Photoacoustic imaging offers tremendous potential for both research and clinical applications because it draws on the advantages of both spectroscopy and ultrasound imaging. To date, researchers have applied the technique primarily for static imaging and spectroscopy of tissue and organs. Recent studies have shown, however, that it also allows dynamic imaging, thus opening up a host of possible new applications. These studies suggest a bright future for photoacoustic imaging, which could contribute to advances in cancer diagnosis and treatment, for example, by allowing noninvasive in vivo imaging at depths not accessible to other optical imaging methods.“Mr. Watson, come here …”The photoacoustic effect, in which the absorption of light results in the generation of sound, was described first by Alexander Graham Bell in 1860. He wanted to take advantage of this phenomenon to develop what he called the “photophone,” which would change sound to light and wirelessly transmit it to a receiver, which would then convert the light back to sound. The photophone never took off, of course, but interest in the photoacoustic effect remained.Although engineers have long used photoacoustic imaging for metal and ceramic surface imaging, the technique was only recently developed for three-dimensional imaging of tissue. Here, investigators irradiate tissue with lasers, inducing transient thermoelastic expansion, which then leads to emission of wideband ultrasonic waves. Because the waves contain tissue-specific information about absorption, the technique allows noninvasive in vivo imaging based on absorption contrasts.Photoacoustic imaging has attracted the attention of researchers and clinicians alike. “One of the key features that people are interested in is the ‘super’ depth capability,” said Lihong Wang, an investigator at Washington University in St. Louis and editor of an upcoming book about the topic, Photoacoustic Imaging and Spectroscopy. Because scattering is dominant in light transport, high-resolution optical imaging methods generally have limited penetration depth – in the skin, typically 1 mm. Photoacoustic imaging makes the most of optical absorption of multiple-scattered photons to facilitate what is known as “super depth penetration.” Here, “if you go beyond 1-mm depth, you can still achieve very high spatial resolution, as determined by the wideband ultrasound signals,” Wang explained.As a result, he continued, the technique could have very broad applications. In the research arena, for example, any lab that uses confocal microscopy could add a photoacoustic microscope “to see beyond what people currently can see.” The super depth capability would contribute to this, of course, but also would the very high contrast afforded by looking at absorption as opposed to scattering or fluorescence, for instance. This contrast – as high as or higher than 10:1, Wang said – enables researchers to look at both total concentration and oxygen saturation of hemoglobin, both very important parameters, by using endogenous photoacoustic contrast only. The researchers demonstrated the technique in mouse and rat models. They showed that the endogenous absorption of hemoglobin in single red blood cells, for example, is high enough to produce photoacoustic (PA) signals that are readily detected using a standard ultrasound transducer. Shown here is a high-resolution optical image of a single cell in natural lymph flow captured in vivo with a color camera (left) and a photoacoustic signal from an individual cell (right).The method also allows them to go places that, without calling on exogenous contrast, might be closed to them. In a recent study, Wang and colleagues showed they could visualize the smallest capillaries with the technique. “Even if you use x-ray, you have to inject iodine-based contrast agents to see these,” he said.The method could also help to advance a range of clinical applications, even in the short term. It could aid in cancer diagnosis by imaging angiogenesis, or formation of new blood vessels, which correlates strongly with the growth of tumors. And by extending the technique to dual-wavelength imaging, doctors and technicians can distinguish between two pigments based on their respective absorption spectra and, thus, detect melanomas.Another possible application is monitoring of chemotherapy. Predicting the outcome of chemotherapy can be difficult: Doctors almost have to run through a major course of it to decide the efficacy of the treatment. Ideally, of course, they would know early on whether it is working and would stop immediately if it weren’t, to avoid any further associated side effects. Wang and co-workers are considering assessing the potential of using photoacoustic imaging to predict treatment efficacy, by obtaining a baseline and monitoring the size of the tumor as a function of time. “That’s a project we’re thinking about pursuing,” he said. Investigators have achieved in vivo lymph flow cytometry by making the most of the natural focusing that occurs in lymphatic vessels and using a photoacoustic technique for detection. Thus they demonstrated how doctors might identify and quantitate normal, malignant, apoptotic and necrotic cells, for example.Listening to cells in lymphaticsIn Arkansas and in the southeastern corner of Russia, researchers are working to develop another application that could aid in cancer diagnosis: in vivo flow cytometry in lymphatics, relying on photoacoustic signals for detection. Researchers previously have reported using flow cytometry for ex vivo detection of bacteria as well as circulating tumor cells, for instance, as markers of cancer progression. But this approach has proved problematic, not least of all because of the small sample volumes of blood (milliliters) or lymph (microliters), which could lead to missing some rare cancer cells circulating in the large 5-l blood volume, and because the extraction and preparation of cells can be time-consuming and can even result in artifacts such as alterations in the expression of proteins on the cell surface.In vivo detection could help to avoid these issues by using vessels as natural flow cells while monitoring blood or lymph volumes in the peripheral vasculatures. Research in this area has yet to demonstrate quantitation of every cell, however. Nor has it fully addressed the issues of translating fluorescence labeling to humans – including the cytotoxicity concerns of currently available fluorescent tags and the phagocytic clearance of labeled tumor cells resulting from an immune response to the tags.During the period from 2004 to 2007, a team led by Vladimir P. Zharov at the University of Arkansas for Medical Sciences, in Little Rock, developed alternatives to in vivo flow cytometry that utilize the principles of photothermal and photoacoustic spectroscopy with near-infrared laser pulses – with either no labeling using intrinsic cell markers such as cytochromes, hemoglobin or melanin, or labeling with conventional contrast agents such as indocyanine green or gold nanoparticles and carbon nanotubes. However, although these techniques showed high sensitivity in the blood flow in vivo, single-cell detection in lymphatics remained just out of reach.In the October 2008 issue of Cytometry Part A, Zharov and colleagues from Saratov State University in Russia, and Prokhorov General Physics Institute in Moscow introduced a new tool: an in vivo noninvasive lymph test that allows them to count cells in the lymph flow. This tool takes advantage of two recent discoveries: a natural cell-focusing that occurs in lymph vessels and photoacoustic effects that can be detected in single cells using conventional ultrasound equipment.When phasic contractions take place, cells line up single file in the vessels beyond the lymph valve, as shown here (The image was captured by a high-speed, black-and-white camera), thus mimicking the hydrodynamic focusing used in conventional ex vivo flow cytometry measurements.Flow cytometry with “natural” hydrodynamic focusingThe researchers first attempted a “mechanical” approach, mimicking the hydrodynamic focusing of cells in ex vivo flow cytometry either by implanting a tube with a small nozzle or by using special catheters. Zharov noted, however, that while these approaches could be applicable in animal studies, they weren’t quite ready to be implemented for lymphatic examinations in humans.This wasn’t nearly the setback it might have been, however, because similar focusing occurs naturally in lymphatic vessels during phasic contractions. When such contractions take place beyond the lymph valve, cells line up in the vessels single file, thus facilitating in vivo flow cytometry measurements.Still, the researchers found that the usual spectral methods for cell identification were not up to the task, because the time that cells spend in the detection volume is very much shorter than in conventional photoacoustic spectroscopy. For this reason, they developed a high-speed, two-color mode in which they irradiate cells with two laser pulses at different wavelengths with a very short (microseconds) time delay between the pulses and then perform time-resolved detection of the laser-induced “double” photoacoustic effects, which serve as spectrally selective cell fingerprints. Detection of the photoacoustic signals is very simple, Zharov said, requiring little more than a miniature ultrasound transducer affixed to the skin near the vessels in question and a slightly modified standard signal acquisition system. With strong signals from pigmented cells or cells labeled with absorbing nanoparticles, even a conventional oscilloscope can be used. Irradiation of the vessels is performed using either an intravital microscope setup, in animal studies, or a fiber with a focusing tip gently attached to the skin above the vessels.In vivo detection with photoacoustic imagingThe researchers demonstrated the technique in rat and mouse models by performing label-free detection and identification of rare erythrocytes and melanoma cells with distinct spectral signatures in the near-infrared range. They showed that endogenous absorption was high enough in these cells to produce photoacoustic signals that they could readily detect with a standard ultrasound transducer.The experiments suggest that the technique, with sensitivity higher even than that of conventional ex vivo assays, could be applied in vivo for early detection of metastasis. And beyond this, they help to shed light on the role of lymph vessels in metastasis – showing, for example, how the vessels transport metastatic cells from the primary tumor to sentinel lymph nodes. Ekaterina Galanzha, a co-investigator in this study, emphasized that the team used conventional blue dye, the absorption of which, in the visible range, does not interfere with the near-infrared absorption of melanoma cells. This allows in vivo real-time identification of lymphatics with simultaneous monitoring of melanoma cell transport through this lymphatic to the lymph nodes and the accumulation of cells there. From a clinical perspective, the method ultimately might allow doctors to identify and quantitate red blood cells, melanoma cells and various types of white blood cells, including immune-related cells in the normal, apoptotic and necrotic stages. Because of their low absorption, these cells can be labeled with at least three “color” nanoparticles with different absorption spectra. In the present study, the researchers demonstrated this in vivo under model conditions using gold nanoshells, carbon nanotubes and gold nanorods.Although the technique is not yet ready for clinical application, Zharov and colleagues are actively working to develop it for that purpose. Currently, they are focusing on detecting metastatic cells in sentinel lymph nodes – in some cases without labeling the cells, as with pigmented melanoma cells. In other situations, they are performing molecular targeting of low-absorbing metastatic cells with multicolored nanoparticles and multispectral lasers with high pulse repetition rates.With the development of a portable device that can be attached to lymph vessels and nodes, the researchers expect a relatively quick translation of the technology for use in humans. By taking advantage of the photoacoustic effect, this device could enable single-cell diagnostics, detection of metastasis and assessment of therapy, among other applications.The researchers demonstrated an in vivo noninvasive lymph test based on flow cytometry that uses photoacoustic effects to perform detection. The cytometer was based on an upright Olympus microscope incorporating photoacoustic, photothermal, fluorescent and transmission digital microscope modules. A tunable pulse laser parametric oscillator made by Lotis TII of Minsk, Belarus, was the source of irradiation. They detected the photoacoustic signals induced by irradiation using an ultrasound transducer made by Panametrics, now Olympus NDT of Waltham, Mass. They recorded the signals using a boxcar technique and an oscilloscope made by Tektronix Inc. of Beaverton, Ore., and then analyzed them using standard and customized software.