Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin

Photoacoustic microscopy (PAM) has been shown to be a valuable tool for quantifying hemoglobin oxygenation within single vessels. Recently, optical-resolution PAM was developed to achieve higher resolution by reducing the laser beam diameter, which increased the light intensity. As intensity increases, saturation of the optical absorption and multiphoton/ multi-step absorption can occur, which, together with the temperature dependence of thermal expansion, result in a non-linear dependence of the photoacoustic signal on the excitation pulse fluence. For hemoglobin, the major absorber in tissue for photoacoustic imaging, these non-linear phenomena have strong wavelength dependence. To enable quantitative photoacoustic measurements at different wavelengths in the presence of nonlinearity, a careful wide range analysis of the intensity-dependent absorption is required. Here, we built a photoacoustic spectrometer, using a tunable nanosecond optical parametric oscillator that operates between 410 nm and 2400 nm as our light source. To reduce uncertainty in our measurements due to inhomogeneous spatial distribution of the optical fluence, we used a flat-top beam illumination and a flat transducer which was mounted in reflection mode, effectively reducing quantitative measurements to a one dimensional problem. Intensity-dependent non-linear spectra of the photoacoustic signals of oxyand deoxy-hemoglobin were obtained. These measurements have the potential to contribute significantly to quantitative functional PAM.