In the current form of multi-parametric photoacoustic microscopy (PAM), imaging hemoglobin concentration and blood flow speed requires dense sampling. Moreover, large-scale recording beyond the focal zone of ultrasonic transducer requires time-consuming mechanical scan of the optical-acoustic dual foci. Thus, the image acquisition time of multi-parametric PAM has been severely limited by the laser repetition rate and the focal diameter of the transducer.
Here, we report an ultrahigh-speed multi-parametric PAM with 1.2-MHz A-line rate for simultaneous real-time imaging of hemoglobin concentration, blood oxygenation, and blood flow in the mouse brain. Capitalizing on the pronounced stimulated Raman scattering in pure silica-core polarization-maintaining single-mode optical fibers, a dual-wavelength (532 and 558 nm) nanosecond laser with 1.2-MHz pulse repetition rate has been developed. Using a weakly focused ultrasonic transducer, we have achieved real-time acquisition of multi-parametric PAM images at a frame rate of 2.2 Hz over the 250-μm-diameter acoustic focal zone. By employing optical-mechanical hybrid scan, 25 dual-wavelength B-scans can be acquired simultaneously within one mechanical-scan trip, leading to a 25-fold improvement of imaging
speed. As a result, the imaging frame rate is improved from 0.08 Hz in the conventional multi-parametric PAM to 2.2 Hz.
The utility of this new PAM technology has been demonstrated in a mouse model of epilepsy by studying the dynamic neurovascular uncoupling during status epilepticus.
NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.
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.