Photoacoustic spectroscopy has the potential to make non-invasive, spatially resolved measurements of absolute chromophore concentrations. This has a wide range of possible applications, for example the mapping of endogenous chromophores such as oxy- (HbO2) and deoxyhaemoglobin (HHb) or externally administered contrast agents designed to target specific tissues or molecular processes. In this study we used near-infrared photoacoustic spectroscopy to determine the absolute concentrations of HbO2 and HHb in a tissue phantom. The phantom consisted of three blood filled capillaries (Ø460microns) suspended at depths between 3mm and 9mm in a 2.5% Intralipid solution which also contained 2% blood in order to simulate the background optical attenuation in biological tissue. The blood oxygen saturation (SO2) of the blood circulating in the capillaries was varied using a membrane oxygenator. At each SO2 level, nanosecond pulses emitted by an OPO laser system that was tuneable over the wavelength range from 740nm to 1040nm illuminated the phantom. The generated photoacoustic waves were recorded using a single Fabry-Perot ultrasound detector and used to obtain a depth profile of the location of the tubes. The amplitudes of the part of the photoacoustic signal that corresponded to the capillaries and the surface of the Intralipid/blood mixture were plotted as a function of wavelength. The output of a diffusion theory based model of the wavelength dependence of the photoacoustic signal amplitude was then fitted to these spectra. This enabled the quantitative determination of absolute HbO2 and HHb concentrations in the capillaries and the Intralipid/blood mixture from which the total haemoglobin concentrations and blood SO2 were calculated. Based on these measurements, the smallest chromophore concentrations that can be detected in biological tissue were estimated.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.