Modulating tissue temperature for high-resolution, in vivo fluorescence tomography
As a molecular imaging modality, optical fluorescence imaging can provide the distribution of molecular probes in vivo using non-ionizing radiation and low-cost instrumentation.1–4 Although fluorescence imaging technology is slowly moving into the clinical arena, it has already found its niche in small imaging research, not only for a better understanding of the fundamental molecular biologic and biochemical nature of various diseases, but also for development of new contrast agents and pathway-specific imaging probes. Nevertheless, the main barrier preventing the widespread use of 3D fluorescence tomography (FT) has been its low resolution and quantitative accuracy.
Small animal fluorescence optical imaging systems became commercially available nearly a decade ago, with their number increasing every year. However, most systems can only provide 2D projection images. Research labs, including ours, have made extensive efforts to develop 3D tomographic, whole-body animal imaging systems. While several commercial FT systems have recently started to appear in the market, high tissue scattering causes blurring in the images and degrades the resolution and quantitative accuracy, especially in 3D tomographic mode.5, 6 Several alternative approaches have been implemented to improve the performance of FT.7–10 We believe our approach is unique in that we use novel temperature-sensitive fluorescent agents together with focused ultrasound.
Our technique is called temperature-modulated FT (TM-FT). It uses temperature modulation of the fluorescence quantum efficiency of temperature-sensitive contrast agents, or what we call thermodots. The medium is irradiated with both light and high-intensity, low-power, focused ultrasound (HIFU) waves. The key benefit of HIFU is the modulation of the medium temperature (∼4°C) for a very short duration (∼1 second) with a very high spatial resolution (<1mm). When the thermodots are present within the HIFU focal zone, the temperature increase in turn alters the quantum efficiency of the agents. As a result, both emitted fluorescence light intensity and lifetime have a detectable change only when thermodots are present within the focal zone (see Figure 1). In another words, TM-FT allows fluorescence imaging with high spatial resolution by scanning a focused ultrasound column over the medium while detecting the variation in the emitted fluorescence signal. Moreover, the HIFU transducer can generate these hot spots up to a depth of 6cm. Since the spatial resolution is mainly determined by the spot size of HIFU, high spatial resolution (<1mm) can be achieved, even for thick tissue.
Our collaborators at InnoSense LLC (Torrance, CA) synthesized the thermodots by encapsulating indocyanine green (ICG) in Pluronic-127 polymeric micelles. In order to verify their temperature response, we recorded the fluorescence intensity and lifetime while increasing the temperature. Later, a phantom study validated the feasibility of the TM-FT method.11, 12 We immersed a 4cm × 10cm × 10cm slab of agar phantom in a water tank. A HIFU transducer (the H102 from Sonic Concepts Inc. of Bothell, WA) with a center frequency of 1.1MHz was mounted on an x-y-z translational stage and used to generate a focused hot spot. The transducer was scanned laterally in both the x and y directions above the phantom. Fluorescence signals were recorded using an FT system that we developed in our lab.13 We averaged four measurements, which yielded an acquisition time of two seconds for a particular transducer position. We embedded a 3mm fluorescence inclusion filled with thermodots into the middle of the phantom. Intralipid and India ink were added in order to set the phantom's reduced scattering and absorption coefficient to 0.005mm−1 and 0.6mm−1, respectively. Figure 2a shows the actual size, position and concentration of the inclusion. During the measurements, HIFU was scanned over an 8mm × 8mm area with 1mm step (see Figure 2b). For each step, the HIFU was turned on for 2 seconds, and the power was adjusted to keep the temperature at the focal spot below 40°C. The change in both fluorescence signal amplitude and phase was mapped to each scanning position. Both amplitude and phase varied significantly when we scanned the HIFU hot spot through the thermodots (see Figure 2c and 2d), resulting in a much improved spatial resolution. As seen from the profiles across the fluorescence inclusion (see Figure 2e and 2f), the full-width half-maximum of the inclusion recovered from both intensity and lifetime maps was the same, 3.2mm.
In summary, we successfully observed a temperature-modulated fluorescence signal in scattering medium with HIFU resolution. This technique has a high potential for clinical applications. For example, components of the thermodots, ICG and Pluronic-127 are U.S. Food and Drug Administration-approved materials. Thermodots can also be modified to target different molecular pathways and processes for true molecular imaging. Besides obtaining fluorescence images at focused ultrasound resolution, TM-FT can also render quantitatively accurate images using a proper reconstruction algorithm. This will be an avenue that we will pursue in the near future.
This research is supported in part by the National Institutes of Health grants R01EB008716, R21/33 CA120175, R01CA1429898, and P30CA062203 and by the Susan G. Komen Foundation training grant KG101442.
Gultekin Gulsen is an associate professor in the Departments of Radiological Sciences, Physics, Biomechanical Engineering, and Electrical Engineering and Computer Science. His team has been working on in vivo, cutting-edge optical imaging techniques for more than a decade. His long-term goal is translating these novel techniques into clinical settings.
University of California, Irvine
Yuting Lin is an assistant project researcher in the Department of Radiological Sciences. She has extensive experience in integrating fluorescence tomography with x-ray, computerized tomography, and magnetic resonance imaging, and a solid theoretical and computational physics background with experience in reconstruction algorithms. Recently, she has been leading the development of the TM-FT technique described in this article.
Tiffany C. Kwong is a second-year physics graduate student who has been involved in the development of the TM-FT technique.
Uma Sampathkumaran, vice president, RD graduated with a PhD in materials science and engineering from the Indian Institute of Technology, Bombay. Her work focuses on designing and applying nanoscale materials for optochemical sensing, bio-imaging, anti-fog, and hard coatings for protective eyewear, as well as refractory aerogels for rare isotope harvesting.
Shaaz Ahmed graduated with a BS in Biochemistry from the University of California, Los Angeles in 2010. Since joining the company in 2011, she has worked in projects focused on developing and using nanoscale materials in optical coatings, imaging, and insulation applications. She is also experienced in materials synthesis techniques.
