螢光奈米鑽石(fluorescent nanodiamond)是一種碳系相關的新穎奈米材料，有著獨特的光學特性與優良的生物相容性，且奈米鑽石表面容易進行化學修飾。這邊我們使用高能量氦離子束轟炸100nm Type Ib的鑽石，再經過高溫焠火後，會創造出大量且帶有負電的氮-空缺(NV-)缺陷中心，缺陷中心便可以在黃綠光的激發下放出遠紅外波段的螢光。優異的光學性質，像是沒有光漂白(no photobleaching)、沒有光閃爍(no photoblinking)與無毒性，使得螢光奈米鑽石不同於一般的螢光探針。因此，螢光奈米鑽石非常適合應用於標記細胞與長時間的追蹤。

我們使用螢光奈米鑽石作為單粒子在細胞膜通道(tunneling nanotubes)與神經突(neurites)中追蹤。細胞經由細胞膜通道與神經突的傳遞溝通行為，被認為與許多疾病有關，像是愛滋病毒的傳遞、普利昂蛋白的感染、阿茲罕默症與帕金森氏症…等。為了研究傳遞的過程，我們使用表面包覆牛血清蛋白(bovine serum albumin)的螢光奈米鑽石去做標記與追蹤。細胞膜通道上的研究用的是HEK293T細胞，神經突則是用N18細胞，我們使用共軛式聚焦顯微鏡拍攝螢光奈米鑽石的傳遞，追蹤與分析影片，並計算出移動速率介於0.05 - 1μm/s。我們成功建立了一個可以追蹤非專一性奈米鑽石標記的技術，同時也為未來追蹤專一性奈米鑽石標記，這種更深入研究細胞傳遞的方式打開了大門。

Fluorescent nanodiamond (FND), a relatively new nanocarbon material, has recently emerged as a novel fluorescent probe for biological applications. The material exhibits unique optical properties and is highly biocompatible with very low toxicity. Also, the surface of FND is easy to be functionalized for specific targeting. In this work, high energy helium ion beam was used to irradiate 100-nm type Ib nanodiamonds, followed by annealing, to create a high density of negatively charged nitrogen-vacancy (NV−) defect centers. The center emits far red fluorescence under excitation by green yellow light. Excellent optical properties such as no photobleaching, no photoblinking and nontoxicity make FND distinct from conventional fluorescent probes for cell labeling and long-term tracking applications.

This work applies FNDs as a single particle tracker in tunneling nanotubes (TNTs) and neurites. The intercellular vesicles transportation through TNTs formed between cells and organelle trafficking in neurites is related to many diseases, such as HIV infection, prion protein infection, Alzheimer's disease and Parkinson's disease. In order to study the transportation, we first coated FNDs with bovine serum albumin (BSA) and then performed single particle tracking of these nanoparticle bioconjugates inside TNTs (HEK293T cells) and neurites (N18 cells) by confocal fluorescence microscopy. We analyzed the transportation of the BSA-coated FNDs individually and obtained an average speed of 0.05 to 1 μm/s in both TNTs and neurites. The success of these experiments opens new ways to explore cellular transports in detail by using specifically labeled FNDs in future experiments.

1 Stephens, D. J. & Allan, V. J. Light Microscopy Techniques for Live Cell Imaging. Science 300, 82-86, doi:10.1126/science.1082160 (2003).
2 Weijer, C. J. Visualizing Signals Moving in Cells. Science 300, 96-100, doi:10.1126/science.1082830 (2003).
3 Smith, A. M. & Nie, S. Chemical analysis and cellular imaging with quantum dots. Analyst 129, 672-677 (2004).
4 Michalet, X. et al. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 307, 538-544, doi:10.1126/science.1104274 (2005).
5 Medintz, I. L., Uyeda, H. T., Goldman, E. R. & Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4, 435-446 (2005).
6 Davies, G. the Institution of Electric Engineers : London. Properties and growth of diamond (1994).
7 Chang, H.-C., Chen, K. & Kwok, S. Nanodiamond as a Possible Carrier of Extended Red Emission. The Astrophysical Journal Letters 639, L63 (2006).
8 Goss, J. P. & Briddon, P. R. Theory of boron aggregates in diamond: First-principles calculations. Physical Review B 73, 085204 (2006).
9 Hui, Y. Y., Cheng, C.-L. & Chang, H.-C. Nanodiamonds for optical bioimaging. Journal of Physics D: Applied Physics 43, 374021 (2010).
10 Tzeng, Y. K. et al. Superresolution imaging of albumin-conjugated fluorescent nanodiamonds in cells by stimulated emission depletion. Angew Chem Int Ed Engl 50, 2262-2265, doi:10.1002/anie.201007215 (2011).
11 Nguyen, T. T.-B., Chang, H.-C. & Wu, V. W.-K. Adsorption and hydrolytic activity of lysozyme on diamond nanocrystallites. Diamond and Related Materials 16, 872-876, doi:10.1016/j.diamond.2007.01.030 (2007).
12 Faklaris, O. et al. Photoluminescent Diamond Nanoparticles for Cell Labeling: Study of the Uptake Mechanism in Mammalian Cells. ACS Nano 3, 3955-3962, doi:10.1021/nn901014j (2009).
13 Mukherjee, S., Ghosh, R. N. & Maxfield, F. R. Endocytosis. Physiological Reviews 77, 759-803 (1997).
14 Chang, Y.-R. et al. Mass production and dynamic imaging of fluorescent nanodiamonds. Nat Nano 3, 284-288, (2008).
15 Mohan, N., Chen, C.-S., Hsieh, H.-H., Wu, Y.-C. & Chang, H.-C. In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis elegans. Nano Letters 10, 3692-3699, doi:10.1021/nl1021909 (2010).
16 Vaijayanthimala, V., Tzeng, Y. K., Chang, H. C. & Li, C. L. The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 20, 425103, doi:10.1088/0957-4484/20/42/425103 (2009).
17 Fu, C.-C. et al. Characterization and application of single fluorescent nanodiamonds as cellular biomarkers. Proceedings of the National Academy of Sciences 104, 727-732, doi:10.1073/pnas.0605409104 (2007).
18 Zhang, B. et al. Receptor-Mediated Cellular Uptake of Folate-Conjugated Fluorescent Nanodiamonds: A Combined Ensemble and Single-Particle Study. Small 5, 2716-2721, doi:10.1002/smll.200900725 (2009).
19 Barnard, A. S. Diamond standard in diagnostics: nanodiamond biolabels make their mark. Analyst 134, 1751-1764 (2009).
20 Holt, K. B. Diamond at the nanoscale: applications of diamond nanoparticles from cellular biomarkers to quantum computing. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 365, 2845-2861, doi:10.1098/rsta.2007.0005 (2007).
21 Yu, S.-J., Kang, M.-W., Chang, H.-C., Chen, K.-M. & Yu, Y.-C. Bright Fluorescent Nanodiamonds:  No Photobleaching and Low Cytotoxicity. Journal of the American Chemical Society 127, 17604-17605, doi:10.1021/ja0567081 (2005).
22 Fang, C.-Y. et al. The Exocytosis of Fluorescent Nanodiamond and Its Use as a Long-Term Cell Tracker. Small 7, 3363-3370, doi:10.1002/smll.201101233 (2011).
23 Christopher M. Breeding, J. E. S. The type classification system of diamonds and its importance in gemology. Gem and Gemology (2009).
24 Chinnery, H. R., Pearlman, E. & McMenamin, P. G. Cutting Edge: Membrane Nanotubes In Vivo: A Feature of MHC Class II+ Cells in the Mouse Cornea. The Journal of Immunology 180, 5779-5783 (2008).
25 Lucas, W. J., Ham, B.-K. & Kim, J.-Y. Plasmodesmata – bridging the gap between neighboring plant cells. Trends in Cell Biology 19, 495-503, doi:10.1016/j.tcb.2009.07.003 (2009).
26 Maeda, S. & Tsukihara, T. Structure of the gap junction channel and its implications for its biological functions. Cellular and Molecular Life Sciences 68, 1115-1129, doi:10.1007/s00018-010-0551-z (2011).
27 Millard, T. H. & Martin, P. Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure. Development 135, 621-626, doi:10.1242/dev.014001 (2008).
28 Pyrgaki, C., Trainor, P., Hadjantonakis, A.-K. & Niswander, L. Dynamic imaging of mammalian neural tube closure. Developmental Biology 344, 941-947, doi:10.1016/j.ydbio.2010.06.010 (2010).
29 Salas-Vidal, E. & Lomelı́, H. Imaging filopodia dynamics in the mouse blastocyst. Developmental Biology 265, 75-89, doi:10.1016/j.ydbio.2003.09.012 (2004).
30 Dubey, G. P. & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell 144, 590-600 (2011).
31 Eugenin, E. A., Gaskill, P. J. & Berman, J. W. Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: A potential mechanism for intercellular HIV trafficking. Cellular Immunology 254, 142-148, doi:10.1016/j.cellimm.2008.08.005 (2009).
32 Gousset, K. et al. Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol 11, 328-336, (2009).
33 Gousset, K. & Zurzolo, C. Tunnelling nanotubes: A highway for prion spreading? Prion 3, 94-98 (2009).
34 Hansen, C. & Li, J.-Y. Beyond α-synuclein transfer: pathology propagation in Parkinson's disease. Trends in Molecular Medicine 18, 248-255, doi:10.1016/j.molmed.2012.03.002 (2012).
35 Schertzer, J. W. & Whiteley, M. Microbial Communication Superhighways. Cell 144, 469-470 (2011).
36 Sowinski, S. et al. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 10, 211-219, (2008).
37 Hsiung, F., Ramirez-Weber, F.-A., David Iwaki, D. & Kornberg, T. B. Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic. Nature 437, 560-563, (2005).
38 Ramírez-Weber, F.-A. & Kornberg, T. B. Cytonemes: Cellular Processes that Project to the Principal Signaling Center in Drosophila Imaginal Discs. Cell 97, 599-607, doi:10.1016/s0092-8674(00)80771-0 (1999).
39 Abel, M. et al. Microstructured platforms to study nanotube-mediated long-distance cell-to-cell connections. Biointerphases 6, 22-31, doi:10.1116/1.3567416 (2011).
40 Abounit, S. & Zurzolo, C. Wiring through tunneling nanotubes – from electrical signals to organelle transfer. Journal of Cell Science 125, 1089-1098, doi:10.1242/jcs.083279 (2012).
41 B. ¨Onfelt, D. M. D. Can membrane nanotubes facilitate communication between immune cells? Biochemical Society Transactions 32 (2004).
42 Baluska, F., Volkmann, D., Barlow, P. W., Gerdes, H.-H. & Rustom, A. 200-207 (Springer New York, 2006).

43 Bukoreshtliev, N. V. et al. Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells. FEBS Letters 583, 1481-1488, doi:10.1016/j.febslet.2009.03.065 (2009).
44 Davis, D. M. & Sowinski, S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nat Rev Mol Cell Biol 9, 431-436 (2008).
45 Domhan, S. et al. Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells. PLoS ONE 6, e21283, doi:10.1371/journal.pone.0021283 (2011).
46 Gerdes, H.-H., Bukoreshtliev, N. V. & Barroso, J. F. V. Tunneling nanotubes: A new route for the exchange of components between animal cells. FEBS Letters 581, 2194-2201 (2007).
47 Gerdes, H.-H. & Carvalho, R. N. Intercellular transfer mediated by tunneling nanotubes. Current Opinion in Cell Biology 20, 470-475, doi:10.1016/j.ceb.2008.03.005 (2008).
48 Gurke, S., Barroso, J. F. & Gerdes, H. H. The art of cellular communication: tunneling nanotubes bridge the divide. Histochemistry and cell biology 129, 539-550 (2008).
49 Gurke, S. et al. Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells. Experimental Cell Research 314, 3669-3683, doi:10.1016/j.yexcr.2008.08.022 (2008).
50 He, K. et al. Intercellular Transportation of Quantum Dots Mediated by Membrane Nanotubes. ACS Nano 4, 3015-3022, doi:10.1021/nn1002198 (2010).
51 He, K. et al. Long-Distance Intercellular Connectivity between Cardiomyocytes and Cardiofibroblasts Mediated by Membrane Nanotubes. Cardiovascular Research, doi:10.1093/cvr/cvr189 (2011).
52 Hurtig, J., Chiu, D. T. & Önfelt, B. Intercellular nanotubes: insights from imaging studies and beyond. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2, 260-276, doi:10.1002/wnan.80 (2010).
53 Koyanagi, M., Brandes, R. P., Haendeler, J., Zeiher, A. M. & Dimmeler, S. Cell-to-Cell Connection of Endothelial Progenitor Cells With Cardiac Myocytes by Nanotubes. Circulation Research 96, 1039-1041, doi:10.1161/01.RES.0000168650.23479.0c (2005).
54 Mi, L. Microscopic observation of the intercellular transport of CdTe quantum dot aggregates through tunneling-nanotubes. Journal of Biomaterials and Nanobiotechnology 02, 172-179, doi:10.4236/jbnb.2011.22022 (2011).
55 Önfelt, B., Nedvetzki, S., Yanagi, K. & Davis, D. M. Cutting Edge: Membrane Nanotubes Connect Immune Cells. The Journal of Immunology 173, 1511-1513 (2004).
56 Rustom, A., Saffrich, R., Markovic, I., Walther, P. & Gerdes, H.-H. Nanotubular Highways for Intercellular Organelle Transport. Science 303, 1007-1010, doi:10.1126/science.1093133 (2004).
57 Vidulescu, C., Clejan, S. & O'Connor, K. C. Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells. Journal of Cellular and Molecular Medicine 8, 388-396, doi:10.1111/j.1582-4934.2004.tb00328.x (2004).
58 Wang, X. & Gerdes, H.-H. Long-distance electrical coupling via tunneling nanotubes. Biochimica et Biophysica Acta (BBA) - Biomembranes, doi:10.1016/j.bbamem.2011.09.002 (2011).
59 Wang, X., Veruki, M. L., Bukoreshtliev, N. V., Hartveit, E. & Gerdes, H.-H. Animal cells connected by nanotubes can be electrically coupled through interposed gap-junction channels. Proceedings of the National Academy of Sciences 107, 17194-17199, doi:10.1073/pnas.1006785107 (2010).
60 Wang, Y., Cui, J., Sun, X. & Zhang, Y. Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ 18, 732-742, (2011).
61 Brunholz, S. et al. Axonal transport of APP and the spatial regulation of APP cleavage and function in neuronal cells. Experimental Brain Research 217, 353-364, doi:10.1007/s00221-011-2870-1 (2012).
62 De Vos, K. J., Grierson, A. J., Ackerley, S. & Miller, C. C. J. Role of Axonal Transport in Neurodegenerative Diseases*. Annual Review of Neuroscience 31, 151-173, doi:doi:10.1146/annurev.neuro.31.061307.090711 (2008).
63 Ho, V. M., Lee, J.-A. & Martin, K. C. The Cell Biology of Synaptic Plasticity. Science 334, 623-628, doi:10.1126/science.1209236 (2011).
64 Koo, E. H. et al. Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport. Proceedings of the National Academy of Sciences 87, 1561-1565 (1990).
65 Scott, David A., Das, U., Tang, Y. & Roy, S. Mechanistic Logic Underlying the Axonal Transport of Cytosolic Proteins. Neuron 70, 441-454, doi:10.1016/j.neuron.2011.03.022 (2011).
66 Yi, J. Y. et al. High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport. The Journal of Cell Biology 195, 193-201, doi:10.1083/jcb.201104076 (2011).

67 Kusumi, A., Sako, Y. & Yamamoto, M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophysical journal 65, 2021-2040 (1993).
68 Neugart, F. et al. Dynamics of Diamond Nanoparticles in Solution and Cells. Nano Letters 7, 3588-3591, doi:10.1021/nl0716303 (2007).
69 Okada, Y. & Hirokawa, N. A Processive Single-Headed Motor: Kinesin Superfamily Protein KIF1A. Science 283, 1152-1157, doi:10.1126/science.283.5405.1152 (1999).