Beyond the infrared, but just before you arrive at the microwaves, awaits an emergent area of opportunity for biomedical research: the terahertz region. Spanning from 0.3 to 3 THz (equivalent to 100 to 1000 µm), this wavelength range allows imaging and analysis of tissues that otherwise are opaque to visible and infrared wavelengths, yet it doesn’t have the risks associated with x-ray scanning. Terahertz imaging is used for proteomics and general drug discovery efforts, including defining the 3-D structures of proteins. It also is helpful for viewing the myriad ways in which proteins fold into various configurations, which affects their biophysical properties. Spectroscopy within the terahertz range piles on the information. It can reveal the different crystalline forms of molecules commonly used – or under consideration – for pharmaceuticals. The same molecule of interest, but with a variant form, may not be as stable; it may solubilize differently, or it may not be absorbed by a patient in the same way as its counterpart. Terahertz spectroscopy can help define these differences, which affect the overall mechanism of action of a promising drug. Terahertz imaging was first demonstrated in the mid-1990s, but despite showing early promise didn’t expand in use quickly because there have been a limited number of sources that could readily and consistently produce pulses at terahertz frequencies. Using these wavelengths for spectroscopy has lagged even more. Primarily used in pharmaceutical research at first, the terahertz range only recently has been investigated for medical imaging and spectroscopy. At University College London, Caroline B. Reid and her colleagues are working with TeraView Ltd. of Cambridge, UK, to explore potential medical applications for terahertz radiation. Reid noted several potential benefits from using this wavelength range. “Terahertz penetrates through many materials, has low scattering compared to visible and near-IR wavelengths, is nonionizing and appears to provide spectral signatures for anything from explosives to cancers,” she said. Reid’s colleagues include Jan G. Laufer, Adam P. Gibson and Jeremy C. Hebden of University College London, Emma Pickwell-MacPherson of the Hong Kong University of Science and Technology in Clearwater Bay, and Vincent P. Wallace of the University of Western Australia in Crawley. They published their findings in the Aug. 21, 2010, issue of Physics in Medicine and Biology. Reid’s group put to the test three terahertz reflection spectroscopy techniques: linear spectral decomposition method (LSDM), spectrally averaged dielectric coefficient method (SADCM) and Debye relaxation coefficient method (DRCM). They tested each method using the same set of reflection data, which was gathered using TeraView’s TPI Imaga1000 (imaging) and TPI Spectra1000 (spectrometry) instruments. The accuracy and resolution of water and lipid concentrations as measured via LSDM, SADCM and DRCM terahertz spectroscopy methods. Reused with permission of Physics in Medicine and Biology. Based on the Beers-Lambert law, LSDM provides a measurement of the concentration of chromophores that react to the emitted terahertz radiation. The concentration is estimated from the wavelength-dependent total attenuation that occurs. The downside of the technique is that it requires previous knowledge of the materials being scanned, especially their absorption coefficient spectra. SADCM is a more empirical method that determines chromophore concentrations by averaging the dielectric coefficients across a specific frequency range within the terahertz band. Terahertz time-domain spectroscopy is well-suited for determining the dielectric coefficients within a sample because it is a phase-sensitive technique. However, Reid said, “SADCM can only be applied to a maximum of three solutes [and] it returns poor resolutions, limiting its potential as a tool for tissue spectroscopy.” Like SADCM, DRCM is an empirical technique. It measures the dielectric relaxation among irradiated solutes within a sample, comparing them to measurements previously taken from phantoms of various compositions. Reid said that DRCM could be used to resolve the concentrations of more than two solutes in a sample, “provided that material-specific variations in the Debye relaxation coefficients can be determined, which are dependent on intramolecular interactions between the solutes.” Reid and her colleagues tested samples comprising various concentrations of lipids in water. Being a polar molecule, water is strongly absorbed – thus easily distinguishable from other molecules – by terahertz radiation. They found that the accuracy of the determined concentrations was strongly dependent on which method was used. Nonetheless, all three proved capable, in principle, of determining material composition through the concentration of the lipid solutes. Shown are the absorption coefficients (left) and indices of refraction (right) garnered from terahertz spectroscopy of various concentrations of lipids in water. Reused with permission of Physics in Medicine and Biology. The sensitivity of all three analytical methods is fixed, Reid said. “In order to improve resolution,” she added, “only the standard deviation of the measurement system may be improved, which may be achieved by increasing the signal-to-noise ratio of the system.” TeraView has developed new generations of its spectroscopy system, which may further the method’s capabilities. “Continued technological advances in the field which increase the output power and signal-to-noise ratio of the systems,” Reid said, “may enable smaller resolutions … and increase the potential of the technique in tissue analysis.” Seeking the cirrhotic liver Imaging techniques such as MRI and ultrasound often can help locate atypical tissue structures, but a biopsy may be required to diagnose the problematic structure. Waiting for the results of a biopsy can be troubling for both patient and physician alike; for example, biopsies are inherently invasive and leave patients subject to complications of surgery. Disease such as liver cirrhosis can be determined using terahertz spectroscopy setups coupled to imaging probes. Photo courtesy of TeraView Ltd. One of Reid’s colleagues, Emma Pickwell-MacPherson, leads her own terahertz research group at the Hong Kong University of Science and Technology. Her team is interested in investigating the contrast mechanisms of various diseases, including liver cirrhosis. In liver cirrhosis, normal tissue is damaged and replaced by scars, fibrosis and nodules, unhealthy features that adversely affect the liver’s ability to function properly. As with many diseases, liver cirrhosis is best treated as soon as possible, which requires early diagnosis. However, Pickwell-MacPherson’s group reported in the Dec. 21, 2010, issue of Physics in Medicine and Biology that current methods are not sensitive or specific enough to detect occult liver injury at either early or even intermediary stages. Using a TeraView TPI Imaga1000 combined with a handheld probe, the researchers tested samples comprising healthy and cirrhotic rat liver tissue. They measured reflections off both samples freshly excised from the animals and from some of the same tissues after fixing them in formalin. They found that both the fresh and the fixed cirrhotic samples exhibited higher refractive indices and absorption coefficients than their healthy counterparts. The formalin-fixed tissues, however, had lower absolute values for both healthy and diseased tissues. Furthermore, they found that cirrhotic liver tissue had a higher water content than healthy tissue. The blue line indicates the absorption coefficient of freshly excised cirrhotic rat tissue, minus that of the fresh healthy tissue. The pink line represents the same calculation for samples that were later fixed in formalin. Thus, the area labeled “A” predominantly represents the difference in absorption due to water, whereas area “B” represents the difference due to structural changes, such as nodules and fibrosis. They also discovered that structural changes in cirrhotic tissues are picked up by the terahertz technique. The diseased rats “had evidence of biliary cirrhosis and nodular liver with intense ductular proliferation, fibrotic bridges and more pronounced inflammatory infiltration,” Pickwell-MacPherson said. “These changes in the diseased tissue structure are the origin of the structural differences that we are seeing.” The investigators concluded that terahertz spectroscopy shows promise for disease diagnosis, especially as a complement to other techniques. “Terahertz imaging and spectroscopy could ultimately be used intra-operatively to help surgeons determine the margins of [a] tumor to be removed,” Pickwell-MacPherson said. “Perhaps, as the technology develops, it will be possible to use a terahertz endoscope for diagnosis of diseases such as colon cancer.”