NANO LETTERS
In Vivo and Scanning Electron Microscopy Imaging of Upconverting Nanophosphors in Caenorhabditis elegans
2006 Vol. 6, No. 2 169-174
Shuang Fang Lim,*,† Robert Riehn,† William S. Ryu,‡ Nora Khanarian,§ Chih-kuan Tung,† David Tank,† and Robert H. Austin† Department of Physics, Princeton UniVersity, Princeton, New Jersey 08544, Lewis-Sigler Institute for IntegratiVe Genomics, Princeton UniVersity, Princeton, New Jersey 08544, and Engineering and Applied Science, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104 Received September 27, 2005; Revised Manuscript Received November 16, 2005
ABSTRACT We show here that upconversion phosphors can be imaged both by infrared excitation and in a scanning electron microscope. We have synthesized and characterized for this work up-converting phosphor nanoparticles nonaggregated nanocrystals of size range 50−200 nm. We have investigated the optical properties of 50−200 nm nanoparticles and found a square dependence of the emitted visible fluorescence on the infrared excitation and verified that under electron excitation similar narrow band emission spectra can be obtained as is seen with IR upconversion. The viability of the nanoparticles for biological imaging was confirmed by imaging the digestive system of the nematode worm Caenorhabditis elegans, and we have confirmed using energy-dispersive X-ray analysis that the up-conversion nanoparticles can be identified in a scanning electron microscope at high spatial resolution.
Upconverting phosphors (UCP) are ceramic materials in which rare earth atoms are embedded in a crystalline matrix. The materials absorb infrared radiation and upconvert to emit in the visible spectrum through a series of real as opposed to virtual levels as in conventional two-photon dyes. The upconversion mechanism can be described as either sequential excitation of the same atom or excitation of two centers and subsequent energy transfer.1-4 The emission of UCPs consists of sharp lines characteristic of atomic transitions in a well-ordered matrix. By use of different rare earth dopants, a large number of distinctive emission spectra can be obtained. The UCP's high IR-visible conversion cross section makes them virtually background-free markers. Note that UCPs emit under IR, visible, UV, and even electron irradiation, as we show in this paper. Fluorescent markers are commonly used for imaging biological samples, which lack intrinsic contrast mechanisms for optical microscopy.5 Traditional organic dyes and fluorescent proteins have been used successfully for in vivo imaging but suffer from a high bleaching rate when used in high-intensity cell imaging studies, thus making long-term experiments unfeasible. Incorporating fluorescent dyes into * Corresponding author.
[email protected]. † Department of Physics, Princeton University. ‡ Lewis-Sigler Institute for Integrative Genomics, Princeton University. § Engineering and Applied Science, University of Pennsylvania. 10.1021/nl0519175 CCC: $33.50 Published on Web 12/23/2005
© 2006 American Chemical Society
nanoparticles can reduce the bleaching problem.6 Unfortunately, their broad emission bands limit the number of colors that can be clearly discriminated within a single experiment during multicolor imaging. The above shortcomings have been overcome by quantum dots.7 However, their toxic constituents and poor biocompatibility have made their use as in vivo markers challenging until recently. An advantage of UCPs is that they are not likely to be toxic, unlike selenium-containing quantum dots.8 The LD50 for rare earth oxides is on the order of 1000 mg/kg,9 while the LD50 values for many selenium oxides is on the order of 1 mg/kg. Previous demonstrations of UCPs for biological imaging have concentrated on using micrometer-sized particles in connection with in vitro immunolabeling of DNA. We demonstrate here in vivo imaging using upconverting nanoparticles.2,3,10 We have synthesized particles with 50-250 nm diamters and characterized their optical and upconverting properties. We have used these phosphors to image the digestive system of the nematode C. elegans and find that labeled individuals survive for days.11 C. elegans was chosen because of the size amenable to optical microscopy. The short life cycle and rapid growth enable quick chartering of genetic mutations.11 The phosphors were prepared by homogeneous precipitation.12-14 An aqueous solution of Y(NO3)3‚6H2O (50 mM),
Figure 1. SEM images of (A) 150 nm and (B) 50 nm sized particles.
Yb(NO3)3‚5H2O (1 mM), Er(NO3)3‚5H2O (0.5 mM), and urea (15 mM) (all Sigma-Aldrich, St. Louis, MO) was heated to boiling with vigorous agitation, which led to thermal hydrolysis. The premixing of the reactants prior to hydrolysis reduced the possibility of any concentration gradient, ensuring that precipitates formed had a narrow size distribution. The reaction was stopped by lowering the temperature of the solution in an ice bath. The size of the precipitates was controlled by the concentration of the salts and the time of the reaction. The resulting precipitate was then washed six times with deionized water, followed by centrifugation after every wash. The product was dried at 150 °C for 2 h, and the crystalline oxide was obtained by annealing at 1000 °C for 2 h. UCPs synthesized under these conditions exhibit green upconversion. A similar synthesis with a different relative rare earth concentration yields red upconversion. We imaged nanoparticles in a secondary-electron microscope (Philips XL30, FEI, Hillsboro, OR) with a 10 kV electron beam after coating the particles with a 5 nm gold film. Energy-dispersive X-ray spectrometry (EDX) was conducted on a PGT-IMIX PTS EDX system in order to perform elemental analysis as well as mapping. N2 wild type C. elegans were grown on nematode growth medium (NGM)-agar plates at 25 °C, which had been seeded with E. coli strain OP50 that had been cultured in 1.05 L Broth. The OP50 strain was cultured in L Broth at 37 °C overnight. A phosphor dispersion consisting of 0.5 mg of phosphor, with a mean particle size of 150 nm, was prepared in 1.0 mL of NGM buffer (3 g of NaCl, 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 25 mL of 1 M KPO4 buffer, 975 mL of deionized (DI) water, Sigma Aldrich). The phosphors were dispersed by sonication and pipetted onto a C. elegans dish that was 72 h old, allowing for 3 h of uptake. For IR imaging purposes, suitable worms were transferred into an Eppendorf tube containing NGM buffer and concentrated by short centrifugation. They were then pipetted onto an agar bed that was afterward sandwiched between two cover slips. A sufficient amount of sodium azide was added in order to immobilize the worms. For SEM imaging, 100 µL of poly-L-lysine solution (0.1% w/v in water and 0.01% Thimerosal, Sigma Aldrich) was applied onto a precleaned glass slide and air-dried over 30 h. Subsequently, another 50 µL of poly-L-lysine was applied over the previously dried layer, followed immediately by transferring of the C. elegans from agar plates under sterile conditions onto the liquid poly-L-lysine layer and allowed to air-dry over 24 h. A final 50 µL aliquot of poly-L-lysine was applied 170
Figure 2. Distribution of particle sizes in Figure 1A with a mean diameter of 154 nm and standard deviation of 40 nm.
onto the C. elegans/poly-L-lysine and air-dried. Dehydration was performed through a series of ethanol/water mixtures, beginning with 25%, 50%, and 100% ethanol (anhydrous, 200 proof, 99.5%, Sigma Aldrich). About 50 ul of ethanol/ water mixture was applied each time, followed by air-drying before the next application. The glass slides were cleaved into 1 cm squares and mounted onto aluminum stubs with the use of carbon tape. Graphite adhesive was also applied to the edges of the substrates in order to enhance charge dissipation. The mounted substrates were then coated with 4 nm thick iridium in order to prevent charging during imaging. Imaging of the C. elegans by upconversion luminescence with IR excitation was performed using a inverted microscope with a 20×, 0.4 NA microscope objective (Nikon, Melville, NY), coupled to an intensified CCD camera (Princeton Instruments, Trenton, NJ). The worms were imaged in both bright-field and epifluorescence geometries. The latter was enabled by a custom-made fluorescence filter set (Chroma technology, Rockingham, VT), and a 20-W infrared LED laser array. The illumination intensity was about 10 W/mm2. The dependence of the luminescence intensity was determined by integrating the emission from one particle in the field of view and varying the illumination intensity. Upconversion luminescence spectra were collected using a fiber-coupled CCD spectrometer (Ocean Optics, Dunedin, FL). We show SEM images of 150 and 50 nm diameter sized, spherical shaped nanoparticles in Figure 1. We have synthesized particles ranging between 50 and 250 nm by varying the time of the nucleation reaction and the concentration of the nitrates used. In Figure 2 we present a histogram of particle sizes after synthesis that gave an average particle size of 150 nm with a standard deviation of 40 nm. We believe that the width of the distribution is, in large part, Nano Lett., Vol. 6, No. 2, 2006
Figure 3. The emission spectra of differently doped 200 nm size nanoparticles, showing (A) red and (B) green emission. Figure 5. Cathodoluminescence spectrum of green Y2O3: Yb, Er nanoparticles obtained at 30 keV acceleration.
Figure 4. The power-law dependence of phosphor luminescence on IR intensity.
due to the annealing step. There appears to be less aggregation with vacuum annealing. Most clusters and aggregates could be removed by centrifugation. Vigorous probe tip sonication also markedly reduces loose aggregate formation. Presently, synthesis has been shifted to flame spray pyrolysis with gradual improvement and upgrades in technical equipment. Optimization of process and materials include better control of parameters such as gas flow rate, heating temperature stability, etc. Downstream processes such as sequential centrifugation and filtration also enhance uniformity and are under development. The upconversion fluorescence spectrum of 200 nm diameter sized particles is shown in Figure 3. We find that the emission spectrum of phosphors synthesized with higher Er dopant concentration is dominated by red spectral lines, while the emission of phosphors synthesized with lower Er levels exhibited strong green emission. Emission bands consist of multiplets of sharp transition lines. We attributed the red transition to the Er3 +:4F9/2 f 4I15/2, and the green transition to Er3 +:4S3/2 f 4I15/2.15-17 The dependence of the fluorescence intensity on the illumination power is plotted in Figure 4. We find a power-law dependence of the luminescence on the IR-illumination intensity, with an exponent of 1.88. The imaging of C. elegans was performed at the high-power end of the presented curve. It is difficult to compare the quantum efficiency of a nonlinear material such as a UCP with a linear material such as a quantum dot or an organic dye since there is no welldefined value for the quantum efficiency for a nonlinear material. We have not measured the luminescence efficiencies ourselves, but note that Page et al.18 have conducted a study using materials similar to ours. They arrive at a maximum power efficiency of 1% (power emitted to power absorbed) for a chemically similar material. We have Nano Lett., Vol. 6, No. 2, 2006
Figure 6. Absorption spectrum of 150 nm size nanoparticles in PBS buffer.
Figure 7. False color two-photon images of C. elegans at 980 nm excitation with red representing the bright field and green for the phosphor emission. The worms were deprived of food over a period of 24 h, showing little or no change at (a) 0 h, (b) 4 h, and (c) 24 h.
calculated from Page’s result that a 150 nm diameter UCP particle can emit up to 104 photons/s before saturation of the intermediate state levels. As it is our aim to develop these materials for highresolution imaging, the cathodoluminescent (CL) properties 171
Figure 9. SEM image of phosphor-fed worm at (A) 336 and (B) 671 times magnification at 20 kV acceleration voltage.
Figure 8. False color two-photon images of C. elegans at 980 nm excitation with red representing the bright field and green for the phosphor emission. The worms were given food immediately after being fed with phosphors, showing decreasing amounts of phosphors at (a) 0 h, (b) 1 h, and (c) 2 h.
of the phosphors was investigated to ascertain if the UCP can be imaged in a scanning electron microscope. The CL spectrum measured at 30 keV electron acceleration of the green phosphors is shown in Figure 5. It is observed from the figure that emission occurs virtually from the same energy levels as during photoluminescent emission, except for differences with regards to relative intensities among the transition lines. We have measured the absorption spectrum of a particle suspension with an average particle size of 150 nm (Figure 6). The apparent absorption curve is the result of both the scattering and actual absorption of IR photons. Several absorption peaks are observed which implies that these materials can be excited both in the visible and in the IR. We successfully inoculated UPC nanoparticles into C. elegans by placing them on an agar plate that has been wetted with a 150 nm sized particle suspension in NGM buffer (Figure 7). We were able to see individual, pointlike UPC particles, and found that the imaging resolution was limited by the combination of the microscope objective and the camera. 172
The phosphors are easily visible in the intestines, with most particles found beyond the pharynx, extending to the rectum. The worms were imaged at fixed time intervals in order to track the movement of the phosphors through their digestive system. Figure 7 shows that the phosphors are retained in the worms when they are deprived of food. This is due to the inhibition of excretion as feeding ceases. These worms can be monitored up to 24 h without any apparent change in the phosphors, as in blinking or bleaching, and in the condition of the worms. On the other hand, when food is made available to the phosphor-fed worms, feeding resumes and the phosphors are secreted in under 2 h, as shown in Figure 8. Thereafter, these worms continue feeding and appear unaffected by the prior ingestion of the phosphors. Hence, it has been demonstrated that UCPs are nonbleaching, biocompatible, and nontoxic, which make them ideal candidates as biolabels. The worms were mounted onto cleaned and pretreated glass slides which ensures sticking of the worm. Systematic dehydration was carried out in a series of ethanol/water mixtures. A 4 nm thick iridium metal coating was sputtered onto the prepared worms prior to SEM imaging. Figure 9 shows SEM images of a phosphor-fed worm at different magnifications. The phosphors typically glow intensely and stably within the worm in both the secondary and backscattered (not shown) imaging mode. The phosphors are observed to glow brightly inside the worm in the SEM image at 20 Nano Lett., Vol. 6, No. 2, 2006
Figure 10. EDX spectrum of (A) worm without phosphor and (B) worm with phosphor.
Figure 11. EDX map of phosphor-fed worm: (A) SEM image, (B) Y map, (C) Yb map, and (D) Er map.
kV acceleration voltage. Figure 10 shows the energydispersive X-ray (EDX) spectra of two worms where worm b had been fed with the phosphors. The Y K, Yb L, and Er L lines were selected for EDX mapping as these peaks occur as discrete and well separated lines, unlike the Yb K and Er K lines that overlap the iridium line. The Y, Yb, and Er maps are in agreement to the SEM image, as shown in Figure 11, showing the presence of upconverting phosphor in the worm. A cross section of the same worm was performed by focused ion beam (FIB) milling and the SEM image shown in Figure 12. Due to the difference in densities between worm material and the phosphors, stalagmite structures were observed after FIB milling. This region shown here corresponds to the pharynx region of the worm. Panels A and B show the stalagmite structures observed at different magnifications, while panel C shows particle-like structures embedded within the worm. The particle-like structures appear to be about 150 nm in diameter and seem brighter than the surrounding tissue. Hence, it implies that the nanoparticles are unaffected physically by the sample Nano Lett., Vol. 6, No. 2, 2006
Figure 12. SEM images of FIB milled worm at the pharynx region (A) at 2735, (B) 5470, and (C) 21879 times magnification.
preparation techniques and also the FIB milling. Since this same region has been shown by SEM to be brighter than the surrounding worm tissue and EDX measurements to contain phosphors, the particles seen here can be directly attributed to phosphors. We have shown that upconverting phosphors can be excited in the UV and IR regions and also by electron impact. These open up new possibilities of using higher resolution imaging techniques such as SEM with UCPs used as biolabels. We are now exploring new synthesis techniques in producing bright, 50 nm diameter or smaller, nonaggregated nanoparticles. Surface functionalization and bioconjugation of the UCP nanoparticles tailored toward specific biological systems are in progress. Acknowledgment. We acknowledge financial support from the NSF and from the Keck Foundation. We thank 173
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NL0519175
Nano Lett., Vol. 6, No. 2, 2006
