title: Three-dimensional imaging of xenograft tumors using optical computed and emission tomography.
abstract: The physical basis and preliminary applications of optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) are introduced, as new techniques with potential to provide unique 3D information on a variety of aspects of tumor structure and function. A particular focus here is imaging tumor micro-vasculature, and the spatial distribution of viable tumor cells, although the techniques have the potential for much wider application. The principle attractiveness of optical-CT and optical-ECT are that high resolution (<20 microm) and high contrast co-registered 3D images of structure and function can be acquired for relatively large intact samples. The unique combination of high contrast and resolution offers advantages over micro-CT and micro-MRI, and the lack of requirement for sectioning offers advantages over confocal microscopy, conventional microscopy, and histological sectioning techniques. Optical-CT/ECT are implemented using in-house custom apparatus and a commercial dissecting microscope capable of both transmission and fluorescence imaging. Basic studies to characterize imaging performance are presented. Negligible geometrical distortion and accurate reconstruction of relative attenuation coefficients was observed. Optical-CT and optical-ECT are investigated here by application to high resolution imaging of HCT116 xenograft tumors, about 1 cc in dimension, which were transfected with constitutive red fluorescent protein (RFP). Tumor microvasculature was stained in vivo by tail vein injection of either passive absorbing dyes or active fluorescent markers (FITC conjugated lectin). Prior to imaging, the tumors were removed (ex vivo) and optically cleared in a key process to make the samples amenable to light transmission. The cleared tumors were imaged in three modes (i) optical-CT to image the 3D distribution of microvasculature as indicated by absorbing dye, (ii) optical-ECT using the FITC excitation and emission filter set, to determine microvasculature as indicated by lectin-endothelial binding, and (iii) optical-ECT using the DSRed2 filter set to determine the 3D distribution of viable tumor as indicated by RFP emission. A clear correlation was observed between the independent vasculature imaging modes (i) and (ii) and postimaging histological sections, providing substantial validation of the optical-CT and optical-ECT techniques. Strong correlation was also observed between the RFP imaging of mode iii, and modes i and ii, supporting the intuitive conclusion that well-perfused regions contain significant viable tumor. In summary, optical-CT and optical-ECT, when combined with new optical clearing techniques, represent powerful new imaging modalities with potential for providing unique information on the structure and function of tumors.
authors.affiliation: Department of Radiation Oncology Physics, and Biomedical Engineering, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Oldham
authors.forename: Harshad
authors.initials: H
authors.lastname: Sakhalkar
authors.forename: Tim
authors.initials: T
authors.lastname: Oliver
authors.forename: Ying Min
authors.initials: YM
authors.lastname: Wang
authors.forename: John
authors.initials: J
authors.lastname: Kirpatrick
authors.forename: Yiting
authors.initials: Y
authors.lastname: Cao
authors.forename: Cristian
authors.initials: C
authors.lastname: Badea
authors.forename: G Allan
authors.initials: GA
authors.lastname: Johnson
authors.forename: Mark
authors.initials: M
authors.lastname: Dewhirst
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000473:pathology*; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013382:Subtraction Technique; D015899:Tomography, Emission-Computed, Single-Photon; D041622:Tomography, Optical / Q000295:instrumentation / Q000379:methods*; D014057:Tomography, X-Ray Computed