Researchers at Texas A&M University in College Station have developed a method for using the photo-acoustic effect to create images. The technique permits functional imaging of oxy- and deoxyhemoglobin, which could be useful for trauma evaluation, for example, where optical absorption reveals information related to hemorrhage and edema. It provides axial resolution of about 15 μm, lateral resolution of 45 μm and an imaging depth of 3 mm. “This technique measures optical contrast based on physiological parameters, such as the total hemoglobin concentration and the oxygen saturation of hemoglobin,” explained Lihong V. Wang from the optical imaging laboratory at the university. The photoacoustic effect is an ultrasonic wave created when tissue is irradiated by a short-pulse laser. Some of the radiation is absorbed and converted partially into heat, which then creates a rise in pressure through thermoelastic expansion. The pressure rise moves through the tissue as a sound wave that can be detected by ultrasonic transducers. “Since ultrasonic waves are much less scattered than optical waves, photoacoustic imaging combines the contrast of optical absorption with the spatial resolution of ultrasound,” Wang said. The technique is a cross between absorption spectroscopy and ultrasound. The researchers picked the absorption band of hemoglobin and exposed tissue to laser light that fell into that band. Then they used the ultrasonic wave to create an image. Ultrasound waves scatter 100 to 1000 times less than light waves, Wang said. “As a result, ultrasound can provide much better resolution than light can for structures located deeper than 1 mm below a tissue’s surface.” To create a functional photoacoustic microscope, the investigators used a tunable dye laser that generated 6-ns pulses and that was pumped by an Nd:YAG laser. The laser beam traveled to the scanning head via an optical fiber, and a photodiode calibrated the laser energy. The beam passed from the fiber through a conical lens that made a ring-shaped illumination pattern. The ring was focused into the tissue where the focal area over-lapped the ultrasonic focal area, although the optical focus was wider. The ring-shaped pattern was intended to reduce the photoacoustic effect generated in the field of view of the ultrasonic transducer. The system was set up to detect the laser-generated photoacoustic signal in reflection mode. Achieving maximum depth The focal diameter of the ultrasonic transducer determined the system’s lateral resolution. “If the laser pulse is sufficiently short, a high-numerical-aperture acoustic lens and a high-center-frequency ultrasonic transducer provide high lateral resolution. A wideband ultrasonic transducer provides high axial resolution,” Wang said. According to Wang’s paper, published in the July issue of Nature Biotechnology, when the center frequency of the transducer exceeds 10 MHz, the penetration depth of the ultrasonic wave — not the penetration of the excitation light — determines the maximum imaging depth. The researchers used a Panametrics 6-mm ultrasonic detector with a 50-MHz central frequency and a 70 percent nominal bandwidth. Coupling the device to a homemade spherically focusing lens with a numerical aperature of 0.44, a focal zone of 0.3 mm and a focal length of 6.7 mm, the group created images with an axial resolution of 15 μm and a lateral resolution of 45 μm at a depth of up to 3 mm in living tissue. This in vivo image shows subcutaneous microvasculature from a 20-g SENCAR mouse at a 584-nm optical wavelength. SENCAR mice are commonly used for experiments with carcinogens. Hemoglobin and melanin are responsible for most optical absorption that creates the photoacoustic effect. As a result, choosing the correct excitation wavelength permits blood detection, which the researchers say enables high-contrast, specific images of the microvasculature. At 584 nm, the laser emitted the wavelength where oxygenated and deoxygenated hemoglobin had the same molecular extinction coefficient. As a result, the image contrast became dependent on the total concentration of hemoglobin but did not reflect changes in oxygenation levels. This could be useful in imaging the blood vessels that characterize rapidly growing tumors and could aid in evaluating the effectiveness of therapies that target angiogenesis. In vivo images of a five-day postinoculation melanoma tumor in a 20-g immunocompromised nude mouse were taken at 584-nm (left) and 764-nm optical wavelengths. A pseudocolored composite of the images from the two wavelengths is shown on the right. The group demonstrated the same technique with dual-wavelength imaging. They used 584-nm light to image blood vessel proliferation in a tumor and then re-imaged the same section with 764-nm light that missed the absorption peaks of hemoglobin and melanin and penetrated deeply into the tumor, providing information about the thickness of a particular tumor. Combining the two images created a high-contrast image capable of resolving individual microvessels roughly 50 μm in diameter. In addition, the dual-wavelength technique can measure oxygen saturation in individual blood vessels. The researchers first tested the oxygen saturation measurements ex vivo using bovine blood and compared their results with a standard optical oxygen saturation measurement. When the results proved to be similar, they used the system to measure normal oxygen saturation in live human tissue, detecting a saturation level of 97 ±2 percent for arterial blood, and 77 ±4 percent for venous blood. They also demonstrated that the system can respond accurately to a shift from normal oxygenation to hypoxic conditions. The system not only created an accurate measurement of overall oxygen saturation, but also can map the saturation levels of individual blood vessels. The group thinks that this that may be useful in conducting functional brain imaging. At present, however, the system is somewhat slow. While a single, one-dimensional depth image takes only about 2 μs, a one-dimensional scan in both the depth and the transverse directions takes about 10 s. It takes a full 18 min to get a two-dimensional image of a 64-square-mm area, and more than 2 hours for the oxygen saturation measurements. Wang and his colleagues acknowledged this and posit that, by increasing the laser repetition rate and using an array of ultrasonic transducers, they can make the system faster. They plan to begin studying the new method in clinical applications such as melanoma imaging, Wang said. Wang’s laboratory has moved to Washington University in St. Louis.