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Biomaterials 29 (2008) 937943 www.elsevier.com/locate/biomaterials

Upconversion uorescence imaging of cells and small animals using lanthanide doped nanocrystals
Dev K. Chatterjeea, Abdul J. Rufaihaha, Yong Zhanga,b,
b

Division of Bioengineering, National University of Singapore, Singapore 117574, Singapore Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, Singapore Received 24 August 2007; accepted 26 October 2007 Available online 3 December 2007

Abstract Upconversion uorescence imaging technique with excitation in the near-infrared (NIR) region has been used for imaging of biological cells and tissues. This has several advantages, including absence of photo-damage to living organisms, very low auto-uorescence, high detection sensitivity, and high light penetration depth in biological tissues. In this report we demonstrate the use of a new upconversion uorophore, lanthanide doped nanocrystals, for imaging of cells and some deep tissues in animal. Polyethyleneimine (PEI) coated NaYF4:Yb,Er nanoparticles were synthesized, which produce very strong upconversion uorescence when excited at 980 nm by a NIR laser. The nanoparticles were shown to be stable in physiologic buffered saline (PBS), non-toxic to bone marrow stem cells, and resistant to photo-bleaching. The nanoparticles delivered into some cell lines or injected intradermally and intramuscularly into some tissues either near the body surface or deep in the body of rats showed visible uorescence, when exposed to a 980 nm NIR laser. To the best of our knowledge, this represents the rst demonstration of use of upconversion uorophores for cellular and tissue imaging. r 2007 Elsevier Ltd. All rights reserved.
Keywords: Nanoparticle; Fluorescence; Surface modication

1. Introduction Fluorescence imaging is a very important technique for biological studies and clinical applications due to high temporal and spatial resolutions [1]. Conventional uorescence imaging is based on single-photon excitation, emitting low energy uorescence when excited by high energy light. Using high energy excitation light has some limitations like DNA damage and cell death caused by long-term irradiation, signicant auto-uorescence from biological tissues resulting in low signal-to-background ratio, and short penetration depth in biological tissues [2]. Upconversion uorescence imaging, on the other hand, involves conversion of two or more low energy photons usually near infrared (NIR)to higher energy visible
Corresponding author. Division of Bioengineering, Faculty of Engineering, Block E3A-04-15, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, Singapore. Tel.: +65 65164871; fax: +65 68723069. E-mail address: biezy@nus.edu.sg (Y. Zhang).

emissions [3]. The upconversion uorophores are generally phosphor nanoparticles with a crystalline matrix doped with lanthanide ions. The rare earth elements used in synthesis of the particles have a lower toxicity than semiconductor elements of quantum dots (QDs, LD50 is approximately a thousand times more than that of QDs) [46]. Infrared excitation is less harmful to cells, minimizes auto-uorescence from biological tissues and penetrates tissues to a greater extent [7]. Upconverting phosphor particles have been used in immunohistochemistry in lateral ow (LF) assay formats, or in immunochromatographic assays for human chorionic gonadotropin (hCG) [1317]. A host of in vitro nucleic acid assays have also been described [18,19]. Furthermore, 150 nm sized particles have been inoculated into live C. elegans and imaged in the intestines of the worms [20]. However, these particles are not suitable for imaging of cells and animals because of their large size and unsuitable surface characteristics. Yb/Er (or Yb/Tm) co-doped NaYF4 nanoparticles have been reported as the most efcient infrared-to-visible upconversion uorescent

0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.10.051

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938 D.K. Chatterjee et al. / Biomaterials 29 (2008) 937943 operating at an acceleration voltage of 200 kV. A small drop of PEI/ thick carbonNaYF4:Yb3+,Er3+ nanoparticle solution was put on a 50 A coated copper grid (300 mesh) and the excess solution was immediately removed. The surface charge of the nanoparticles was measured by zeta potential measurements in phosphate buffer (Zetasizer NanoZS, Malvern Inst., Malvern, UK). Size distribution was also measured using dynamic light scattering (DLS) with the Zetasizer to determine the presence of aggregates after the attachment of FA. Fluorescence spectra were obtained with a SpectroPro 2150i spectrophotometer (Roper Scientic Acton Research, MA) equipped with a 1200 g mm1 grating and a 980 nm diode laser. The stability of the nanoparticles was tested by incubating the nanoparticles in PBS at 37 1C and 4% CO2 and testing for peak uorescence at different time points. To test photostability under laser illumination, the NIR laser current was set at maximum (1.9 A) and then the beam further focused using a convex lens. The particles were placed at this focal point, and emission values monitored continuously for 7 h. To perform elemental analysis, 10 ml of a solution of nanoparticles (concentration 0.044 mg ml1) was sent for analysis using the inductively coupled plasma-mass spectrometry.

material [21]. Colloidal Yb/Er and Yb/Tm co-doped NaYF4 nanoparticles with strong upconversion uorescence seven orders of magnitude higher than that of CdSeZnS QDs have been prepared [5,6]. These nanoparticles are usually synthesized in organic solvents or at high temperatures [2224]. Ethylenediamine tetraacetic acid (EDTA) has been used as a chelating agent to control the growth of NaYF4 nanocrystals, and thermal decomposition of mixed triuoroacetates has also been used to produce high quality cubic- and hexagonal-phase NaYF4 nanocrystals [5,2426]. Very recently, we reported a method to use polyvinylpyrrolidone (PVP) as a chelating agent and surfactant to control size and stability of the nanoparticles but the surfactants used to control the nanoparticle growth do not lend themselves to easy modication with biomolecules, and further surface modications of the nanoparticles are usually required [27]. Coating of the nanoparticles with a layer of silica is sometimes preferred but it is difcult to directly make silica coatings on hydrophobic nanoparticles unless they are previously made hydrophilic or some specially designed silane precursors are used [28]. Furthermore, it is very difcult to make uniform and thin silica coatings on individual nanoparticles and silica is easily coated on the aggregates of nanoparticles. Recently, a simple one-pot synthetic method was developed by our group using polyethyleneimine (PEI) to coat the nanoparticles and control the crystal growth [29]. PEI is a thermally stable and hydrophilic polymer with primary, secondary and tertiary amino groups, which render the nanoparticles water soluble. The amino groups can be used for conjugation of biomolecules to the nanoparticles. In this work, we explore the use of the PEI/NaYF4 nanoparticles for upconversion uorescence imaging of cells and animals in vitro and in vivo.
2. Materials and methods 2.1. Materials
PEI, FA (approximately 98%), dimethyl sulfoxide (DMSO), N-hydroxy-succinimide (NHS), 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC), sodium chloride (NaCl, X99.0%), yttrium chloride hexahydrate (YCl3 6H2O, 99.99%), ytterbium oxide (Yb2O3, 99.99%), erbium oxide (Er2O3, 99.99+%), thulium oxide (Tm2O3, 99.99%) and ammonium uoride (NH4F, 98+%) were purchased from Sigma-Aldrich (Singapore). All the reagents were used as received without further purication. PEI stock solution (5 wt%) was prepared by dissolving PEI in DI water. YCl3 and NaCl stock solutions (0.2 M) were prepared by dissolving YCl3 6H2O and NaCl in DI water, respectively. YbCl3, ErCl3, and TmCl3 stock solutions (0.2 M) were prepared by dissolving in hydrochloric acid. Green emitting (540 nm peak) QDs with carboxylated surfaces were purchased from American Dye Sources, Inc. (product ADS540QD).

2.3. Cell viability test

Bone marrow derived stem cells were collected from rats and used to assess the cell biocompatibility of the PEI/NaYF4 nanoparticles. Mitochondrial function was assessed using the Cell Titer 96 aqueous one solution assay (Promega, Madison, WI). The reagent contains tetrazolium compound [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salth; MTS] and an electron coupling reagent (penazine ethosulfate; PES). The cells were seeded onto a 96-well plate at a density of 5 103 cells per well. After 2 days of culture, different concentrations of nanoparticles were added to the wells. The cells were incubated at various time points and then 20 ml of the reagent was directly added to the wells. After 2 h of incubation at 37 1C, absorbance at 490 nm was measured with a standard microplate reader (Bio-Tek Instruments, USA). The quantity of the formazan product formed as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in the culture. Each experiment was done in triplicate. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by [A]expt/[A]control 100, where [A]expt is the absorbance of the test sample and [A]control is the absorbance of control sample.

2.4. Biodistribution study

Female Wistar rats weighing about 200250 g were used in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institute of Health, USA. Approval was also obtained from International Animal Care and Use Committee (IACUC), National University of Singapore. The rats were anaesthetized with ketamine xylazine mixture (ketamine 75 mg kg1, xylazine 10 mg kg1) by intraperitoneal injection and then injected intravenously with the PEI/NaYF4 nanoparticles at a concentration of 10 mg ml1 as determined by ICP-MS. The rats were then euthanized at pre-determined time points; 0.5, 24 h, and 7 days. The heart, lung, spleen, kidney, liver, and blood were collected, weighed, incubated at 37 1C overnight in digestion buffer solution containing 22.4% KOH and 2% Tween-80 while the blood sample was incubated in solution containing 89.2% potassium hydroxide (KOH) and 2% of Tween-80. Yttrium (Y) content in the samples were then determined using ICP-MS as a means of determining nanoparticle concentration.

2.2. Synthesis and characterization of PEI/NaYF4 nanoparticles

A one-pot synthetic method was used to synthesize PEI/NaYF4 nanoparticles as previously described [29]. Transmission electron microscope (TEM) measurements were carried out on a JEOL 2010 TEM

2.5. Cell imaging

The human colon adenocarcinoma cell line HT29 and ovarian carcinoma cell line OVCAR3 were cultured in 25 cm2 asks in a medium made up of DMEM, foetal bovine serum (FBS), and antibiotics in a ratio

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D.K. Chatterjee et al. / Biomaterials 29 (2008) 937943 of 100:10:1, and incubated in a 100% humidied incubator with 5% CO2 at 37 1C according to established procedure. The cells were collected and grown on glass coverslips for 24 h. PEI/NaYF4:Yb,Er nanoparticles (5 mg ml1) were added and the cells were incubated for 24 h at 37 1C and 4% CO2, then imaged in bright eld and under infrared excitation using a Nikon confocal microscope. Infrared excitation was with a specially tted continuous wave infrared laser source (500 mW output power) and images captured using the Evolution MP Cooled Camera Kit. 939

2.6. Animal imaging

Wistar rats were anaesthetized and fur was clipped at the groin, back, and abdomen; 100 ml of PEI/NaYF4:Yb,Er nanoparticles (4.4 mg ml1) and QD was injected subcutaneously at these regions in different animals. Luminescence was observed in a darkened room by excitation with a 980 nm VA-II DPSS laser (current set 1.0 A) and recorded using CCDbased digital camera (Sony) with heat lters to eliminate NIR scatter. The depth of injection was estimated from needle penetration. Animal-to-laser distance was xed using a ruler attached to the laser head. At the end of the experiments, the animals were euthanized according to standard approved protocol.

3. Results and discussion 3.1. Synthesis and characterization of PEI/NaYF4 nanoparticles PEI coated NaYF4:Yb,Er and NaYF4:Yb,Tm nanoparticles were synthesized using high molecular weight (25 kDa) PEI as surfactant as previously reported, with modication [29]. The nanoparticles formed a clear, aggregate free solution in water. TEM image (Fig. 1) showed that these are well-dispersed spherical nanoparticles with a mean diameter of about 50 nm and a relatively narrow size distribution as determined by DLS measurement. The nanoparticles were positively charged as determined by zeta potential measurement. Zetasizer measurements showed a single peak with narrow size distribution, demonstrating absence of aggregates. When excited with a 980 nm NIR laser, the nanoparticles in buffered saline emitted strong upconversion uorescence.

The NaYF4:Yb,Er nanoparticles emitted uorescence at 653 nm (red) and 540 nm (green) with a full-width at halfmaximum (FWHM) about 24 nm for the red emission peak and 16 nm for the green emission peak. These nanoparticles were chosen for subsequent experiments. Elemental analysis of the nanoparticle suspension was done using inductively coupled plasma-mass spectrometry and the following results (in parts per million) were obtained: Y 20.24, Na 2.31, F 2.44, Yb 4.82, Er 1.446. This demonstrated the preponderance of Y in the nanoparticles core matrix. The ratio of energy donor ions (Yb) to emitter ions (Er) is about 3.3. It may be noted that during synthesis, the corresponding ratio was 8.7. Thus, there is a differential doping of the elements to the nanocrystalline matrix. The large preponderance of Y in the nal nanoparticle composition is especially important for highly sensitive detection of the particles in tissues. Since Y is normally absent in tissues or present in quantities too minute to be detected, using ICP for detection of Y in a digested tissue sample is a very sensitive method to test for the presence of the nanoparticles. The nanoparticles demonstrated very strong resistance to photo-bleaching. Dried sample of PEI/NaYF4 nanoparticles was continually exposed to a 980 nm focused laser beam and the maximum emission intensity was recorded over nearly 7 h with no reduction in emission intensity (Fig. 2(a)). The intensity of exposure was greater than the upper limit of measurement of a standard photo-meter. The nanoparticles were also stored in PBS and the emission intensity with NIR excitation tested at intervals. There was no reduction in emission intensity observed on storage over 3 weeks (Fig. 2(b)). 3.2. Cell biocompatibility and tissue biodistribution Bone marrow derived stem cells were treated with different concentrations of PEI/NaYF4 nanoparticles for 2448 h, to determine the effect of both period of

a Intensity (a.u.)

b 400 500 600 Wavelength (nm) 700

Fig. 1. TEM images of the NaYF4:Yb,Er nanoparticles coated with 25 kD PEI. Fluorescence spectra of Tm (a) and Er (b) doped PEI/NaYF4 nanoparticles (insert, photographs of the nanoparticles when excited by a NIR laser at 980 nm).

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Fluorescence Intensity (%)

100 80 60 40 20 0 0 1 2 3 4 5 Time (h) 6 7

Fluorescence Intensity (%)

100 80 60 40 20 0 0 4 8 12 Time (day) 16 20

Fig. 2. Dried PEI/NaYF4 nanoparticles continually exposed to 980 nm laser as a function of exposure time (a) and uorescence intensity of the nanoparticles in PBS as a function of incubation time (b).

100 80 60 40 20 0 0 5 10 15 20 Concentration (g/ml) 25 day 1 day 2 Concentration of Yttrium (mg/g) Cell viability (%)

25

30min post-injection 24hr post-injection 7days post-injection

20

15

10

Fig. 3. Viability of bone marrow stem cells from rats after incubation with PEI/NaYF4 nanoparticles with different concentrations for 1 and 2 days.

0 Heart Lung Spleen Kidney Liver Blood

Fig. 4. Biodistribution of PEI/NaYF4 nanoparticles in organs of rat harvested after tail-vein injection.

incubation as well as concentration of the nanoparticles. The viability of non-treated cells is assumed to be 100%. With a nanoparticle concentration of 1 mg ml1, incubation of the stem cells with the nanoparticles for 24 and 48 h did not change the cell viability. When the nanoparticle concentration was increased to 25 mg ml1, the cell viability still remained above 90% (Fig. 3). No sub-cellular apoptotic changes or signicant cell death was seen in cells after incubation with the nanoparticles and imaged under NIR over a period of 5 days. The nanoparticles appear to be non-toxic to bone marrow derived stem cells when used in certain range of concentrations and within limited time periods of incubation. Stem cell research has attracted a lot of interest and stem cells have been demonstrated to have a number of potential uses. However, the cells are very sensitive to the environment and an excellent method to demonstrate toxicity in a material. The PEI/NaYF4 nanoparticles have showed no toxic effect on the cells within reasonable concentrations. The results showed that the nanoparticles could be a better alternative to conventional uorescent probes for continuous imaging of stem cells due to their unique optical properties and non-toxic nature. Female Wistar rats were injected with the

nanoparticles intravenously and the amount of Y in heart, lung, spleen, kidney, liver, and blood were measured (Fig. 4). The nanoparticles had a rapid accumulation in lungs immediately after injection, but the amount of the nanoparticles was already signicantly reduced in all tissues with the highest concentration in the spleen, by 24 h post-injection. By 7 days the nanoparticles were undetectable in the rats. The half hour distribution prole is important for imaging since most imaging of tumors will take place within this time period after injection. The lung nding is an interesting and important point that will need to be investigated in depth later. However, a complete biodistribution study is beyond the scope of the present report. The present study is to give an overall idea of the retention and time period of removal of the particles by different organs in the short and longer term. The results indicate that there are no potentially damaging retention proles and that these particles can be safely used for imaging in small mammals.

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3.3. Confocal uorescence imaging of cancer cells To use the nanoparticles for imaging of cancer cells, folic acid was covalently conjugated to the PEI/NaYF4 nanoparticles. The method of cross-linking folic acid to PEI involves a simple condensation reaction between carboxyl groups of folic acid and amino groups of PEI and the substitution of folic acid by other biomolecules such as antibodies or peptides can be carried out without signicant alteration of the synthesis protocol. Folic acid coated PEI/NaYF4 nanoparticles were incubated in physiological conditions with human HT29 adenocarcinoma cells and human OVCAR3 ovarian carcinoma cells. Both cell lines are known to express abnormally high levels of folate receptors on the cell surface. After different incubation time periods, the cells were washed and imaged in bright eld and under NIR radiation using a confocal microscope equipped with a 980 nm NIR laser (Fig. 5). NIR-to-visible upconversion uorescence was clearly observed from both cells, demonstrating that the nanoparticles are useful for live cell imaging. The images also showed the attachment of the nanoparticles onto the surface of the cells. While the binding of the nanoparticles to the surface of the cells is rapid and extensive uorescence can be detected within an hour, uptake into cells through the internalization of the bound receptornanoparticle complex is a much slower process and can usually be detected in signicant amount only after 2448 h of incubation. Because biological samples have very low absorption to 980 nm NIR light, the autouorescence (background noise) from cells is very low and as such the detection is sensitive. To determine non-specic binding of the nanoparticles, retention fraction of nanoparticles on cells after incubation under different conditions were tested. Folic acid coated

nanoparticles were found to be retained more than uncoated nanoparticles by HT29 cells. This retention is antagonized by excess free folic acid in the medium, proving that specic binding takes place through the folic acid receptor (data not shown). 3.4. Fluorescence imaging of deep tissues in animal To demonstrate the effectiveness of using the nanoparticles for imaging of deep tissues in animal, anaesthetized Wistar rats were injected subcutaneously at the groin and upper leg regions with 100 ml of PEI/NaYF4:Yb,Er nanoparticles (4.4 mg ml1). The depth of injection was estimated from needle penetration. Nanoparticles injected subcutaneously into the groin and upper leg of Wistar rats showed visible uorescence from a depth of upto 10 mm (Fig. 6). The uorescence intensity varied with tissues, the muscles with skin removed showing a much stronger uorescence from deep injections than intact skin at similar depths. Studies using green-emitting QD showed uorescence only through the translucent skin of the rat foot. Much can be done to improve the animal experiments: to develop a mobile imaging platform to record whole body scans of the animals; to optimize laser power for maximal luminescence with minimal tissue damage; to continuously image subcutaneous and muscular tissues in real time, etc. To the best of our knowledge, this represents the rst study of two-photon imaging of live cells and deep tissues in animal using such lanthanide doped nanoparticles. In the clinical setting, these nanoparticles may nd use in dermal or sub-dermal imaging or photodynamic therapy of pathologic conditions like melanomas, warts, and burns, as well as for mucosal and sub-mucosal pathologies. Continuous live imaging of tissues in small animal models

Fig. 5. Bright eld, confocal, and superimposed images of live human ovarian carcinoma cells (OVCAR3, top row) and human colonic adenocarcinoma cells (HT29, bottom row), with PEI/NaYF4 nanoparticles attached. The nanoparticles were surface modied with folic acid.

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Fig. 6. In vivo imaging of rat: quantum dots (QDs) injected into translucent skin of foot (a) show uorescence, but not through thicker skin of back (b) or abdomen (c); PEI/NaYF4:Yb,Er nanoparticles injected below abdominal skin (d), thigh muscles (e), or below skin of back (f) show luminescence. QDs on a black disk in (a, b) are used as the control.

can be utilized in monitoring of tumors and exploration of pathologies without unnecessary sacrice of animals particularly important where temporal series of data are necessary. 4. Conclusion The development of upconverting phosphor reporter particles has added a powerful tool to modern detection technologies. These materials absorb two or more photons of incident energy and discharge the added energy as emission with higher wavelengths than absorbed radiation, the so called upconversion process. While this technology has been known for several decades now, the use of nanosized upconverting particles in biology is a relatively recent phenomenon. Since most biological tissues have minimal absorbance of NIR light, most such reporter labels have been constructed to absorb in the NIR range and emit in the visible range. We report the physical characterization, biocompatibility, tissue distribution, and application of polyethylene-imine (PEI) coated NaYF4: Yb3+,Er3+ upconversion nanoparticles for in vitro imaging of cancer cells and in vivo imaging in tissues. We

demonstrate high uorescent detection sensitivity of the particles in these conditions using continuous wave infrared laser stimulation. To the best of our knowledge, this represents the rst report of in vivo imaging of upconversion nanoparticles in small mammals. Future work will involve targeted localization of these particles to tumors in vivo in small animals as a prelude to use in higher animals and humans. Acknowledgments We acknowledge the nancial support from Singapore A*STAR BMRC (Grant number R397-000-624-305) and National University of Singapore. References
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