We report wide-field polygon-scanning optical-resolution photoacoustic microscopy (OR-PAM) that achieves 1-MHz A-line rate of oxygen saturation in vivo. We develop a polygon-scanning imaging probe. Using stimulated Raman scattering and optical delay in fiber, we develop a dual-wavelength pulsed laser that has proper wavelengths, sufficient pulse energy, high pulse repetition rate, and sub-microseconds wavelength switching time for fast imaging of oxygen saturation. The OR-PAM system offers a 478-Hz B-scan rate and a 1-Hz volumetric imaging rate over a 12×5 mm2 scanning area. We demonstrate dynamic imaging of oxygen saturation of fast response to epinephrine injection in the mouse ear. Results show fast hemodynamic and functional changes in centimeter-sized areas.
Optical-resolution photoacoustic microscopy (OR-PAM) can image the blood oxygen saturation (sO2) in vivo without labeling. OR-PAM assumes a linear relationship between the photoacoustic amplitude and the optical absorption coefficient and ignores the wavelength-dependent optical fluence attenuation in tissue. However, strong scattering in biological tissues may significantly change the optical energy deposition, leading to inaccurate sO2 measurement. Here, we report fluence-compensated OR-PAM to correct the sO2 imaging. In a narrow optical spectrum, we assume the scattered fluence is linearly related to the optical wavelength. Using three optical wavelengths, we can compensate for the scattering-induced photoacoustic signal change and thus improve the accuracy of sO2 measurement. We use a Monta Carlo model to validate the linear assumption of the scattered fluence. In in vivo experiments, we demonstrate that the optical fluence compensation can effectively improve the sO2 accuracy. The compensated arterial sO2 values are in the range of 0.95 ~ 0.99, which is consistent with normal physiological values. Compared with the uncompensated ones, the accuracy has been improved greatly. Enabled by the accurate sO2 imaging tool, we can reliably observe the sO2 gradient in the vascular network. We expect this new technique will further broaden the preclinical and clinical applications of functional OR-PAM.
Optical-resolution photoacoustic microscopy (OR-PAM) has been developed for anatomical, functional, and molecular imaging but usually requires multiple scanning for different contrasts. We present five-wavelength OR-PAM for simultaneous imaging of hemoglobin concentration, oxygen saturation, blood flow speed, and lymphatic vessels in single raster scanning. We develop a five-wavelength pulsed laser via stimulated Raman scattering. The five pulsed wavelengths, i.e., 532, 545, 558, 570, and 620 / 640 nm, are temporally separated by several hundreds of nanoseconds via different optical delays in fiber. Five photoacoustic images at these wavelengths are simultaneously acquired in a single scanning. The 532- and 620 / 640-nm wavelengths are used to image the blood vessels and dye-labeled lymphatic vessels. The blood flow speed is measured by a dual-pulse method. The oxygen saturation is calculated and compensated for by the Grüneisen-relaxation effect. In vivo imaging of hemoglobin concentration, oxygen saturation, blood flow speed, and lymphatic vessels is demonstrated in preclinical applications of cancer detection, lymphatic clearance monitoring, and functional brain imaging.
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