Near‐Infrared Molecular Probes for In Vivo Imaging

Xuan Zhang1, Sharon Bloch1, Walter Akers1, Samuel Achilefu1

1 Department of Radiology, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.27
DOI:  10.1002/0471142956.cy1227s60
Online Posting Date:  April, 2012
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Cellular and tissue imaging in the near‐infrared (NIR) wavelengths between 700 and 900 nm is advantageous for in vivo imaging because of the low absorption of biological molecules in this region. This unit presents protocols for small animal imaging using planar and fluorescence lifetime imaging techniques. Included is an overview of NIR fluorescence imaging of cells and small animals using NIR organic fluorophores, nanoparticles, and multimodal imaging probes. The development, advantages, and application of NIR fluorescent probes that have been used for in vivo imaging are also summarized. The use of NIR agents in conjunction with visible dyes and considerations in selecting imaging agents are discussed. We conclude with practical considerations for the use of these dyes in cell and small animal imaging applications. Curr. Protoc. Cytom. 60:12.27.1‐12.27.20. © 2012 by John Wiley & Sons, Inc.

Keywords: in vivo imaging; in vitro imaging; near‐infrared fluorescent probe; confocal microscopy; fluorescence lifetime imaging

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Table of Contents

  • Introduction
  • Basic Protocol 1: Planar NIR Fluorescence Imaging
  • Basic Protocol 2: NIR Fluorescence Lifetime Imaging
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Planar NIR Fluorescence Imaging

  • Mice (15 to 25 g)
  • Anesthesia (e.g., 2% isoflurane by nosecone or injectable such as 100 mg/kg ketamine, 10 mg/kg xylazine cocktail)
  • Fluorescent molecular probes (e.g., ProSense750, Perkin Elmer; IRDye 800CW 2‐DG, LiCor Biosciences)
  • CCD‐based preclinical fluorescence reflectance imaging (FRI) system (e.g., In‐Vivo MS FX PRO Carestream Health)
  • Broadband (Xenon) light source
  • Excitation bandpass filters
  • Emission bandpass filters
  • Light‐tight imaging chamber with animal support systems (anesthesia, warming, positioning)
  • Digital camera, typically cooled to reduce electronic noise
  • Software for acquisition and analysis of images (e.g., Molecular Imaging Software, Carestream Health)
  • Warming pad for recovery
  • Clear disposable petri dishes

Basic Protocol 2: NIR Fluorescence Lifetime Imaging

  • Mice (15 to 25 g)
  • Anesthesia (e.g., 2% isoflurane by nosecone or injectable such as 100 mg/kg ketamine, 10 mg/kg xylazine cocktail)
  • Fluorescent molecular probes
  • Preclinical time‐domain diffuse optical imaging system (e.g., Optix MX2, Advanced Research Technologies)
  • 670‐nm and 780‐nm laser excitation source
  • 693‐nm longpass filter and 850‐ ± 12 nm bandpass filter
  • Fast PMT with time‐correlated single photon counting detection
  • Software for acquisition and analysis of images (e.g., Optiview, ART)
  • Warming pad for recovery
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Literature Cited

Literature Cited
   Achilefu, S. 2010. The insatiable quest for near‐infrared fluorescent probes for molecular imaging. Angew. Chem. Int. Ed. 49:9816‐9818.
   Achilefu, S., Dorshow, R.B., Bugaj, J.E., and Rajagopalan, R. 2000. Novel receptor‐targeted fluorescent contrast agents for in vivo tumor imaging. Invest. Radiol. 35:479‐485.
   Achilefu, S., Bloch, S., Markiewicz, M.A., Zhong, T.X., Ye, Y.P., Dorshow, R.B., Chance, B., and Liang, K.X. 2005. Synergistic effects of light‐emitting probes and peptides for targeting and monitoring integrin expression. Proc. Natl. Acad. Sci. U.S.A. 102:7976‐7981.
   Akers, W.J., Kim, C., Berezin, M., Guo, K., Fuhrhop, R., Lanza, G.M., Fischer, G.M., Daltrozzo, E., Zumbusch, A., Cai, X. Wang, L.V., and Achilefu, S. 2011. Noninvasive photoacoustic and fluorescence sentinel lymph node identification using dye‐loaded perfluorocarbon nanoparticles. ACS Nano. 5:173‐182.
   Akers, W.J., Zhang, Z., Berezin, M., Ye, Y., Agee, A., Guo, K., Fuhrhop, R.W., Wickline, S.A., Lanza, G.M., and Achilefu, S. 2010. Targeting of alpha(nu)beta(3)‐integrins expressed on tumor tissue and neovasculature using fluorescent small molecules and nanoparticles. Nanomedicine 5:715‐726.
   Alian, W., Andersson‐Engels, S., Svanberg, K., and Svanberg, S. 1994. Laser‐induced fluorescence studies of meso‐tetra(hydroxyphenyl)chlorin in malignant and normal tissues in rats. Br. J. Cancer 70:880‐885.
   Altinoğlu, E.I., Russin, T.J., Kaiser, J.M., Barth, B.M., Eklund, P.C., Kester, M., and Adair, J.H. 2008. Near‐infrared emitting fluorophore‐doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. ACS Nano. 2:2075‐2084.
   Andersson‐Engels, S., Ankerst, J., Johansson, J., Svanberg, K., and Svanberg, S. 1993. Laser‐induced fluorescence in malignant and normal tissue of rats injected with benzoporphyrin derivative. Photochem. Photobiol. 57:978‐983.
   Arunkumar, E., Forbes, C.C., Noll, B.C., and Smith, B.D. 2005. Squaraine‐derived rotaxanes: Sterically protected fluorescent near‐IR dyes. J. Am. Chem. Soc. 127:3288‐3289.
   Arunkumar, E., Fu, N., and Smith, B.D. 2006. Squaraine‐derived rotaxanes: Highly stable, fluorescent near‐IR dyes. Chemistry 12:4684‐4690.
   Bakshi, M.S., Sharma, P., Banipal, T.S., Kaur, G., KanjiroTorigoe, Petersen N.O., and Possmayer, F. 2007. Lamellar phase supported synthesis of colloidal gold nanoparticles, nanoclusters, and nanowires. J. Nanosci. Nanotechnol. 7:916‐924.
   Ballou, B., Ernst, L.A., and Waggoner, A.S. 2005. Fluorescence imaging of tumors in vivo. Curr. Med. Chem. 12:795‐805.
   Berezin, M.Y., Akers, W.J., Guo, K., Fischer, G.M., Daltrozzo, E., Zumbusch, A., and Achilefu, S. 2009. Long fluorescence lifetime molecular probes based on near infrared pyrrolopyrrole cyanine fluorophores for in vivo imaging. Biophys. J. 97:L22‐L24.
   Bremer, C., Tung, C.H., and Weissleder, R. 2001. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7:743‐748.
   Bugaj, J.E., Achilefu, S., Dorshow, R.B., and Rajagopalan, R. 2001. Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor‐targeted dye‐peptide conjugate platform. J. Biomed. Opt. 6:122‐133.
   Cai, W., Shin, D.W., Chen, K., Gheysens, O., Cao, Q., Wang, S.X., Gambhir, S.S., and Chen, X. 2006. Peptide‐labeled near‐infrared quantum dots for imaging tumor vasculature in living subjects. Nano. Lett. 6:669‐676.
   Cheng, Z., Wu, Y., Xiong, Z.M., Gambhir, S.S., and Chen, X.Y. 2005. Near‐infrared fluorescent RGD peptides for optical imaging of integrin alpha(v)beta 3 expression in living mice. Bioconjugate Chem. 16:1433‐1441.
   Chopra, A. 2004. Humanized anti‐type 1 insulin‐like growth factor receptor monoclonal antibody conjugated to Alexa 680. In: Molecular Imaging and Contrast Agent Database (MICAD) [database online]. Bethesda (MD): National Library of Medicine (US), NCBI; 2004‐2012. Available from:
   Conway, C.L., Walker, I., Bell, A., Roberts, D.J., Brown, S.B., and Vernon, D.I. 2008. In vivo and in vitro characterisation of a protoporphyrin IX‐cyclic RGD peptide conjugate for use in photodynamic therapy. Photochem. Photobiol. Sci. 7:290‐298.
   Cooper, M.E., Gregory, S., Adie, E., and Kalinka, S. 2002. pH‐sensitive cyanine dyes for biological applications. J. Fluorescence 12:425‐429.
   Costantino, L., Gandolfi, F., Tosi, G., Rivasi, F., Vandelli, M.A., and Forni, F. 2005. Peptide‐derivatized biodegradable nanoparticles able to cross the blood‐brain barrier. J. Control. Rel. 108:84‐96.
   Cubeddu, R., Pifferi, A., Taroni, P., Torricelli, A., Valentini, G., Comelli, D., D'Andrea, C., Angelini, V., and Canti, G. 2000. Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine. Photochem. Photobiol. 72:690‐695.
   Diagaradjane, P., Orenstein‐Cardona, J.M., Colon‐Casasnovas, N.E., Deorukhkar, A., Shentu, S., Kuno, N., Schwartz, D.L., Gelovani, J.G., and Krishnan, S. 2008. Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe. Clin. Cancer Res. 14:731‐741.
   Dijkgraaf, I., Beer, A.J., and Wester, H.J. 2009. Application of RGD‐containing peptides as imaging probes for alphavbeta3 expression. Front Biosci. 14:887‐899.
   Dorozhkin, S.V. and Epple, M. 2002. Biological and medical significance of calcium phosphates. Angew. Chem. Int. Ed. Engl. 41:3130‐3146.
   Dougherty, T.J., Gomer, C.J., and Weishaupt, K.R. 1976. Energetics and efficiency of photoinactivation of murine tumor cells containing hematoporphyrin. Cancer Res. 36:2330‐2333.
   Escobedo, J.O., Rusin, O., Lim, S., and Strongin, R.M. 2010. NIR dyes for bioimaging applications. Curr. Opin. Chem. Biol. 14:64‐70.
   Fischer, G.M., Isomaki‐Krondahl, M., Gottker‐Schnetmann, I., Daltrozzo, E., and Zumbusch, A. 2009. Pyrrolopyrrole cyanine dyes: A new class of near‐infrared dyes and fluorophores. Chemistry 15:4857‐4864.
   Fischer, G.M., Jungst, C., Isomaki‐Krondahl, M., Gauss, D., Moller, H.M., Daltrozzo, E., and Zumbusch, A. 2010. Asymmetric PPCys: Strongly fluorescing NIR labels. Chem. Commun. 46:5289‐5291.
   Funovics, M., Weissleder, R., and Tung, C.H. 2003. Protease sensors for bioimaging. Anal. Bioanal. Chem. 377:956‐963.
   Gao, J., Chen, K., Xie, R., Xie, J., Lee, S., Cheng, Z., Peng, X., and Chen, X. 2010. Ultrasmall near‐infrared non‐cadmium quantum dots for in vivo tumor imaging. Small 6:256‐261.
   Ghoroghchian, P.P., Frail, P.R., Susumu, K., Blessington, D., Brannan, A.K., Bates, F.S., Chance, B., Hammer, D.A., and Therien, M.J. 2005. Near‐infrared‐emissive polymersomes: self‐assembled soft matter for in vivo optical imaging. Proc. Natl. Acad. Sci. U.S.A. 102:2922‐2927.
   Gompels, L.L., Madden, L., Lim, N.H., Inglis, J.J., McConnell, E., Vincent, T.L., Haskard, D.O., and Paleolog, E.M. 2011. In vivo fluorescence imaging of E‐selectin: Quantitative detection of endothelial activation in a mouse model of arthritis. Arthritis Rheum. 63:107‐117.
   Gurfinkel, M., Thompson, A.B., Ralston, W., Troy, T.L., Moore, A.L., Moore, T.A., Gust, J.D., Tatman, D., Reynolds, J.S., Muggenburg, B., Nikula, K., Pandey, R., Mayer, R.H., Hawrysz, D.J., and Sevick‐Muraca, E.M. 2000. Pharmacokinetics of ICG and HPPH‐car for the detection of normal and tumor tissue using fluorescence, near‐infrared reflectance imaging: A case study. Photochem. Photobiol. 72:94‐102.
   Hama, Y., Koyama, Y., Urano, Y., Choyke, P.L., and Kobayashi, H. 2007. Simultaneous two‐color spectral fluorescence lymphangiography with near infrared quantum dots to map two lymphatic flows from the breast and the upper extremity. Breast Cancer Res Treat. 103:23‐28.
   Hardman, R. 2006. A toxicologic review of quantum dots: Toxicity depends on physicochemical and environmental factors. Environ. Health Perspect. 114:165‐172.
   He, X., Gao, J., Gambhir, S.S., and Cheng, Z. 2010a. Near‐infrared fluorescent nanoprobes for cancer molecular imaging: Status and challenges. Trends Mol. Med. 16:574‐583.
   He, X., Wang, K., and Cheng, Z. 2010b. In vivo near‐infrared fluorescence imaging of cancer with nanoparticle‐based probes. WIREs Nanomed. Nanobiotechnol. 2:349‐366.
   He, X.X., Gao, J.H., Gambhir, S.S., and Cheng, Z. 2010. Near‐infrared fluorescent nanoprobes for cancer molecular imaging: Status and challenges. Trends Mol. Med. 16:574‐583.
   Hewett, J., Nadeau, V., Ferguson, J., Moseley, H., Ibbotson, S., Allen, J.W., Sibbett, W., and Padgett, M. 2001. The application of a compact multispectral imaging system with integrated excitation source to in vivo monitoring of fluorescence during topical photodynamic therapy of superficial skin cancers. Photochem. Photobiol. 73:278‐282.
   Hilderbrand, S.A. and Weissleder, R. 2010. Near‐infrared fluorescence: Application to in vivo molecular imaging. Curr. Opin. Chem. Biol. 14:71‐79.
   Hilderbrand, S.A., Kelly, K.A., Niedre, M., and Weissleder, R. 2008. Near infrared fluorescence‐based bacteriophage particles for ratiometric pH imaging. Bioconjug. Chem. 19:1635‐1639.
   Hong, H., Zhang, Y., and Cai, W. 2010. In vivo imaging of RNA interference. J. Nucl. Med. 51:169‐172.
   Huang, C.C., Chen, C.T., Shiang, Y.C., Lin, Z.H., and Chang, H.T. 2009. Synthesis of fluorescent carbohydrate‐protected Au nanodots for detection of Concanavalin A and Escherichia coli. Anal. Chem. 81:875‐882.
   Jin, T., Fujii, F., Komai, Y., Seki, J., Seiyama, A., and Yoshioka, Y. 2008. Preparation and characterization of highly fluorescent, glutathione‐coated near infrared quantum dots for in vivo fluorescence imaging. Int. J. Mol. Sci. 9:2044‐2061.
   Johnson, J.R., Fu, N., Arunkumar, E., Leevy, W.M., Gammon, S.T., Piwnica‐Worms, D., and Smith, B.D. 2007. Squaraine rotaxanes: Superior substitutes for Cy‐5 in molecular probes for near‐infrared fluorescence cell imaging. Angew. Chem. Int. Ed. Engl. 46:5528‐5531.
   Ke, S., Wen, X., Gurfinkel, M., Charnsangavej, C., Wallace, S., Sevick‐Muraca, E.M., and Li, C. 2003. Near‐infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts. Cancer Res. 63:7870‐7875.
   Kim, S., Lim, Y.T., Soltesz, E.G., De Grand, A.M., Lee, J., Nakayama, A., Parker, J.A., Mihaljevic, T., Laurence, R.G., Dor, D.M., Cohn, L.H., Bawendi, M.G., and Frangioni, J.V. 2004. Near‐infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 22:93‐97.
   Kobayashi, H., and Choyke, P.L. 2011. Target‐cancer‐cell‐specific activatable fluorescence imaging probes: Rational design and in vivo applications. Acc. Chem. Res. 44:83‐90.
   Kobayashi, H., Koyama, Y., Barrett, T., Hama, Y., Regino, C.A.S., Shin, I.S., Jang, B.S., Le, N., Paik, C.H., Choyke, P.L., and Urano, Y. 2007. Multimodal nanoprobes for radionuclide and five‐color near‐infrared optical lymphatic imaging. ACS Nano. 1:258‐264.
   Komatsu, T., Kikuchi, K., Takakusa, H., Hanaoka, K., Ueno, T., Kamiya, M., Urano, Y., and Nagano, T. 2006. Design and synthesis of an enzyme activity‐based labeling molecule with fluorescence spectral change. J. Am. Chem. Soc. 128:15946‐15947.
   Kosaka, N., Mitsunaga, M., Longmire, M.R., Choyke, P.L., and Kobayashi, H. 2011. Near infrared fluorescence‐guided real‐time endoscopic detection of peritoneal ovarian cancer nodules using intravenously injected indocyanine green. Int J Cancer. 129:1671‐1677.
   Kundu, K., Knight, S.F., Willett, N., Lee, S., Taylor, W.R., and Murthy, N. 2009. Hydrocyanines: A class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. Angew. Chem. Int. Ed. Engl. 48:299‐303.
   Lakowicz, J.R. 1988. Principles of frequency‐domain fluorescence spectroscopy and applications to cell membranes. Subcell. Biochem. 13:89‐126.
   Lee, C.‐H., Cheng, S.‐H., Wang, Y.‐J., Chen, Y.‐C., Chen, N.‐T., Souris, J., Chen, C.‐T., Mou, C.‐Y., Yang, C.‐S., and Lo, L.‐W. 2009. Near‐infrared mesoporous silica nanoparticles for optical imaging: Characterization and in vivo biodistribution. Adv. Funct. Mat. 19:215‐222.
   Lee, H., Akers, W.J., Cheney, P.P., Edwards, W.B., Liang, K., Culver, J.P., and Achilefu, S. 2009. Complementary optical and nuclear imaging of caspase‐3 activity using combined activatable and radio‐labeled multimodality molecular probe. J. Biomed. Opt. 14:040507.
   Lee, H., Akers, W., Bhushan, K., Bloch, S., Sudlow, G., Tang, R., and Achilefu, S. 2011. Near‐infrared pH‐activatable fluorescent probes for imaging primary and metastatic breast tumors. Bioconjug. Chem. 22:777‐784.
   Leung, K. 2004a. Anti‐vascular cell adhesion molecule‐1 monoclonal antibody M/K‐2.7 microbubbles. In: Molecular Imaging and Contrast Agent Database (MICAD) [database online]. Bethesda (MD): National Library of Medicine (US), NCBI; 2004‐2012. Available from:
   Leung, K. 2004b. IRDye 800‐labeled anti‐epidermal growth factor receptor affibody. In: Molecular Imaging and Contrast Agent Database (MICAD) [database online]. Bethesda (MD): National Library of Medicine (US), NCBI; 2004‐2012. Available from:
   Li, L., Qian, H., and Ren, J. 2005. Rapid synthesis of highly luminescent CdTe nanocrystals in the aqueous phase by microwave irradiation with controllable temperature. Chem. Commun. 528‐530.
   Licha, K. 2002. Contrast agents for optical imaging. Topics Curr. Chem. 222:1‐29.
   Licha, K., Riefke, B., Ntziachristos, V., Becker, A., Chance, B., and Semmler, W. 2000. Hydrophilic cyanine dyes as contrast agents for near‐infrared tumor imaging: Synthesis, photophysical properties and spectroscopic in vivo characterization. Photochem. Photobiol. 72:392‐398.
   Linder, K.E., Metcalfe, E., Nanjappan, P., Arunachalam, T., Ramos, K., Skedzielewski, T.M., Tweedle, M.F., Nunn, A.D., and Swenson, R.E. 2011. Synthesis, in vitro evaluation and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and black hole quencher‐3 (BHQ‐3). Bioconjug Chem. 22:1287‐1297.
   Liu, Z., Tabakman, S., Welsher, K., and Dai, H. 2009. Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery. Nano Res. 2:85‐120.
   Ma, N., Marshall, A.F., and Rao, J. 2010. Near‐infrared light emitting luciferase via biomineralization. J. Am. Chem. Soc. 132:6884‐6885.
   Mankoff, D.A., Link, J.M., Linden, H.M., Sundararajan, L., and Krohn, K.A. 2008. Tumor receptor imaging. J. Nucl. Med. 49:S149‐S163.
   Matsumura, Y. and Maeda, H. 1986. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 46:6387‐6392.
   Meek, S.T., Nesterov, E.E., and Swager, T.M. 2008. Near‐infrared fluorophores containing benzo[c]heterocycle subunits. Org. Lett. 10:2991‐2993.
   Morgan, N.Y., English, S., Chen, W., Chernomordik, V., Russo, A., Smith, P.D., and Gandjbakhche, A. 2005. Real time in vivo non‐invasive optical imaging using near‐infrared fluorescent quantum dots. Acad. Radiol. 12:313‐323.
   Murari, K., Zhang, Y.Y., Li, S.P., Chen, Y.P., Li, M.J., and Li, X.D. 2011. Compensation‐free, all‐fiber‐optic, two‐photon endomicroscopy at 1.55 mu m. Optics Lett. 36:1299‐1301.
   Nesterov, E.E., Skoch, J., Hyman, B.T., Klunk, W.E., Bacskai, B.J., and Swager, T.M. 2005. In vivo optical imaging of amyloid aggregates in brain: design of fluorescent markers. Angew. Chem. Int. Ed. Engl. 44:5452‐5456.
   O'Connell, M.J, Bachilo, S.M., Huffman, C.B., Moore, V.C., Strano, M.S., Haroz, E.H., Rialon, K.L., Boul, P.J., Noon, W.H., Kittrell, C., Ma, J., Hauge, R.H., Weisman, R.B., and Smalley, R.E. 2002. Band gap fluorescence from individual single‐walled carbon nanotubes. Science 297:593‐596.
   Ochsner, M. 1997. Photophysical and photobiological processes in the photodynamic therapy of tumours. J. Photochem. Photobiol. B 39:1‐18.
   Ogawa, M., Kosaka, N., Choyke, P.L., and Kobayashi, H. 2009. In vivo molecular imaging of cancer with a quenching near‐infrared fluorescent probe using conjugates of monoclonal antibodies and indocyanine green. Cancer Res. 69:1268‐1272.
   Owens, S.L. 1996. Indocyanine green angiography. Br. J. Ophthalmol. 80:263‐266.
   Peng, Q., Warloe, T., Berg, K., Moan, J., Kongshaug, M., Giercksky, K.E., and Nesland, J.M. 1997. 5‐Aminolevulinic acid‐based photodynamic therapy. Clinical research and future challenges. Cancer 79:2282‐2308.
   Peng, X., Chen, H., Draney, D.R., Volcheck, W., Schutz‐Geschwender, A., and Olive, D.M. 2009. A nonfluorescent, broad‐range quencher dye for Forster resonance energy transfer assays. Anal. Biochem. 388:220‐228.
   Peng, Z.A. and Peng, X. 2001. Formation of high‐quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J. Am. Chem. Soc. 123:183‐184.
   Povrozin, Y.A., Markova, L.I., Tatarets, A.L., Sidorov, V.I., Terpetschnig, E.A., and Patsenker, L.D. 2009. Near‐infrared, dual‐ratiometric fluorescent label for measurement of pH. Anal. Biochem. 390:136‐140.
   Qian, H., Qiu, X., Li, L., and Ren, J. 2006. Microwave‐assisted aqueous synthesis: A rapid approach to prepare highly luminescent ZnSe(S) alloyed quantum dots. J. Phys. Chem. B 110:9034‐9040.
   Rao, J.H., Dragulescu‐Andrasi, A., and Yao, H.Q. 2007. Fluorescence imaging in vivo: Recent advances. Curr. Opin. Biotechnol. 18:17‐25.
   Sameiro, M. and Goncalves, T. 2009. Fluorescent labeling of biomolecules with organic probes. Chem. Rev. 109:190‐212.
   Sampath, L., Kwon, S., Ke, S., Wang, W., Schiff, R., Mawad, M.E., and Sevick‐Muraca, E.M. 2007. Dual‐labeled trastuzumab‐based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer. J. Nucl. Med. 48:1501‐1510.
   Santra, S., Wang, K., Tapec, R., and Tan, W. 2001. Development of novel dye‐doped silica nanoparticles for biomarker application. J. Biomed. Opt. 6:160‐166.
   Santra, S., Dutta, D., Walter, G.A., and Moudgil, B.M. 2005. Fluorescent nanoparticle probes for cancer imaging. Technol. Cancer Res. Treat. 4:593‐602.
   Schenke‐Layland, K., Vasilevski, O., Opitz, F., Konig, K., Riemann, I., Halbhuber, K.J., Wahlers, T., and Stock, U.A. 2003. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J. Struct. Biol. 143:201‐208.
   Schenke‐Layland, K., Riemann, I., Stock, U.A., and Konig, K. 2005. Imaging of cardiovascular structures using near‐infrared femtosecond multiphoton laser scanning microscopy. J. Biomed. Opt. 10:024017.
   Sevick‐Muraca, E.M., Houston, J.P., and Gurfinkel, M. 2002. Fluorescence‐enhanced, near infrared diagnostic imaging with contrast agents. Curr. Opin. Chem. Biol. 6:642‐650.
   Shao, X., Zheng, W., and Huang, Z. 2010. Polarized near‐infrared autofluorescence imaging combined with near‐infrared diffuse reflectance imaging for improving colonic cancer detection. Opt. Express. 18:24293‐24300.
   Smith, A.M., Duan, H., Mohs, A.M., and Nie, S. 2008. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60:1226‐1240.
   Smith, B.R., Cheng, Z., De, A., Koh, A.L., Sinclair, R., and Gambhir, S.S. 2008. Real‐time intravital imaging of RGD‐quantum dot binding to luminal endothelium in mouse tumor neovasculature. Nano. Lett. 8:2599‐2606.
   So, M.K., Loening, A.M., Gambhir, S.S., and Rao, J. 2006a. Creating self‐illuminating quantum dot conjugates. Nat. Protoc. 1:1160‐1164.
   So, M.K., Xu, C., Loening, A.M., Gambhir, S.S., and Rao, J. 2006b. Self‐illuminating quantum dot conjugates for in vivo imaging. Nat. Biotechnol. 24:339‐343.
   Tang, B., Yu, F., Li, P., Tong, L., Duan, X., Xie, T., and Wang, X. 2009. A near‐infrared neutral pH fluorescent probe for monitoring minor pH changes: Imaging in living HepG2 and HL‐7702 cells. J. Am. Chem. Soc. 131:3016‐3023.
   Tavares, A.J., Chong, L., Petryayeva, E., Algar, W.R., and Krull, U.J. 2011. Quantum dots as contrast agents for in vivo tumor imaging: Progress and issues. Anal. Bioanal. Chem. 399:2331‐2342.
   Tosi, G., Bondioli, L., Ruozi, B., Badiali, L., Severini, G.M., Biffi, S., De Vita, A., Bortot, B., Dolcetta, D., Forni, F., and Vandelli, M.A. 2011. NIR‐labeled nanoparticles engineered for brain targeting: In vivo optical imaging application and fluorescent microscopy evidences. J. Neural Transm. 118:145‐153.
   Umezawa, K., Nakamura, Y., Makino, H., Citterio, D., and Suzuki, K. 2008. Bright, color‐tunable fluorescent dyes in the visible‐near‐infrared region. J. Am. Chem. Soc. 130:1550‐1551.
   Umezawa, K., Matsui, A., Nakamura, Y., Citterio, D., and Suzuki, K. 2009. Bright, color‐tunable fluorescent dyes in the Vis/NIR region: Establishment of new “tailor‐made” multicolor fluorophores based on borondipyrromethene. Chemistry 15:1096‐1106.
   Urano, Y., Asanuma, D., Hama, Y., Koyama, Y., Barrett, T., Kamiya, M., Nagano, T., Watanabe, T., Hasegawa, A., Choyke, P.L., and Kobayashi, H. 2009. Selective molecular imaging of viable cancer cells with pH‐activatable fluorescence probes. Nat. Med. 15:104‐109.
   Wang, K., Wang, K., Li, W., Huang, T., Li, R., Wang, D., Shen, B., and Chen, X. 2009. Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near‐infrared optical imaging. Acta Radiol. 50:1095‐1103.
   Xiao, Y., Gao, X., Taratula, O., Treado, S., Urbas, A., Holbrook, R.D., Cavicchi, R.E., Avedisian, C.T., Mitra, S., Savla, R., Wagner, P.D., Srivastava, S., and He, H. 2009. Anti‐HER2 IgY antibody‐functionalized single‐walled carbon nanotubes for detection and selective destruction of breast cancer cells. BMC Cancer 9:351.
   Xie, J., Zheng, Y., and Ying, J.Y. 2009. Protein‐directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 131:888‐889.
   Xing, Y., Xia, Z., and Rao, J. 2009. Semiconductor quantum dots for biosensing and in vivo imaging. IEEE Trans. Nanobiosci. 8:4‐12.
   Yang, Y., Lowry, M., Xu, X., Escobedo, J.O., Sibrian‐Vazquez, M., Wong, L., Schowalter, C.M., Jensen, T.J., Fronczek, F.R., Warner, I.M., and Strongin, R.M. 2008. Seminaphthofluorones are a family of water‐soluble, low molecular weight, NIR‐emitting fluorophores. Proc. Natl. Acad. Sci. U.S.A. 105:8829‐8834.
   Yazdanfar, S., Joo, C., Zhan, C., Berezin, M.Y., Akers, W.J., and Achilefu, S. 2010. Multiphoton microscopy with near infrared contrast agents. J. Biomed. Opt. 15:030505.
   Ye, Y., Bloch, S., Xu, B., and Achilefu, S. 2008. Novel near‐infrared fluorescent integrin‐targeted DFO analogue. Bioconjug. Chem. 19:225‐234.
   Ye, Y.P., Bloch, S., Xu, B.G., and Achilefu, S. 2006. Design, synthesis, and evaluation of near infrared fluorescent multimeric RGD peptides for targeting tumors. J. Med. Chem. 49:2268‐2275.
   Ying, L.Q. and Branchaud, B.P. 2011. Facile synthesis of symmetric, monofunctional cyanine dyes for imaging applications. Bioconjug. Chem. 22:865‐869.
   Zhang, Z. and Achilefu, S. 2005. Design, synthesis and evaluation of near‐infrared fluorescent pH indicators in a physiologically relevant range. Chem. Commun. 5887‐5889.
   Zhang, Z., Fan, J., Cheney, P.P., Berezin, M.Y., Edwards, W.B., Akers, W.J., Shen, D., Liang, K., Culver, J.P., and Achilefu, S. 2009. Activatable molecular systems using homologous near‐infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo. Mol. Pharm. 6:416‐427.
   Zhao, H., Cui, K., Muschenborn, A., and Wong, S.T. 2008. Progress of engineered antibody‐targeted molecular imaging for solid tumors (review). Mol. Med. Report. 1:131‐134.
   Zheng, J., Petty, J.T., and Dickson, R.M. 2003. High quantum yield blue emission from water‐soluble Au8 nanodots. J. Am. Chem. Soc. 125:7780‐7781.
   Zheng, J., Nicovich, P.R., and Dickson, R.M. 2007. Highly fluorescent noble‐metal quantum dots. Annu. Rev. Phys. Chem. 58:409‐431.
   Zheng, Q., Xu, G., and Prasad, P.N. 2008. Conformationally restricted dipyrromethene boron difluoride (BODIPY) dyes: Highly fluorescent, multicolored probes for cellular imaging. Chemistry 14:5812‐5819.
   Zou, P., Xu, S., Povoski, S.P., Wang, A., Johnson, M.A., Martin, E.W. Jr., Subramaniam, V., Xu, R., and Sun, D. 2009. Near‐infrared fluorescence labeled anti‐TAG‐72 monoclonal antibodies for tumor imaging in colorectal cancer xenograft mice. Mol. Pharm. 6:428‐440.
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