. Fluctuations in the PAM signal acquired at this wavelength encode both the flow and Brownian motion of red blood cells (RBCs).…”

“…2,3 To this end, we have developed multiparametric PAM, which enables simultaneous imaging of C Hb , sO 2 , and CBF at the same spatial scale. 4 However, the speed of multiparametric PAM has been severely limited by the pulse repetition rate of the dual-wavelength photoacoustic excitation and the scanning mechanism. By employing two commercially available high-repetition single-wavelength lasers with distinct pulse durations (3 ns and 3 ps) and a customized water-immersible microelectromechanical system (MEMS) scanning mirror, Yao et al 5 have markedly improved the speed of PAM and demonstrated functional imaging of the mouse brain with 100-kHz A-line rate.…”