Five-wavelength optical-resolution photoacoustic microscopy of blood and lymphatic vessels

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 Gruneisen-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.