Large-scale, high-resolution imaging of cerebral hemodynamics is essential for brain research. Uniquely capable of comprehensive quantification of cerebral hemodynamics and oxygen metabolism in rodents based on the endogenous hemoglobin contrast, multiparametric photoacoustic microscopy (PAM) is ideally suited for this purpose. However, the out-of-focus issue due to the uneven surface of the rodent brain results in inaccurate PAM measurements and presents a significant challenge to cortex-wide multiparametric recording. We report a large-scale, high-resolution, multiparametric PAM system based on real-time surface contour extraction and scanning, which avoids the prescan and offline calculation of the contour map required by previously reported contour-scanning strategies. The performance of this system has been demonstrated in both phantoms and the live mouse brain through a thinned-skull window. Side-by-side comparison shows that the real-time contour scanning not only improves the quality of structural images by addressing the out-of-focus issue but also ensures accurate measurements of the concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF) over the entire mouse cortex. Furthermore, quantitative analysis reveals how the out-of-focus issue impairs the measurements of CHb, sO2, and CBF.
Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of quantifying the cerebral hemodynamics and oxygen metabolism at the microscopic level. However, the limited depth of focus of conventional PAM is insufficient to encompass the depth variation of the mouse brain when imaging a large area. For instance, the surface contour of the mouse cortex is dome-shaped and spans several hundred microns along the depth direction. When out of focus, the resolution and sensitivity of PAM quickly degrades. Moreover, quantitative measurements (e.g., blood oxygenation and flow) are no longer accurate with the compromised resolution and sensitivity. Here, we report automated contour-scan multi-parametric PAM, which enables simultaneous imaging of blood perfusion, oxygenation and flow with high resolution and sensitivity over the entire mouse cortex. Different from the traditional contour-scan method that requires three steps (pre-scan, off-line calculation of the contour map, and contour scan), our technique can perform high-resolution wide-field contour scan without the first two steps, thereby significantly reducing the acquisition time. We first tested the feasibility of this technique by imaging a plastic ball coated with black ink. Then, we quantitatively analyzed the influence of out-of-focus on the measurement of blood flow in a vessel-mimicking phantom. Finally, we demonstrated cortex-wide multi-parametric PAM in the live mouse brain with high resolution and sensitivity.
General anesthetics are known to have profound effects on cerebral hemodynamics and neuronal activities. However, it remains a challenge to directly assess anesthetics-induced hemodynamic and oxygen-metabolic changes from the true baseline under wakefulness at the microscopic level, due to the lack of an enabling technology for high-resolution functional imaging of the awake mouse brain. To address this challenge, we have developed head-restrained photoacoustic microscopy (PAM), which enables simultaneous imaging of the cerebrovascular anatomy, total concentration and oxygen saturation of hemoglobin (CHb and sO2), and blood flow in awake mice. From these hemodynamic measurements, two important metabolic parameters, oxygen extraction fraction (OEF) and the cerebral metabolic rate of oxygen (CMRO2), can be derived. Side-by-side comparison of the mouse brain under wakefulness and anesthesia revealed multifaceted cerebral responses to isoflurane, a volatile anesthetic widely used in preclinical research and clinical practice. Key observations include elevated cerebral blood flow (CBF) and reduced oxygen extraction and metabolism.
Enabling simultaneous high-resolution imaging of the total concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF), multiparametric photoacoustic microscopy (PAM) holds the potential to quantify the cerebral metabolic rate of oxygen at the microscopic level. However, its imaging speed has been severely limited by the pulse repetition rate of the dual-wavelength photoacoustic excitation and the scanning mechanism. To address these limitations, we have developed a new generation of multiparametric PAM. Capitalizing on a self-developed high-repetition dual-wavelength pulsed laser and an optical–mechanical hybrid-scan configuration, this innovative technique has achieved an unprecedented A-line rate of 300 kHz, leading to a 20-fold increase in the imaging speed over our previously reported multiparametric PAM that is based on pure mechanical scanning. The performance of the high-speed multiparametric PAM has been examined both in vitro and in vivo. Simultaneous PAM of microvascular CHb, sO2, and CBF in absolute values over a ∼3-mm-diameter brain region of interest can be accomplished within 10 min.