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Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of simultaneous high-resolution mapping of blood oxygenation and flowin vivo. However, its speed has been limited by the dense sampling required for blood flow quantification. To overcome this limitation, we have developed a high-speed multi-parametric PAM system, which enables simultaneous acquisition of ∼500 densely sampled B-scans by superposing the rapid optical scanning across the line-shaped focus of a cylindrically focused ultrasonic transducer over the conventional mechanical scan of the optical-acoustic dual foci. A novel, to the best of our knowledge, optical-acoustic combiner (OAC) is designed and implemented to accommodate the short working distance of the transducer, enabling convenient confocal alignment of the dual foci in reflection mode. A resonant galvanometer (GM) provides stabilized high-speed large-angle scanning. This new system can continuously monitor microvascular blood oxygenation (sO₂) and flow over a 4.5 × 3 mm²area in the awake mouse brain with high spatial and temporal resolutions (6.9 µm and 0.3 Hz, respectively).

 

Multi-parametric photoacoustic microscopy (PAM) has emerged as a promising new technique for high-resolution quantification of hemodynamics and oxygen metabolism in the mouse brain. In this work, we have extended the scope of multi-parametric PAM to longitudinal, cortex-wide, awake-brain imaging with the use of a long-lifetime (24 weeks), wide-field (5 × 7 mm 2 ), light-weight (2 g), dual-transparency ( i.e., light and ultrasound) cranial window. Cerebrovascular responses to the window installation were examined in vivo, showing a complete recovery in 18 days. In the 22-week monitoring after the recovery, no dura thickening, skull regrowth, or changes in cerebrovascular structure and function were observed. The promise of this technique was demonstrated by monitoring vascular and metabolic responses of the awake mouse brain to ischemic stroke throughout the acute, subacute, and chronic stages. Side-by-side comparison of the responses in the ipsilateral (injury) and contralateral (control) cortices shows that despite an early recovery of cerebral blood flow and an increase in microvessel density, a long-lasting deficit in cerebral oxygen metabolism was observed throughout the chronic stage in the injured cortex, part of which proceeded to infarction. This longitudinal, functional-metabolic imaging technique opens new opportunities to study the chronic progression and therapeutic responses of neurovascular diseases. 

A widely used cerebrovascular stimulus and common pathophysiologic condition, hypercapnia is of great interest in brain research. However, it remains controversial how hypercapnia affects brain hemodynamics and energy metabolism. By using multi-parametric photoacoustic microscopy, the multifaceted responses of the awake mouse brain to different levels of hypercapnia are investigated. Our results show significant and vessel type-dependent increases of the vessel diameter and blood flow in response to the hypercapnic challenges, along with a decrease in oxygen extraction fraction due to elevated venous blood oxygenation. Interestingly, the increased blood flow and decreased oxygen extraction are not commensurate with each other, which leads to reduced cerebral oxygen metabolism. Further, time-lapse imaging over 2-hour chronic hypercapnic challenges reveals that the structural, functional, and metabolic changes induced by severe hypercapnia (10% CO 2 ) are not only more pronounced but more enduring than those induced by mild hypercapnia (5% CO 2 ), indicating that the extent of brain’s compensatory response to chronic hypercapnia is inversely related to the severity of the challenge. Offering quantitative, dynamic, and CO 2 level-dependent insights into the hemodynamic and metabolic responses of the brain to hypercapnia, these findings might provide useful guidance to the application of hypercapnia in brain research. 

Improving the imaging speed of multi-parametric photoacoustic microscopy (PAM) is essential to leveraging its impact in biomedicine. However, to avoid temporal overlap, the A-line rate is limited by the acoustic speed in biological tissues to a few megahertz. Moreover, to achieve high-speed PAM of the oxygen saturation of hemoglobin, the stimulated Raman scattering effect in optical fibers has been widely used to generate 558 nm from a commercial 532 nm laser for dual-wavelength excitation. However, the fiber length for effective wavelength conversion is typically short, corresponding to a small time delay that leads to a significant overlap of the A-lines acquired at the two wavelengths. Increasing the fiber length extends the time interval but limits the pulse energy at 558 nm. In this Letter, we report a conditional generative adversarial network-based approach that enables temporal unmixing of photoacoustic A-line signals with an interval as short as$\sim <\#comment/>38\mathrm{n}\mathrm{s}$, breaking the physical limit on the A-line rate. Moreover, this deep learning approach allows the use of multi-spectral laser pulses for PAM excitation, addressing the insufficient energy of monochromatic laser pulses. This technique lays the foundation for ultrahigh-speed multi-parametric PAM.

 

Understanding and measuring parameters responsible for the pathogenesis of sepsis-induced AKI (SI-AKI) is critical in developing therapies. Blood flow to the kidney is heterogeneous, partly due to the existence of dynamic networks of capillaries in various regions, responding differentially to oxygen demand in cortex versus medulla. High energy demand regions, especially the outer medulla, are susceptible to hypoxia and subject to damage during SI-AKI. Proximal tubule epithelial cells in the cortex and the outer medulla can also undergo metabolic reprogramming during SI-AKI to maintain basal physiological status and to avoid potential damage. Current data on the assessment of renal hemodynamics and oxygen metabolism during sepsis is limited. Preclinical and clinical studies show changes in renal hemodynamics associated with SI-AKI, and in clinical settings, interventions to manage renal hemodynamics seem to help improve disease outcomes in some cases. Lack of proper tools to assess temporospatial changes in peritubular blood flow and tissue oxygen metabolism is a barrier to our ability to understand microcirculatory dynamics and oxygen consumption and their role in the pathogenesis of SI-AKI. Current tools to assess renal oxygenation are limited in their usability as these cannot perform continuous simultaneous measurement of renal hemodynamics and oxygen metabolism. Multi-parametric photo-acoustic microscopy (PAM) is a new tool that can measure real-time changes in microhemodynamics and oxygen metabolism. Use of multi-parametric PAM in combination with advanced intravital imaging techniques has the potential to understand the contribution of microhemodynamic and tissue oxygenation alterations to SI-AKI. 

Capable of imaging blood perfusion, oxygenation, and flow simultaneously at the microscopic level, multi-parametric photoacoustic microscopy (PAM) has quickly emerged as a powerful tool for studying hemodynamic and metabolic changes due to physiological stimulations or pathological processes. However, the low scanning speed poised by the correlation-based blood flow measurement impedes its application in studying rapid microvascular responses. To address this challenge, we have developed a new, to the best of our knowledge, multi-parametric PAM system. By extending the optical scanning range with a cylindrically focused ultrasonic transducer (focal zone,$76\text{µ<\#comment/>}\mathrm{m}\times <\#comment/>4.5\mathrm{m}\mathrm{m}$) for simultaneous acquisition of 500 B-scans, the new system is 112 times faster than our previous multi-parametric system that uses a spherically focused transducer (focal diameter, 40 µm) and enables high-resolution imaging of blood perfusion, oxygenation, and flow over an area of$4.5\times <\#comment/>1{\mathrm{m}\mathrm{m}}^2$at a frame rate of 1 Hz. We have demonstrated the feasibility of this system in the living mouse ear. Further development of this system into reflection mode will enable real-time cortex-wide imaging of hemodynamics and metabolism in the mouse brain.