Abstract. Obtaining absolute chromophore concentrations from photoacoustic images obtained at multiple wavelengths is a nontrivial aspect of photoacoustic imaging but is essential for accurate functional and molecular imaging. This topic, known as quantitative photoacoustic imaging, is reviewed here. The inverse problems involved are described, their nature (nonlinear and ill-posed) is discussed, proposed solution techniques and their limitations are explained, and the remaining unsolved challenges are introduced.

A multiwavelength backward-mode planar photoacoustic scanner for 3D imaging of soft tissues to depths of several millimeters with a spatial resolution in the tens to hundreds of micrometers range is described. The system comprises a tunable optical parametric oscillator laser system that provides nanosecond laser pulses between 600 and 1200 nm for generating the photoacoustic signals and an optical ultrasound mapping system based upon a Fabry-Perot polymer film sensor for detecting them. The system enables photoacoustic signals to be mapped in 2D over a 50 mm diameter aperture in steps of 10 microm with an optically defined element size of 64 microm. Two sensors were used, one with a 22 microm thick polymer film spacer and the other with a 38 mum thick spacer providing -3 dB acoustic bandwidths of 39 and 22 MHz, respectively. The measured noise equivalent pressure of the 38 microm sensor was 0.21 kPa over a 20 MHz measurement bandwidth. The instrument line-spread function (LSF) was measured as a function of position and the minimum lateral and vertical LSFs found to be 38 and 15 microm, respectively. To demonstrate the ability of the system to provide high-resolution 3D images, a range of absorbing objects were imaged. Among these was a blood vessel phantom that comprised a network of blood filled tubes of diameters ranging from 62 to 300 microm immersed in an optically scattering liquid. In addition, to demonstrate the applicability of the system to spectroscopic imaging, a phantom comprising tubes filled with dyes of different spectral characteristics was imaged at a range of wavelengths. It is considered that this type of instrument may provide a practicable alternative to piezoelectric-based photoacoustic systems for high-resolution structural and functional imaging of the skin microvasculature and other superficial structures.

Photoacoustic imaging allows absorption-based high-resolution spectroscopic in vivo imaging at a depth beyond that of optical microscopy. Until recently, photoacoustic imaging has largely been restricted to visualizing the vasculature through endogenous haemoglobin contrast, with most non-vascularized tissues remaining invisible unless exogenous contrast agents are administered. Genetically encodable photoacoustic contrast is attractive as it allows selective labelling of cells, permitting studies of, for example, specific genetic expression, cell growth or more complex biological behaviours in vivo. In this study we report a novel photoacoustic imaging scanner and a tyrosinase-based reporter system that causes human cell lines to synthesize the absorbing pigment eumelanin, thus providing strong photoacoustic contrast. Detailed threedimensional images of xenografts formed of tyrosinase-expressing cells implanted in mice are obtained in vivo to depths approaching 10 mm with a spatial resolution below 100 μm. This scheme is a powerful tool for studying cellular and genetic processes in deep mammalian tissues.O ptical techniques such as fluorescence or bioluminescence imaging are widely used to visualize biological tissues in vivo 1-4 . However, strong optical scattering fundamentally limits the penetration depth or spatial resolution. Microscopy and other techniques that utilize ballistic photons 4 can provide cellular resolution, but only to sub-millimetre penetration depths, while diffuse optical methods such as fluorescence optical tomography 1 can provide greater penetration depths (on the scale of centimetres) but with only limited spatial resolution (on the scale of millimetres). Photoacoustic imaging (PAI) offers the prospect of overcoming these limitations 5-8 . Here, ultrasound waves generated by the absorption of laser light by tissue chromophores are used to produce images of biological tissues based on optical absorption. Because acoustic waves are scattered much less than photons in soft tissues, PAI avoids the depth and spatial resolution limitations of purely optical imaging techniques: depths of a few centimetres with scalable spatial resolution ranging from tens to hundreds of micrometres (depending on depth) are readily achievable.Although strong absorption by haemoglobin enables the acquisition of exquisite three-dimensional photoacoustic (PA) images of the vasculature 9-14 , most cells and tissues are relatively weakly absorbing at visible and near-infrared wavelengths and are thus indistinguishable in the absence of exogenous contrast. The latter can be provided by nanoparticle-or dye-based targeted contrast agents 15-17 , but these can present challenges in achieving effective specific targeting and clearance. The use of reporter genes to provide genetically encoded exogenous PA contrast would avoid these limitations and has the further advantage of providing opportunities to study more complex biological behaviours such as cell growth dynamics and intracellular processes such as gene expressi...