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.

Distinguishing early-stage tumors from normal tissues is of great importance in cancer diagnosis. We report fiber-based confocal visible/near-infrared (NIR) optical-resolution photoacoustic microscopy that can image tumor microvasculature, oxygen saturation, and nanoprobes in a single scanning. We develop a cost-efficient single laser source that provides 532, 558, and 1064 nm pulsed light with sub-microseconds wavelength switching time. Via dual-fiber illumination, we can focus the three beams to the same point. The optical and acoustic foci are confocally aligned to optimize the sensitivity. The visible and NIR wavelengths enable simultaneous tumor imaging with three different contrast modes. Results show obvious angiogenesis, significantly elevated oxygen saturation, and accumulated nanoparticles in the tumor regions, which offer comprehensive information to detect the tumor. This approach also allows us to identify feeding and draining vessels of the tumor and thus to determine local oxygen extraction fraction. In the tumor region, the oxygen extraction fraction significantly decreases along with tumor growth, which can also assist in tumor detection and staging. Fiber-based confocal visible/NIR photoacoustic microscopy offers a new tool for early detection of cancer.