Journal of Biomedical Optics
Thayer School of Engineering
Geisel School of Medicine
Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX.
Fluorescence tomography provides a tool for preclinical molecular contrast agent assessment in oncology.1, 2, 3, 4 Systems have advanced in complexity to where noncontact imaging,5 automated boundary recovery,6 and sophisticated internal tissue shapes can be included in the recovered images. The translation of this work to humans will require molecular contrast agents that are amenable to regulatory approval and maintain tumor specificity in humans, where often nonspecific uptake of molecular imaging agents can decrease their utility. In this study, a new fluorescence tomography system coupled to microCT7 was used to illustrate diagnostic detection of orthotopic glioma tumors that were not apparent in the microCT images, using endogenous fluorescent contrast from protoporphyrin IX (PpIX).
Glioma tumors provide significant endogenous fluorescence from PpIX,8, 9, 10, 11 and this is enhanced when the subject imaged has been administered aminolevulinic acid (ALA). The endogenous production process of PpIX is known to stem from the administered, ALA bypassing the regulatory inhibition of ALA synthase, allowing the heme synthesis pathway to proceed uninhibited. Since there is a limited supply of iron in the body, this process produces overabundance of PpIX rather than heme, and many tumors have been shown to have high yields of PpIX. Clinical trials with PpIX fluorescence guided resection of tumors have shown significant promise,12 and yet deep tissue imaging with PpIX fluorescence has not been exploited in clinical use. Early studies have shown that detection of these tumors with PpIX is feasible,13, 14 but no tomographic imaging has been used. This limitation in development has largely been caused by problems in wavelength filtering and low signal intensity, as well as background fluorescence from the skin limiting sensitivity to deeper structures. In the system developed and used here, this feasibility is demonstrated by imaging a human xenograft glioma model.
To solve the sensitivity problem and study the ability to diagnostically image PpIX in vivo, time-correlated single-photon counting was used in the fluorescence tomography system, which provides maximum sensitivity. Figure 1a shows the system designed to match up with a microCT, allowing both x-ray structural and optical functional imaging sequentially. Lens-coupled detection of signals is acquired from the mouse using five time-resolved photomultiplier tubes (H7422P-50, Hamamatsu, Japan) with single-photon counting electronics (SPC-134 modules, Becker and Hickl GmbH, Germany). The system has fan-beam transmission geometry similar to a standard CT scanner, with single source delivery of a1-mW" role="presentation">1-mW
pulsed diode laser light at 635nm" role="presentation">635nm , collimated to a 1-mm" role="presentation">1-mm effective area on the animal. The five detection lenses were arranged in an arc, each with 22.5-deg" role="presentation">22.5-deg angular separation, centered directly on the opposite side of the animal with long working distance pickup,7 allowing noncontact measurement of the diffuse light through the animal. The diffuse intensity signals collected at each of the five channels were then translated via 400-μm" role="presentation">400-μm fibers and split using beamsplitters to be directed toward the fluorescence (95%) and transmission (5%) channel detectors. A 650-nm" role="presentation">650-nm long-pass filter was used in the fluorescence channels to isolate the signal, and in the transmitted intensity signals, a neutral density filter (2 OD) was used to attenuate the signals. This latter filtering was necessary to ensure that the fluorescence and transmission. Intensity signals fell within the same dynamic range, allowing a single 1s" role="presentation">1s acquisition for each detector. Scans were then performed by rotating the fan-beam around the specimen to 32 locations. A GE eXplore Locus SP scanner (GE Healthcare, London, Ontario, Canada) that incorporated a detector with 94-micronpixel" role="presentation">94-micronpixel resolution, a 80-kV" role="presentation">80-kV peak voltage, and a tube current of 450μAs" role="presentation">450μAs , was used in acquiring the microCT data, as displayed in Fig. 2 . In this example, since soft tissue was being imaged, the CT data was largely used to image the exterior of the animal, although in future studies, it could be used to isolate the cranium region as well.
Kepshire DS, Gibbs-Strauss SL, O'Hara JA, Hutchins M, Mincu N, Leblond F, Khayat M, Dehghani H, Srinivasan S, Pogue BW. Imaging of glioma tumor with endogenous fluorescence tomography. J Biomed Opt. 2009 May-Jun;14(3):030501. doi: 10.1117/1.3127202. Erratum in: J Biomed Opt. 2009 May-Jun;14(3):039802. Gibbs-Strauss, Summer L [corrected to Gibbs-Struass, Summer L]. PMID: 19566285; PMCID: PMC2810635.
Dartmouth Digital Commons Citation
Kepshire, Dax S.; Gibbs-Strauss, Summer L.; O'Hara, Julia A.; Hutchins, Michael; Mincu, Niculae; Leblond, Frederic; Khayat, Mario; Dehghani, Hamid; Srinivasan, Subhadra; and Pogue, Brian W., "Imaging of Glioma Tumor with Endogenous Fluorescence Tomography" (2009). Dartmouth Scholarship. 3635.