Mass production and dynamic imaging of fluorescent nanodiamonds YI-REN CHANG 1† , HSU-YANG LEE 1† , KOWA CHEN 1 , CHUN-CHIEH CHANG 2 , DUNG-SHENG TSAI 1 , CHI-CHENG FU 1 , TSONG-SHIN LIM 1‡ , YAN-KAI TZENG 1 , CHIA-YI FANG 1,3 , CHAU-CHUNG HAN 1 , HUAN-CHENG CHANG 1,3 * AND WUNSHAIN FANN 1,2,4 * 1 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan 2 Department of Physics, National Taiwan University, Taipei 106, Taiwan 3 Department of Chemistry, National Taiwan Normal University, Taipei 106, Taiwan 4 Institute of Polymer Science and Engineering, National Taiwan University, Taipei 106, Taiwan † These authors contributed equally to this work ‡ Present address: Department of Physics, Tunghai University, Taichung 407, Taiwan *e-mail: fann@gate.sinica.edu.tw; hcchang@po.iams.sinica.edu.tw Published online: 27 April 2008; doi:10.1038/nnano.2008.99 Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility 1 , excellent photostability 1,2 and facile surface functionalizability 2,3 . Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications 1,2,4 . Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions. The fluorescence is sufficiently bright and stable to allow three-dimensional tracking of a single particle within the cell by means of either one- or two-photon-excited fluorescence microscopy. The excellent photophysical characteristics are maintained for particles as small as 25 nm, suggesting that fluorescent nanodiamond is an ideal probe for long-term tracking and imaging in vivo, with good temporal and spatial resolution. Serving as an in vivo nanoprobe, fluorescent nanodiamonds (FNDs) have two major advantages over the commonly used fluorescent beads 5 and quantum dots 6,7 . First, the emission from FNDs is exceptionally stable; no photobleaching or fluorescence intermittency are observed, even for a single nitrogen vacancy (N-V) defect centre 8,9 . Second, diamond nanoparticles are non- toxic to a number of cell types, as has been documented by cell viability assays 1,10–13 . In recent work 7 , the axonal transport of nerve growth factor signals was studied using quantum dots for live tracking. It was found that dots made of materials such as CdSe were immune to photobleaching in live cells; however, the photoblinking characteristic of these semiconductor fluorophores rendered their utility for continuous three-dimensional tracking difficult. In contrast, FND is perfectly photostable, allowing the tracking and imaging of a single FND particle in a cell for hours. Although FNDs are well suited for biomedical use, the nanomaterial has not yet received widespread exploitation because of difficulties in its mass production. Conventionally, the N-V defect centres in diamond are produced by bombarding the material with a high-energy (typically 2 MeV) electron beam from a van de Graaff accelerator, followed by annealing at elevated temperatures (typically 800 8C) 4,8,9,14,15 . This requires highly sophisticated and costly equipment, which therefore hinders the easy availability of FNDs. We present a practical method to scale up the production of FNDs using a home-built prototype device composed of a high-fluence, medium-energy He þ beam. Compared with previously used methods 1,2 , the setup has boosted FND yield by nearly two orders of magnitude and can be installed and operated safely in ordinary laboratories. High-brightness FNDs were produced through radiation- damage of synthetic type Ib diamond powders (mean sizes of 35 and 140 nm) using 40-keV He þ bombardment at a dose of 1  10 13 ions cm 22 . The merit of using He þ as the damage agent is threefold. First, helium atoms are chemically inert, and embedding these atoms in a diamond lattice through neutralization of the stopped He þ ions does not appreciably change the photophysical properties of the FNDs produced. Second, a 40-keV He þ ion can create 40 vacancies as it penetrates diamond 16 , in contrast to the 0.1 and 13 vacancies generated by 2-MeV e 2 and 3-MeV H þ , respectively 15,17 . This remarkably high damaging efficacy reduces the ion dosage required for irradiation. Third, high-fluence 40-keV He þ beams can be readily generated by radio-frequency ion sources. The current typically delivered by these sources is 10 mA( 6  10 13 ions s 21 ), which is more than two orders of magnitude higher than that of a 3-MeV H þ beam emanating from a tandem particle accelerator 1,17 . These factors together make it possible and practical to produce bright FNDs on a large scale (see Supplementary Information, Figs S1 and S2). Figure 1a shows an ensemble emission spectrum (l max ¼ 680 nm) of 35-nm FNDs prepared by He þ irradiation. The spectrum, acquired for particles suspended in water (inset in Fig. 1a) and excited with a continuous-wave (cw) 532-nm laser (see Supplementary Information, Fig. S3), reveals two types of N-V centres inside the material: (N-V) 0 with a zero-phonon line at 575 nm and (N-V) 2 with a zero-phonon line at 638 nm (ref. 14) Comparing it with the spectrum of another sample LETTERS nature nanotechnology | VOL 3 | MAY 2008 | www.nature.com/naturenanotechnology 284 © 2008 Nature Publishing Group