Exploring a fluorescent blood pool agent in photoacoustic imaging

Objectives Oxy- and deoxyhemoglobin give rise to a photoacoustic signal and are particularly popular sources of endogenous contrast, which can be manipulated with an oxygen breathing challenge. However, comparison of the oxy- and deoxyhemoglobin signal may present difficulties if signal changes are caused by both their ratio (i.e. SO2) and concentrations (i.e. blood volume). To validate multispectral analysis algorithms, we have used a blood pool agent to independently determine blood volume. Genhance™ 750 is a fluorescent blood pool imaging agent with long vascular retention, which has not been reported previously for photoacoustic imaging, but which possesses many attractive properties. We have now explored the use of Genhance as a photoacoustic imaging contrast agent in healthy nude mice.

Methods A phantom study was first performed to obtain the absorption spectrum. Genhance 750 was diluted in PBS to 50 μg/ml and inserted into a standard agarose-lipid phantom. The phantom was imaged using an iThera MSOT 256 system. The MSOT signal intensity was obtained from 680 to 875 nm at 5 nm increments for ROIs placed on the phantom to determine the photoacoustic absorption spectrum. Healthy nude mice were anesthetized (2% isoflurane/air) and imaged with an IV tail catheter secured in situ. Photoacoustic tomography was performed at the aforementioned wavelengths to assess Genhance as well as oxy- and deoxyhemoglobin signal. After allowing 15 minutes for the animal to reach thermal equilibrium in the imaging system, Genhance (50 μg in 100 μl PBS) was injected through the IV catheter. Spectral images were acquired continuously every 20 s before and up to one hour after injection. The animals breathed medical air throughout the experiment. Reconstruction was performed using back-projection and model based algorithm for comparison. Multispectral analysis was performed using linear regression. ROIs were placed on both kidneys; spleen, spine and skin.

Results Genhance gave rise to a strong photoacoustic signal in the phantom with a peak absorption wavelength of 745 nm, similar to the manufacturer’s excitation spectrum. The spectrum of Genhance is well separated from oxy- and deoxyhemoglobin absorption maxima, allowing for effective spectral decomposition. The kidneys, spleen, spine and other anatomical structures were clearly visible before and after injection of Genhance. Most Genhance enhancement appeared in kidneys and blood vessels surrounding the spine. The spleen and skin also showed some nominal Genhance signal. The intensity of Genhance showed a transient peak associated with first pass inflow and then stabilized after about 20 mins, with minimal further changes in the spleen and spinous blood vessels over the next 30 mins.

Conclusions Genhance can be used as a photoacoustic imaging contrast agent and doesn’t interfere with the decomposition of oxy- and deoxyhemoglobin concentration. Genhance should be given 20 minutes prior to the imaging window for stable intensity. $$graphic_CDFDDF96-99A9-4D9B-B684-2DB5458CE2A2$$