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.
Optical-resolution photoacoustic microscopy can measure oxygen saturation () in vivo, offering an important tool to assess tissue oxygenation and health condition. Limited by available wavelengths for fast OR-PAM, the accuracy of sO2 imaging may be degraded by absorption saturation due to high absorption in the blood. Here, we report a nonlinear model to solve the saturation problem and increase the accuracy of measurement. The absorption saturation is analyzed by comparing a nonlinear and linear photoacoustic model using numerical simulation, which shows the nonlinear model has an improved accuracy than the linear model when the absorption is high. Phantom experiments on bovine blood further validate the accuracy of the nonlinear sO2 measurement method. In vivo experiments are conducted in the mouse ear. The values in a pair of arteries and veins are calculated using both linear and nonlinear methods, showing that the nonlinear method measures the arterial value closer to normal physiological condition than the conventional linear model. The nonlinear model requires the use of three or more wavelengths (532nm, 545nm, and 558nm in this work). As a result, we demonstrate the saturation effect in OR-PAM can be compensated via a nonlinear model, which may advance the application of functional optical-resolution photoacoustic microscopy.
In fast functional photoacoustic microscopy (FPAM), the detection and monitoring of the oxygen saturation are important to monitor tissue functionality and disease progress. FPAM needs multi-wavelength pulsed laser sources with high pulse repetition rates, sufficient pulse energies and short wavelength switching time. Here, we develop a multi-wavelength pulsed laser source based on the stimulated Raman-scattering effect. The new laser is based on a 532-nm 1-MHz pulsed laser. The 532-nm laser pulse is split into two beams: one pumps a 5-m optical fiber to excite a 558-nm wavelength via stimulated Raman scattering; the other one propagates through a 50-m optical fiber to delay the pulse by 220 nano second so that the excitation wavelengths can be separated in time for fast functional photoacoustic imaging. The two beams are spatially combined and coupled into an optical fiber for photoacoustic excitation. Consequently, the new laser source can generate 2 million pulses per second, switch wavelengths in 220 ns, and provide hundreds of nano-Joules pulse energy for each wavelength. Using this laser source, we demonstrate optical-resolution photoacoustic imaging of microvascular structure and oxygen saturation in the mouse ear. The ultrashort wavelength switching time enables oxygen saturation imaging of flowing single red blood cells.
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