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The establishment of multiplex photoacoustic molecular imaging to characterize heterogeneous tissues requires the use of a tunable, thermally stable contrast agent targeted to specific cell types. We have developed a multiplex photoacoustic imaging technique which uses targeted silica-coated gold nanorods to distinguish cell inclusions in vitro. This paper describes the use of tunable targeted silica-coated gold nanorods (SiO(2)-AuNRs) as contrast agents for photoacoustic molecular imaging. SiO(2)-AuNRs with peak absorption wavelengths of 780 nm and 830 nm were targeted to cells expressing different cell receptors. Cells were incubated with the targeted SiO(2)-AuNRs, incorporated in a tissue phantom, and imaged using multiwavelength photoacoustic imaging. We used photoacoustic imaging and statistical correlation analysis to distinguish between the unique cell inclusions within the tissue phantom.

We describe 3D single particle tracking of gold nanoparticles (AuNPs) moving along collagen fibers in aqueous environment with two-photon excitation conditions. The photoacoustic effect at the collagen fiber caused by the irradiation with ultrashort, near-infrared laser pulses propels the particles adsorbed to the surface of the collagen fibers. We report the tracking of individual AuNPs in three dimensions with high spatial and temporal resolution, of few nanometers and milliseconds, respectively. Due to the emission signal caused by the interaction between the AuNPs and the weak chromophores in the collagen fiber, the trajectories of individual AuNPs reveal the fiber topography with nanometric resolution. The intensity along the trajectory shows that we are sensitive to the distribution of the weak chromophores on the fiber.

Modulated tone-burst light was employed to measure non-radiative relaxation time of fluorophores with biomedical importance through photoacoustic effect. Non-radiative relaxation time was estimated through the frequency dependence of photoacoustic signal amplitude. Experiments were performed on solutions of new indocyanine green (IR-820), which is a near infrared dye and has biomedical applications, in two different solvents (water and dimethyl sulfoxide (DMSO)). A 1.5 times slower non-radiative relaxation for the solution of dye in DMSO was observed comparing with the aqueous solution. This result agrees well with general finding that non-radiative relaxation of molecules in triplet state depends on viscosity of solvents in which they are dissolved. Measurements of the non-radiative relaxation time can be used as a new source of contrast mechanism in photoacoustic imaging technique. The proposed method has potential applications such as imaging tissue oxygenation and mapping of other chemophysical differences in microenvironment of exogenous biomarkers.

We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy (TAUM), which fuses photoacoustic microscopy with non-linear microscopy. Photoacoustic microscopy is well known for its ability to image chromophores deep (> 1 mm) in scattering media with spatial resolutions in the 10s of microns. Non-linear microscopy is well known for its exquisite spatial resolution in three dimensions. This superior spatial resolution is attributed to the fact that the collected signal has a non-linear dependence on the light intensity. This dependence confines the signal to a very small focal volume, producing optically resolved voxels. Transient absorption is a non-linear process often used to map the excited state lifetimes of molecules exhibiting low fluorescence quantum efficiency. This sensitivity to non-radiative transitions makes transient absorption an attractive process to combine with photoacoustic imaging. We have built a prototype transient absorption ultrasonic microscope, implementing off-axis photoacoustic detection to allow the use of a high-quality, high numerical aperture objective. This high-quality, commercial lens is required to provide the tight focusing needed to optimize non-linear effects. We have demonstrated the increased spatial resolution of TAUM by imaging Rhodamine 6G in a capillary tube. The capillary cross-section is fully resolved, suggesting an axial resolution of < 10 microns. A 6 MHz transducer was used in this experiment, which results in an axial resolution of ~ 400 microns when used in a traditional photoacoustic microscope. Boasting the superior penetration depth and absorption contrast offered by photoacoustic emission and complemented by spatial resolutions comparable to confocal microscopy, we believe that Transient Absorption Ultrasonic Microscopy has excellent potential for producing volumetric images with cellular/subcellular resolution in three dimensions deep inside living tissue.