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 $1200\text{\hspace{0.17em} nm}$ 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 $50\text{\hspace{0.17em} mm}$ diameter aperture in steps of $10\text{\hspace{0.17em} μm}$ with an optically defined element size of $64\text{\hspace{0.17em} μm}$. Two sensors were used, one with a $22\text{\hspace{0.17em} μm}$ thick polymer film spacer and the other with a $38\text{\hspace{0.17em} μm}$ thick spacer providing $-3\text{\hspace{0.17em} dB}$ acoustic bandwidths of 39 and $22\text{\hspace{0.17em} MHz}$, respectively. The measured noise equivalent pressure of the $38\text{\hspace{0.17em} μm}$ sensor was $0.21\text{\hspace{0.17em} kPa}$ over a $20\text{\hspace{0.17em} MHz}$ 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 $15\text{\hspace{0.17em} μm}$, 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 $300\text{\hspace{0.17em} μm}$ 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 three-dimensional 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.

The use of a novel all-optical photoacoustic scanner for imaging the development of tumor vasculature and its response to a therapeutic vascular disrupting agent is described. The scanner employs a Fabry-Perot polymer film ultrasound sensor for mapping the photoacoustic waves and an image reconstruction algorithm based upon attenuation-compensated acoustic time reversal. The system was used to noninvasively image human colorectal tumor xenografts implanted subcutaneously in mice. Label-free three-dimensional in vivo images of whole tumors to depths of almost 10 mm with sub-100-micron spatial resolution were acquired in a longitudinal manner. This enabled the development of tumor-related vascular features, such as vessel tortuosity, feeding vessel recruitment, and necrosis to be visualized over time. The system was also used to study the temporal evolution of the response of the tumor vasculature following the administration of a therapeutic vascular disrupting agent (OXi4503). This revealed the well-known destruction and recovery phases associated with this agent. These studies illustrate the broader potential of this technology as an imaging tool for the preclinical and clinical study of tumors and other pathologies characterized by changes in the vasculature.

A new approach based on pulsed photoacoustic spectroscopy for non-invasively quantifying tissue chromophore concentrations with high spatial resolution has been developed. The technique is applicable to the quantification of tissue chromophores such as oxyhaemoglobin (HbO(2)) and deoxyhaemoglobin (HHb) for the measurement of physiological parameters such as blood oxygen saturation (SO(2)) and total haemoglobin concentration. It can also be used to quantify the local accumulation of targeted contrast agents used in photoacoustic molecular imaging. The technique employs a model-based inversion scheme to recover the chromophore concentrations from photoacoustic measurements. This comprises a numerical forward model of the detected time-dependent photoacoustic signal that incorporates a multiwavelength diffusion-based finite element light propagation model to describe the light transport and a time-domain acoustic model to describe the generation, propagation and detection of the photoacoustic wave. The forward model is then inverted by iteratively fitting it to measurements of photoacoustic signals acquired at different wavelengths to recover the chromophore concentrations. To validate this approach, photoacoustic signals were generated in a tissue phantom using nanosecond laser pulses between 740 nm and 1040 nm. The tissue phantom comprised a suspension of intralipid, blood and a near-infrared dye in which three tubes were immersed. Blood at physiological haemoglobin concentrations and oxygen saturation levels ranging from 2% to 100% was circulated through the tubes. The signal amplitude from different temporal sections of the detected photoacoustic waveforms was plotted as a function of wavelength and the forward model fitted to these data to recover the concentrations of HbO(2) and HHb, total haemoglobin concentration and SO(2). The performance was found to compare favourably to that of a laboratory CO-oximeter with measurement resolutions of +/-3.8 g l(-1) (+/-58 microM) and +/-4.4 g l(-1) (+/-68 microM) for the HbO(2) and HHb concentrations respectively and +/-4% for SO(2) with an accuracy in the latter in the range -6%-+7%.

The application of a photoacoustic imaging instrument based upon a Fabry-Perot polymer film ultrasound sensor to imaging the superficial vasculature is described. This approach provides a backward mode-sensing configuration that has the potential to overcome the limitations of current piezoelectric based detection systems used in superficial photoacoustic imaging. The system has been evaluated by obtaining non-invasive images of the vasculature in human and mouse skin as well as mouse models of human colorectal tumours. These studies showed that the system can provide high-resolution 3D images of vascular structures to depths of up to 5 mm. It is considered that this type of instrument may find a role in the clinical assessment of conditions characterized by changes in the vasculature such as skin tumours and superficial soft tissue damage due to burns, wounds or ulceration. It may also find application in the characterization of small animal cancer models where it is important to follow the tumour vasculature over time in order to study its development and/or response to therapy.