Near-infrared (NIR)-to-Visible upconversion fluorescent nanoparticles emit visible light upon NIR-light excitation, and are well suited for bioimaging, compared to the commonly used downconversion fluorescent materials. These nanoparticles have advantages such as minimum photodamage to living organisms, weak background fluorescence, high detection sensitivity, and high light-penetration depth in tissues. However, development of upconversion fluorescent nanoparticles is still in its infancy, and such materials have yet to be used for bioimaging applications due to their unsuitable surface properties, poor dispersibility in water, and limited colors. In this work, facile and user-friendly methods are developed to synthesize uniform NaYF 4 nanospheres with strong upconversion fluorescence and core-shell silica/NaYF 4 nanospheres with uniform silica coating on the surface. Multicolor upconversion nanospheres are produced by encapsulating organic dyes or quantum dots (QDs) in the silica shell, and upconversion fluorescence is generated based on fluorescence resonance energy transfer (FRET) from the NaYF 4 nanospheres to these organic dyes or QDs. Use of the upconversion nanospheres for imaging of cells is also demonstrated, the first report in the field using such nanoparticles for bioimaging.
Fluorescence imaging is a very important technique for biological studies and clinical applications. It has been used for in vivo imaging due to its high temporal and spatial resolutions. [1] Conventional fluorescence imaging is based on single-photon excitation, emitting low-energy fluorescence when excited by a high-energy light. It has some limitations, such as causing DNA damage and cell death due to long-term irradiation with UV or short-wavelength light, significant auto-fluorescence from biological tissues, resulting in low signal-to-noise ratios, and short penetration depth of short-wavelength excitation light in biological tissues. [2] Two-photon fluorescence imaging (TPFI) is a novel technique that generates high-energy visible photons from low-energy radiation in NIR region. [3] NIR radiation is less harmful to cells, minimizes auto-fluorescence from biological tissues, and penetrates tissues to a greater extent. [4] So far, most commercially available two-photon fluorophores are organic dyes that exhibit relatively low two-photon absorption crosssections, low fluorescence quantum yield, and photochemical instability (photobleaching). Some efforts have been made to prepare better organic fluorophores with tailored properties. [5] Some inorganic materials, such as semiconductor quantum dots (QDs) and metal nanospheres or nanorods, have also been developed and used for two-photon imaging of cells. [6][7][8][9] However, TPFI requires nearly simultaneous absorption of two coherent NIR photons, and therefore, the efficiency is usually low, and expensive pulsed lasers are usually required. Photon upconversion is an alternative process for converting NIR radiation to visible radiation. It is based on sequential (not simultaneous) absorption of photons, and as such its efficiency is much higher compared to two-photon absorption, and continuous wave (CW) lasers can be used for excitation. Typical laser power densities are 1-10 3 W cm Γ2 for upconversion and 10 6 -10 9 W cm Γ2 for two-photon absorption. Pulsed lasers are normally used for downconversion fluorescent materials. Although the average power density is on the order of 50-400 mW cm Γ2 , the peak power density could be quite high. Upconversion fluorescent materials can be excited by CW lasers. Biological cells and tissues have very weak absorption in the NIR region, and as such increasing the laser power does not cause any significant heat damage.
Various inorganic crystals doped with lanthanide ions have been synthesized, producing strong NIR-to-vis upconversion fluorescence. These are very promising materials for bioimaging, because the rare-earth elements used in their synthesis have lower toxicity than the semiconductor elements of QDs (LD 50 is approximately a thousand times higher than that of QDs), while the upconversion fluorescence is much stronger than that of QDs. [10][11][12] These materials have been used in immunohistochemistry in lateral flow (LF) assay formats, and in immunochromatographic assays of human chorionic gonadotropin (hCG). [13][14][15][16][17] Hosting of in vitro nucleic acid assays has also been described. [18,19] Furthermore, 150 nm sized particles have been inoculated into live Caenorhabditis elegans, and imaged in the intestines of the worms; however, no biological materials were used for the particle surface COMMUNICATION [