Chao-Kang Liao

In vivo Photoacoustic Molecular Imaging with Simultaneous Multiple Selective Targeting Using Antibody-Conjugated Gold Nanorods

The use of gold nanorods for photoacoustic molecular imaging with simultaneous multiple targeting is reported. Multiple targeting is done by utilizing the tunable optical absorption property of gold nanorods. This technique allows multiple molecular signatures to be obtained by simply switching laser wavelength. HER2 and EGFR were chosen as the primary target molecules for examining two cancer cells, OECM1 and Cal27. Both in vitro and in vivo mouse model imaging experiments were performed, with contrast enhancement of up to 10 dB and 3.5 dB, respectively. The potential in improving cancer diagnosis is demonstrated.

Photoacoustic Imaging of Multiple Targets Using Gold Nanorods

Photoacoustic (PA) imaging has been used mainly for anatomical and functional imaging. Although functionalized nanoparticles also have been developed for PA molecular imaging, only single targeting has been demonstrated. In this study, PA imaging of multiple targets using gold nanorods is demonstrated experimentally using HER2 and CXCR4 as target molecules. The two corresponding monoclonal antibodies were conjugated to two types of gold nanorod with different aspect ratios. Gold nanorods with mean aspect ratios of 5.9 and 3.7 exhibited peak optical absorptions at 1000 and 785 nm, respectively. Appropriate selection of laser irradiation wavelength enhances PA signals by 7-12 dB and allows signals from gold nanorods corresponding to specific bindings to be distinguished. This approach potentially allows the expression levels of different oncogenes of cancer cells to be revealed simultaneously.

Optoacoustic imaging with synthetic aperture focusing and coherence weighting

Optoacoustic imaging takes advantage of high optical contrast and low acoustic scattering and has found several biomedical applications. In the common backward mode a laser beam illuminates the image object, and an acoustic transducer located on the same side as the laser beam detects the optoacoustic signal produced by thermoelastic effects. A cross-sectional image is formed by laterally scanning the laser beam and the transducer. Although the laser beam width is generally narrow to provide good lateral resolution, strong optical scattering in tissue broadens the optical illumination pattern and thus degrades the lateral resolution. To solve this problem, a combination of the synthetic aperture focusing technique with coherence weighting is proposed. This method synthesizes a large aperture by summing properly delayed signals received at different positions. The focusing quality is further improved by using the signal coherence as an image quality index. A phantom comprising hair threads in a 1% milk solution was imaged with an optoacoustic imaging system. The results show that the proposed technique improved lateral resolution by 400-800% and the signal-to-noise ratio by 7-23 dB over conventional techniques.

Nanorod-based flow estimation using a high-frame-rate photoacoustic imaging system

A quantitative flow measurement method that utilizes a sequence of photoacoustic images is described. The method is based on the use of gold nanorods as a contrast agent for photoacoustic imaging. The peak optical absorption wavelength of a gold nanorod depends on its aspect ratio, which can be altered by laser irradiation (we establish a wash-in flow estimation method of this process). The concentration of nanorods with a particular aspect ratio inside a region of interest is affected by both laser-induced shape changes and replenishment of nanorods at a rate determined by the flow velocity. In this study, the concentration is monitored using a custom-designed, high-frame-rate photoacoustic imaging system. This imaging system consists of fiber bundles for wide area laser irradiation, a laser ultrasonic transducer array, and an ultrasound front-end subsystem that allows acoustic data to be acquired simultaneously from 64 transducer elements. Currently, the frame rate of this system is limited by the pulse-repetition frequency of the laser (i.e., 15 Hz). With this system, experimental results from a chicken breast tissue show that flow velocities from 0.125 to 2 mms can be measured with an average error of 31.3%.

Multiple targeting in photoacoustic imaging using bioconjugated gold nanorods

Cancer cells presented altered surface molecules to encourage their growth and metastasis. Expression of oncogeneic surface molecules also play important roles in the prediction of clinical outcome and treatment response of anti-cancer drugs. It is thus conceivable that imaging of cancer lesions while simultaneously obtaining their pathogenic information at molecular level of as many oncogenic proteins as possible is of great clinical significance. Gold nanoparticles have been used as a contrast agent for photoacoustic imaging. In addition, gold nanoparticles can be bioconjugated to probe certain molecular processes. An intriguing property of gold nanoparticles is its ability to tailor its optical properties. For example, size effects on the surface plasmon absorption of spherical gold nanoparticles have shown that the peak optical absorption red-shifts with the increasing particle size. In addition, the optical absorption spectrum of cylindrical gold nanoparticles (i.e., gold nanorods) exhibits a strong absorption band that is directly related to the aspect ratio. With these unique characteristics, selective targeting can be achieved in photoacoustic molecular imaging. Specifically, gold nanorods with different aspect ratios can be bioconjugated to different antibodies. Multiple targeting and simultaneous detection can then be achieved by using laser irradiation at the respective peak optical absorption wavelength. In this study, photoacoustic multiple targeting using gold nanorods is experimentally demonstrated. We have chosen Her2 and CXCR4 as our primary target molecule as Her2 expression is associated with growth characteristics and sensitivity to Herceptin chemotherapy. On the other hand, CXCR4 expression predict the organ-specific metastatic potential of the cancer cells for clinical intervention in advance. Monoclonal antibody (mAb) against Her2/neu was conjugated to nanorods with several different aspect ratios. The agarose gel is suitable for photoacoustic signal acquisition. A wavelength tunable Ti-Sapphire laser was used for laser irradiation and a 1 MHz ultrasound transducer was used for acoustic detection. The optical wavelength of the laser was tuned between 800 nm and 940 nm, corresponding to gold nanorods of an aspect ratio ranging from 3.7 to 5.9. The results clearly show the potential of photoacoustic molecular imaging with multiple targeting in revealing different oncogene expression levels of the cancer cells.

Simulations of optoacoustic wave propagation in light-absorbing media using a finite-difference time-domain method

Optoacoustic (OA) imaging is an emerging technology that combines the high optical contrast of tissues with the high spatial resolution of ultrasound. Taking full advantage of OA imaging requires a better understanding of OA wave propagation in light-absorbing media. Current simulation methods are mainly based on simplified conditions such as thermal confinement, negligible viscosity, and homogeneous acoustic properties throughout the image object. In this study a new numerical approach is proposed based on a finite-difference time-domain (FDTD) method to solve the general OA equations, comprising the continuity, Navier-Stokes, and heat-conduction equations. The FDTD code was validated using a benchmark problem that has an approximate analytical solution. OA experiments were also conducted and data were in good agreement with those predicted by the FDTD method. Characteristics of simulated OA waveforms and OA images were discussed. The simulator was also employed to study wavefront distortion in OA breast imaging.

Photoacoustic Flow Measurements with Gold Nanoparticles

The hypothesis that quantitative blood flow measurements are feasible with the time-intensity based method in photoacoustic imaging using gold nanoparticles as contrast agent is experimentally tested. The in vitro results show good linearity between the measurements and the theory, thus suggesting the potential of relative photoacoustic flow measurements with gold nanoparticles

Time-intensity based optoacoustic flow measurements with gold nanoparticles

The indicator-dilution theory has been used for flow rate measurements in various imaging modalities, including magnetic resonance imaging, computed tomography and ultrasound. The experimental procedure generally involves the injection of a dose of indicator (i.e., the contrast agent), after which the concentration of the agent is monitored as a function of time; it is therefore also known as the time-intensity method. Although the time-intensity method has been widely applied to other imaging modalities, it has not been demonstrated with optoacoustic imaging. In this study, we experimentally test the hypothesis that quantitative blood flow measurements are feasible with the time-intensity based method in optoacoustic imaging. Gold nanospheres (broad band absorption spectrum peaks at 520 nm) were used as the optoacoustic contrast agent. The imaging system consisted of a frequency-doubled Nd:YAG laser operating at 532 nm for optical illumination, and an ultrasonic single crystal transducer with a center frequency of 3.5 MHz and a focal depth of 7 cm for detection. The volumetric flow rate ranged from 0.23 to 4.29 ml/sec, and the volume of the mixing chamber was from 30 to 80 ml. Results show good agreement between the measured mean transit times and the predicted time constants (correlation coefficient higher than 0.88), thus demonstrating the feasibility of the time-intensity based flow measurement technique. In addition to describing the method and experimental results, issues regarding the system sensitivity and estimation of the dilution transfer function are also discussed.

In vivo Photoacoustic Imaging with Multiple Selective Targeting Using Bioconjugated Gold Nanorods

In this study, photoacoustic imaging is utilized to probe information from oncogene surface molecules of cancer cell with the aid of specific targeting. The ultimate goal is to provide prediction of clinical outcome and treatment response of anti-cancer drugs. Different from single targeting in most research, we accomplished multiple targeting to obtain a molecular profile potentially representing tumor characteristics or to locate the heterogeneous population in one lesion. By conjugating different antibodies to gold nanorods corresponding to different peak absorption bands, multiple targeting and simultaneous detection with photoacoustic imaging can be achieved with laser irradiation at the respective peak optical absorption wavelength. Her2 and EGFR were chosen as our primary target molecules. The targeting complex was evaluated in two types of oral cancer cells, OECM1 and Cal27. The OECM1 cell line overexpresses Her2 but has low expression of EGFR, while Cal27 cell line expresses both antibodies. Also, the targeting efficacy to OECM1 can be further improved by using mixed nanoprobes. The cancer cells were induced on the back of the mice by subcutaneous injection. The captured images show that both cancer cells exhibit a higher photoacoustic response (maximum 3 dB) than control groups with specific targeting, thus demonstrating the feasibility of multiple selective targeting with bioconjugated gold nanorods. Images of multiple targeting with mixed nanoprobes of OECM1 cells also reveal further enhancement of targeting (4 dB). The results showed potential of in vivo photoacoustic molecular imaging, providing a better guidance for diagnosis and treatment of cancer.

A high frame rate photoacoustic imaging system and its applications to perfusion measurements

A high frame rate photoacoustic imaging system is described. Applications of this system to perfusion measurements are also presented as a demonstration of its potential usage. The system consists of an ultrasound front-end sub-system for acquisition of acoustic array data. The ultrasound front-end sub-system is also known as the DiPhAS (digital phased array system) which is capable of simultaneously acquiring radio frequency data from 64 transducer channels at a rate up to 40 MSamples/sec per channel. In this study, an ultrasonic linear array with a 5 MHz center frequency was employed as part of the integrated photoacoustic probe. The photoacoustic probe also had two linear light guides mounted on the sides of the ultrasonic array for broad laser irradiation from a Q-switched Nd:YAG pulsed laser. After the acquired ultrasound array data were transferred to a personal computer via a high speed digital I/Q card, dynamic focusing and image reconstruction were done off-line. The 64-channel array data can be acquired and transferred every 4 milliseconds, thus making the frame rate of the system up to 250 Hz. The actual frame rate of the current system is limited by the pulse repetition frequency of the laser at 15 Hz. To demonstrate capabilities of the system, photoacoustic perfusion measurements with gold nanorods were performed. A previously proposed time-intensity based flow estimation technique utilizing the shape transitions of gold nanorods under laser irradiation was employed. Good estimation results were achieved and potential of this high frame rate photoacoustic imaging system is clearly demonstrated.