Picosecond absorption relaxation-central to many disciplines-is typically measured by ultrafast ͑femtosecond or picosecond͒ pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural-including strongly scattering and nonfluorescent-materials. © 2010 American Institute of Physics. ͓doi:10.1063/1.3500820͔Picosecond absorption relaxation is central to many optical phenomena in physics, chemistry, biology, and medicine-such as absorption saturation, 1-3 photosynthesis, 4 and photolysis. [5][6][7] Typically, relaxation times are studied via an ultrafast ͑femtosecond or picosecond͒ pump-probe technique by measuring either fluorescence in a reflection configuration or absorption in a transmission configuration. 8,9 In the reflection-based approach, the molecules are excited by a laser pulse through the transition ͉0͘ → ͉i͘. A probe pulse with a variable delay time relative to the pump pulse monitors the time evolution of the population density N i ͑t͒. 8 When applied to molecules with low fluorescence quantum yield, this approach is noisy. In the transmission-based approach, the relaxation time is extracted by measuring pump-induced changes in probe-beam transmittivity ⌬T = T͑I p ͒ − T͑0͒, where T͑I p ͒ and T͑0͒ are the probe beam transmittivities with and without a pump of intensity I p . 9 In both approaches, the time resolution is limited by the pulse width of both the pump and the probe pulses. Hence, not only is an expensive picosecond or femtosecond laser required, but also the detection is susceptible to pulse broadening in dispersive media. Alternatively, intensity correlation of nanosecond laser pulses can be used. 10,11 Working in transmission mode, this technique is not suitable for optically thick media. All of the current techniques are limited to optically thin and weakly scattering materials or require artificial sample preparation.Photoacoustic microscopy and photoacoustic computed tomography, usually based on nanosecond laser excitation, are effective functional and molecular imaging tools in vivo. In the photoacoustic phenomenon, light is absorbed by a material and converted to heat. The subsequent thermoelastic expansion generates a detectable acoustic wave. 12 Photoacoustic sensing presents an exquisite sensitivity due to its inherent background-free nature. Its relative sensitivity to absorption reaches the theoretical limit of 100%, by far the highest among all optical imaging modalities. Most quantitative photoacoustic studies have assumed a linear dependence between ...