The development of probes for single-molecule imaging has dramatically facilitated the study of individual molecules in cells and other complex environments. Single-molecule probes ideally exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-molecule probes are imperfect in several of these aspects, and none have been shown to possess all of these characteristics. Here we show that individual lanthanidedoped upconverting nanoparticles (UCNPs)-specifically, hexagonal phase NaYF 4 (-NaYF4) nanocrystals with multiple Yb 3؉ and Er 3؉ dopants-emit bright anti-Stokes visible upconverted luminescence with exceptional photostability when excited by a 980-nm continuous wave laser. Individual UCNPs exhibit no on/off emission behavior, or ''blinking,'' down to the millisecond timescale, and no loss of intensity following an hour of continuous excitation. Amphiphilic polymer coatings permit the transfer of hydrophobic UCNPs into water, resulting in individual watersoluble nanoparticles with undiminished photophysical characteristics. These UCNPs are endocytosed by cells and show strong upconverted luminescence, with no measurable anti-Stokes background autofluorescence, suggesting that UCNPs are ideally suited for single-molecule imaging experiments.bio-imaging ͉ fluorescence ͉ nanoparticle ͉ single molecule ͉ phosphorescence L anthanide-doped nanocrystals have recently shown promise as imaging probes due to their ability to upconvert lowenergy near-infrared (NIR) radiation into higher-energy visible luminescence (1, 2). Unlike Stokes-shifted luminescence from organic-and protein-based fluorophores (3), semiconductor quantum dots (4-6), fluorescent latex (7) and silica (8) nanobeads, carbon nanotubes (9, 10), or newly developed nanodiamonds (11), this anti-Stokes luminescence circumvents competition from autofluorescent background signals in biological systems. The NIR-to-visible upconversion is based on sequential energy transfers between lanthanide dopants or excited-state absorption involving their real metastable-excited states with lifetimes as long as several milliseconds, a process orders of magnitude more efficient than the 2-photon absorption process typically used in multiphoton microscopy (12). For example, the UNCPs described here are approximately 10 5 times more efficient at upconversion than many commonly used 2-photon fluorescence (TPF) dyes, including rhodamine 6G and fluorescein (13). This increased efficiency permits use of a continuous wave (CW) laser to generate the upconverted luminescence, resulting in virtually zero 2-photon photoluminescence background from biomolecules, since powerful pulsed-laser excitation is generally required for generating measurable multiphoton absorption. Use of NIR excitation also minimizes the possible photodamage in biological systems and permits deeper tissue penetration and whole-animal imaging.While single-molecule imaging has become increasingly routine in biol...