Long-term real-time tracking of lanthanide ion doped upconverting nanoparticles in living cells.

Recently, there has been great interest in employing nanoparticles for various biological applications. Nanoparticles can be synthesized in a controlled manner such that they have desirable sizes, shapes, and optical or magnetic properties. In addition, one may provide nanoparticles with biological functions through chemical surface modifications and conjugation of ligands. Such intrinsic and extrinsic properties of nanoparticles enable them to be used as excellent biological imaging probes and diagnostic/therapeutic agents at the cellular level. Among the various nanoparticle systems developed thus far, semiconductor nanocrystals or quantum dots (QDs) are most widely used. QDs are extremely bright and photostable, and exhibit excellent spectral properties (i.e., broad absorption and narrow emission bands) suited for multicolor detection. However, the drawbacks such as photoblinking, the presence of nonradiant dark particles, and potential cytotoxicity limit their applicability. In recent years, several alternative types of luminescent nanoparticles have been introduced for biological applications. For example, nanodiamonds (NDs) with nitrogen vacancy centers were found to be highly photoluminescent while exhibiting no photoblinking and photobleaching, 11] and even useful as the imaging probe for super-resolution optical microscopy. However, applying NDs for biological imaging has limitations, especially in the case of long-term tracking studies, since the excitation in the blue or green region (typically 488 or 532 nm) might result in fatal photodamage to cells or low penetration depth into tissues. In contrast, single-walled carbon nanotubes (SWNTs) were shown to be appropriate for biological imaging in that the excitation and emission lie in the near-infrared (NIR) spectral range. However, being longer than 100 nm typically, SWNTs are considered to be too large to be used as biolabels. Meanwhile, lanthanide ion doped upconverting nanoparticles (UCNPs), which emit in the visible range upon absorption of NIR photons, have attracted great attention owing to their unique optical properties. First, two-photon upconversion of NIR excitation to the emission of a visible photon is so efficient that a tiny continuous-wave (CW) diode laser (980 nm) with the output of tens of milliwatts is sufficient as the excitation source. Second, by employing NIR excitation, one can suppress cellular autofluorescence, induce little photodamage to living cells, and achieve relatively deep penetration into tissues. Finally, UCNPs exhibit neither photoblinking on the millisecond and second time scales nor photobleaching even with hours of continuous excitation, 21] their cytotoxicity is very low, 22] and the inclusion or doping of Gd ions in the host materials endows UCNPs with an additional modality for magnetic resonance imaging (MRI). 23] As a result, UCNPs became one of the most promising nanoparticle systems for biological imaging and there are continuing efforts to improve their properties (e.g., increasing luminescence intensity and reducing the particle size) by designing new synthetic strategies. Herein, we report the first real-time tracking study with UCNPs at the single vesicle level in living cells. Thanks to the remarkable photostability of UCNPs and the noninvasiveness of NIR excitation, we were able to visualize the intracellular movements of UCNPs for as long as 6 h without interruption. We first assessed the benefits of using NIR radiation as the excitation source to demonstrate the feasibility of long-term live-cell imaging with UCNPs. The UCNPs (hexagonal-phase NaYF4 co-doped with Yb 3+ and Er, ca. 30 nm in diameter) coated by amphiphilic PEG–phospholipids (PEG = poly(ethylene glycol)) were internalized into HeLa cells and imaged on a home-made epi-fluorescence microscope setup (Methods section and Figures S1 and S2 in the Supporting [*] Dr. S. H. Nam, Y. M. Bae, Dr. H. M. Kim, Dr. K. T. Lee, Dr. Y. D. Suh Laboratory for Advanced Molecular Probing (LAMP) NanoBio Fusion Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600 (Korea) Fax: (+ 82)42-860-7164 E-mail: ktlee@krict.re.kr ydsuh@krict.re.kr Y. I. Park, Dr. J. H. Kim, Prof. Dr. T. Hyeon National Creative Research Initiative Center for Oxide Nanocrystalline Materials, World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2) School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744 (Korea) Y. M. Bae, Prof. Dr. J. S. Choi Department of Biochemistry, Chungnam National University Daejeon 305-764 (Korea) [] These authors contributed equally to this work.