tocFluorescent Protein Tomography Scanner for Small Animal ImagingGiannis Zacharakis*, Jorge Ripoll, Ralph Weissleder, and VasilisI. I NTRODUCTIONII. T HEORYFig.€1. Changes in percentage in the absorption-dependent coeffiFig.€2. (a) Schematic representation of the experimental setup sIII. M ATERIALS AND M ETHODSA. Optical Scanning SetupB. Measurements and Image ReconstructionC. Reconstruction ParametersD. Phantoms and Animal ProceduresIV. R ESULTSA. Stability and Reproducibility Measurements of the Optical SysFig.€3. The algorithmic structure used for image reconstruction.B. Tomographic LocalizationFig.€4. Images representing cross sections of two glass tubes coFig.€5. Images comparing trans- with epiillumination. A thin glaC. Tomography and Quantification in TissuesFig.€6. Quantification accuracy of the modality when imaging fluFig.€7. Coronal slices of the reconstructed images correspondingV. D ISCUSSIONR. Weissleder and V. Ntziachristos, Shedding light into live molF. R. Wouters, P. J. Verveer, and P. I. H. Bastiaens, Imaging biM. Yang, E. Baranov, P. Jiang, F. Sun, X. Li, L. Li, S. HasegawaE. B. Brown, R. B. Campell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. M. Yang, E. Baranov, A. R. Moossa, S. Penman, and R. M. Hoffman,R. M. Hoffman, Green fluorescent protein imaging of tumour growtD. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. KilmeV. Ntziachristos, A. G. Yodh, M. Schnall, and B. Chance, ConcurrB. Pogue, S. P. Poplack, T. McBride, W. Wells, K. Osterman, U. OY. Bluestone, G. Abdoulaev, C. H. Schmitz, R. L. Barbour, and A.V. Ntziachristos, C. Tung, C. Bremer, and R. Weissleder, FluoresA. D. Klose and A. H. Hielscher, Iterative reconstruction schemeR. Aronson and N. Corngold, Photon diffusion coefficient in an aJ. Ripoll, D. Yessayan, G. Zacharakis, and V. Ntziachristos, ExpV. Ntziachristos and R. Weissleder, Experimental three-dimensionR. Aronson, Boundary conditions for the diffusion of light, J. OM. S. Patterson, B. Chance, and B. C. Wilson, Time-resolved reflA. P. Soubret, J. Ripoll, D. Yessayan, and V. Ntziachristos . ThV. Ntziachristos and R. Weissleder, CCD-based scanner for tomogrC. Kak and M. Slaney, Principles of Computerized Tomographic ImaJ. P. Culver, V. Ntziachristos, M. J. Holboke, and A. G. Yodh, OA. P. Soubret, J. Ripoll, and V. Ntziachristos, Accuracy of fluoE. E. Graves, J. Ripoll, R. Weissleder, and V. Ntziachristos, A J. C. Hedben, Evaluating the spatial resolution performance of aT. Troy, D. Jekic-McMullen, L. Sambucetti, and B. Rice, QuantitaM. Yang, L. Li, P. Jiang, A. R. Moossa, S. Penman, and R. M. HofT. Zimmermann, J. Rietdorf, and R. Pepperkok, Spectral imaging aJ. Ripoll, V. Ntziachristos, R. Carminati, and M. Nieto-VesperinJ. Ripoll, R. B. Schulz, and V. Ntziachristos, Free-Space propag878 IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 24, NO. 7, JULY 2005Fluorescent Protein Tomography Scannerfor Small Animal ImagingGiannis Zacharakis*, Jorge Ripoll, Ralph Weissleder, and Vasilis NtziachristosAbstract—Microscopy of fluorescent proteins has enabled un-precedented insights into visualizing gene expression in living sys-tems. Imaging deeper into animals, however, has been limited dueto the lack of accurate imaging methods for the visible. We presenta novel system designed to perform tomographic imaging of fluo-rescent proteins through whole animals. The tomographic methodemployed a multiangle, multiprojection illumination scheme, whiledetection was achieved using a highly sensitive charge-coupled de-vice camera with appropriate filters. Light propagation was mod-eled using a modified solution to the diffusion equation to accountfor the high absorption and high scattering of tissue at the visiblewavelengths. We show that the technique can quantitatively detectfluorescence with sub millimeter spatial resolution both in phan-toms and in tissues. We conclude that the method could be appliedin tomographic imaging of fluorescent proteins for in vivo targetingof different diseases and abnormalities.Index Terms—Diffusion theory, fluorescence tomography, vis-ible light, whole-body imaging.I. INTRODUCTIONFLUORESCENCE proteins (FPs) have become essentialreporter molecules for tagging gene-expression in cellsand applied in a large variety of biomedical applications. Theycan be engineered so that they are expressed in specific cellsonly, and they exhibit constant emission independent on the cellfunction and proliferation covering the green and red spectralrange (500–630 nm) [1]–[3]. Engineered FPs are typicallydetected microscopically by epifluorescence, or confocal andmultiphoton microscopy [4], [5]. Microscopy is a powerfulmodality for studying FP expression with high resolution butis limited in depths of a few hundred micrometersin vivo.Imaging at the animal level would be very important in manyapplications such as in monitoring tumor growth and metastasisof various types of cancer (i.e., prostate, brain, breast, andManuscript received July 15, 2004; revised December 21, 2004.This work was supported in part by the National Institutes of Healthunder Grants RO1 EB 000750-1 and R33 CA 91807. The work ofJ. Ripoll was supported by the European Union under Integrated Project“Molecular Imaging” LSHG-CT-2003-503259 and STREP “TRANS-REG”LSHG-CT-2004-502950. The Associate Editor responsible for coordinatingthe review of this paper and recommending its publication was J. Basilion.Asterisk indicates corresponding author.*G. Zacharakis is with the Center for Molecular Imaging Research, Massa-chusetts General Hospital, Harvard Medical School, Charlestown, MA 02129USA (e-mail: [email protected]).J. Ripoll is with the Institute of Electronic Structure and Laser, Foundationfor Research and Technology-Hellas, GR 711 10 Heraklion, Greece (e-mail:[email protected]).R. Weissleder is with the Center for Molecular Imaging Research, Massachu-setts General Hospital, Harvard Medical School, Charlestown, MA 02129 USA.V. Ntziachristos is with the Center for Molecular Imaging Research, Mass-achusetts General Hospital, Harvard Medical School, Charlestown, MA 02129USA (e-mail: [email protected]).Digital Object Identifier 10.1109/TMI.2004.843254lung cancer), in cell trafficking (i.e., lymphocytes homing totumor sites and cancer cells metastasizing to lymph nodes), inangiogenesis imaging, and in many others [5], [6]. However,whole animal imaging is currently limited to planar illumi-nation (reflectance) imaging, i.e., a photography using