Monochromatic Near-Infrared to Near-Infrared Upconversion

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Monochromatic Near-Infrared to Near-Infrared Upconversion Nanoparticles for High-Contrast Fluorescence Imaging Jiayin Zhang,† Hua Zhao,‡ Xitian Zhang,§ Xuanzhang Wang,§ Hong Gao,*,§ Zhiguo Zhang,*,† and Wenwu Cao*,†,∥ †

Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin 150080, China School of Materials and Engineering, Harbin Institute of Technology, Harbin 150001, China § Key Laboratory for Photonic and Electronic Bandgap Materials, Ministry of Education, Harbin Normal University, Harbin 150025, China ∥ Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡

S Supporting Information *

ABSTRACT: To develop a fluorescent bioprobe for high-contrast deep tissue fluorescence imaging, monochromatic 800 nm upconversion emissions based on NaYF4: Yb3+, Tm3+ upconversion nanoparticles are investigated. The ratio of I800 to I470, which is used to describe the monochromaticity, showing exponential growth with the increase of Tm3+ doping concentration in NaYF4: Yb3+, Tm3+ nanoparticles, can reach as high as 757 at 4% Tm3+. At such a doping level, the absolute quantum efficiency can reach 3.9 × 10−3 as measured by a calibrated integrating sphere, which is sufficient for fluorescence imaging. Highcontrast fluorescence phantom imaging was obtained by adjusting monochromaticity of 800 nm upconversion emission under the excitation of a 980 nm diode laser.

1. INTRODUCTION Fluorescence imaging plays an important role for monitoring physiological processes in living cells, tissues, and organisms because of its high resolution and high sensitivity.1−7 The properties of fluorescent bioprobes directly affect the quality of fluorescence imaging. A sensitive fluorescent bioprobe should have the following properties:8 (i) deep penetration of excitation and emission lights with reduced scattering and absorption in tissues; (ii) high quantum efficiency and strong photostability; (iii) nontoxicity. Traditional fluorescent materials, such as organic dyes and quantum dots, are based on a downconversion process. The excitation and emission are in the visible range, leading to low tissue penetration and poor contrast due to light scattering and tissue autofluorescence.9 Furthermore, they are not suitable for long-term living cell imaging because of photodamage caused by high energy excitation. Upconversion nanoparticles (UCNPs) with nearinfrared (NIR) to visible luminescence have been studied for vivo imaging in recent years, which show enhanced contrast and improved penetration owing to the NIR excitation within the “optical transmission window” of tissues from 800 to 1000 nm.10−14 However, the emissions fall into the visible range, which are difficult to be detected because of scattering, thus affecting the penetration depth and the contrast of fluorescence imaging. In addition, some emissions in the visible range can cause autofluorescence,15,16 which also depress the quality of fluorescence imaging. It is, therefore, desirable to design both © 2014 American Chemical Society

the excitation and emission within the NIR range, which could achieve the goal of deep penetration and high-contrast imaging. NaYF4: Yb3+, Tm3+ UCNPs as promising fluorescent bioprobes can realize the NIR to NIR upconversion luminescence. Nyk et al. have high-contrast bioimaging in vitro and in vivo using NIR to NIR Tm3+ and Yb3+ doped NaYF4.17 More recently, Chen et al. reported high-contrast deep tissue bioimaging based on NIR to NIR of NaYF4: Tm3+/ CaF2 core/shell UCNPs.18 However, impurity band emissions (mixtures of all the band emissions except the emission of 800 nm), especially at 470 nm, still exist in their nanoparticles, which would bring scattering and reduce the contrast of fluorescence imaging. In addition, it could serve as the excitation source built-in tissue, producing autofluorescence, leading to less ideal bioimaging. Although Wong et al. has explored pure NIR to NIR upconversion nanoparticles based on criteria of I800/I470 with some degree of improvement (I800/ I470 = 105),15 the specific link between monochromaticity and doping concentration have not been optimized and the fluorescence imaging with pure NIR to NIR upconversion nanoparticles has not been investigated. In this work, we have developed uniform NaYF4: Ybx3+, Tmy3+ (x = 20%, y = 0.3%, 1%, 2%, 3%, 4%) nanoparticles with Received: November 8, 2013 Revised: December 22, 2013 Published: January 9, 2014 2820

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particle size of ∼18 nm. With the increase of Tm3+ doping concentration, the monochromaticity of 800 nm upconversion emission was investigated. The prominent physical mechanism to dominate the monochromaticity of 800 nm emission has been analyzed. The quantum efficiencies of nanoparticles with the emission at 470 and 800 nm were also measured. Furthermore, the advantage offered by the fluorescence imaging with monochromatic 800 nm emission of the NaYF4: 20% Yb3+, 4% Tm3+ UCNPs was tested in phantom imaging; a much higher contrast image was obtained.

2. EXPERIMENTAL SECTION 2.1. Sample Preparation. All the samples were synthesized by solvothermal method. The synthesis process can be described as follows: Yttrium chloride, ytterbium chloride, and thulium chloride in typical stoichiometric amounts mixed with 6 mL of oleic acid and 15 mL of 1-octadecene were added into a 50 mL round-bottom flask. N2 was used as a shielding gas. The reactions were stirred vigorously at 160 °C for 1 h to get a clear solution. Thereafter, the solution was cooled down to 50 °C, followed by the addition of NaOH (0.1 g) and NH4F (0.148 g) in 3 mL of methanol and stirring for 30 min. Then the methanol and water were evaporated at 100 °C under a strong stream of N2, followed by heating the solution to 310 °C, with constant stirring for 90 min. Finally, the mixture was cooled down to room temperature and precipitated by ethanol. The synthesized nanoparticles were washed several times to remove the impurities and collected by centrifugation. After purification, nanoparticles were dispersed into cyclohexane. 2.2. Characterizations. X-ray diffraction (XRD) was obtained using Rigaku D/MAX-2600/PC with Cu Kα radiation (λ = 1.5406 Å) at the scanning step at 0.02°. Transmission electron microscope (TEM) image was obtained using FEI Tecnai TF20. The fluorescence spectra were measured at room temperature using 60 mW power-controlled 980 nm laser diode as an excitation source. A monochromator (Zolix Instrument SBP 300) coupled with photomultiplier (Zolix Instrument PMTH-S1-CR131) was used to collect the fluorescence. All fluorescence spectra were measured using the same measuring system, which was calibrated by a standard lamp. The timeresolved spectra of 2F5/2 → 2F7/2 (980 nm) of Yb3+ and 1G4 → 3 H6 (470 nm) of Tm3+ transitions in NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) nanoparticles were measured under the excitation of 808 and 980 nm in pulse mode, respectively. The spectrometer used is the same as that for the measurement of fluorescence spectra. For the detection, photomultiplier (Zolix Instrument PMTH-S1-CR131) and NIR sensitive detector (Zolix Instrument D InGaAs 2600TE) were employed for the decay profile measurements of 1G4 → 3H6 (470 nm) of Tm3+ and 2F5/2 → 2F7/2 (980 nm) of Yb3+ transitions, respectively. The decay profiles were recorded by a Tektronix TDS 5052 digital oscilloscope.

Figure 1. (a) TEM image of NaYF 4 : 20% Yb 3+ , 4% Tm 3+ nanoparticles. (b) HRTEM image of NaYF4: 20% Yb3+, 4% Tm3+ nanoparticles. (c) Corresponding XRD pattern.

nm). The X-ray powder diffraction (XRD) analysis of NaYF4: 20% Yb3+, 4% Tm3+ was performed to get the crystal structure of the as-synthesized products. The XRD pattern shown in Figure 1c corresponds well with the JCPDS card 16-334 of βNaYF4, which indicates that the products are pure hexagonal phase nanocrystals. Figure 2a presents the fluorescence spectrum of NaYF4: 20% Yb3+, 4% Tm3+ UCNPs. Only one peak at NIR 800 nm can be observed, which is an ideal case for high-contrast deep tissue fluorescence imaging. To understand the prominent physical mechanism to produce the monochromatic 800 nm emission, a series of emission spectra with different Tm3+ doping concentration are measured (Figure 2b). Here, only the main emission band of 800 nm and the impurity emission band of 470 nm are considered; other impurity emission bands at 650, 720, and 700 nm can be neglected because they are much lower in intensity compared with that of the emission of 470 nm and they do not bring noticeable autofluorescence and scattering in tissues. When the 800 nm emission is normalized, we found that the relative intensity of the 470 nm emission decreases

3. RESULTS AND DISCUSSION The NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) UCNPs (Figure S1 in the Supporting Information) were synthesized by solvothermal method. Parts (a) and (b) of Figure 1 show typical TEM and HRTEM images of NaYF4: 20% Yb3+, 4% Tm3+, respectively. The average diameter of uniform UCNPs is about 18 nm (Figure 1a). Figure 1b shows the NaYF4: 20% Yb3+, 4% Tm3+ nanoparticles with good crystallinity as indicated by (1120̅ ) lattice fringes (d112̅0 = 0.297 2821

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Figure 3. Energy levels diagram of the Yb3+ and Tm3+ system and the proposed energy transitional mechanisms of the upconversion process.

(1G4−3F4) and 720 nm (1G4−3H5) also depend on the population of 1G4, so they are also quenched. Because of the thermal distribution between 3F2 and 3F3,20 the emission of 700 nm (3F3−3H6) still exists. To understand the monochromaticity of the 800 nm emission theoretically, rate equations in the steady states based on the process of energy transitions are given as follows (Figure 3): dN1 N = 0 = W0NYb1N0 − W1NYb1N1 − 1 dt τ1

(1.1)

dN2 N = 0 = W1NYb1N1 − W2NYb1N2 − 2 dt τ2

(1.2)

dN3 N = 0 = W2NYb1N2 − 3 dt τ3

(1.3)

dNYb1 =0 dt = ρσ YbNYb0 − W0NYb1N0 − W1NYb1N1 − W2NYb1N2 N − Yb1 τYb1 (1.4)

Figure 2. (a) Upconversion emission spectrum of NaYF4: Yb3+, Tm3+ with the doping concentration of Tm3+ at 4%. (b) Fluorescent spectra of NaTmxYb0.2Y0.8−xF4 (x = 0.003, 0.01, 0.02, 0.03, 0.04). (C) Intensity ratio of 800 and 470 nm emissions in NaTmxYb0.2Y0.8−xF4 (x = 0.003, 0.01, 0.02, 0.03, 0.04).

where Ni (i = 0, 1, 2, 3) are populations of the 3H6, 3F4, 3H4, and 1G4 states of the Tm3+ ions, respectively; NYb0 and NYb1 are the populations of ground and exited states of Yb3+, respectively; τx are the decay times of relevant levels; Wi are the energy transfer rates from the excited state of Yb3+ to the Ni levels of Tm3+; ρ denotes the laser photon number density; σYb is the absorption cross section of Yb3+. In the steady state equations above, all terms about upconversion would be neglected in eq 1.4 because of their contribution being much less than that of the pump laser. The upconversion terms W1NYb1N1 in eqs 1.1 and W2NYb1N2 in eq 1.2 are also neglected owing to the fact that the rates of energy transmission in the upconversion processes are much less than the decay rates of relevant energy levels. In this way, according to eqs 1.1−1.4, the populations (Ni) of N2 and N3 become

with the increase of Tm3+ doping concentration. The ratio of I800/I470 shows exponential growth with the increasing Tm3+ doping concentration (Figure 2c). It can reach as high as 757 in NaYF4: 20% Yb3+, 4% Tm3+, which is 7 times higher than that of GdF3: Yb3+, Tm3+ excited by 980 nm with the power of 90 mW.15 The emissions of 470 and 800 nm arise from the 1G4 → 3 H6 and 3H4 → 3H6 transitions of Tm3+ (Figure 3), respectively.19 With the increase of Tm3+ doping concentration, the distance between the Tm3+ ions becomes closer and thus the cross-relaxation between 1G4 → 3H4 and 3F4 → 3F2 is enhanced. In this way, the population of 1G4 would decrease, leading to the 470 nm (1G4 → 3H6) emission being quenched, while the population of 3H4 would increase, resulting in the emission of 800 nm (3H4 → 3H6) being preserved and a high I800/I470 ratio. Furthermore, impurity emissions of 650 nm 2822

N2 = ρ2 σ Yb 2W0W1N0(NYb0τYb1)2 τ1τ2 ∝ I800

(2.1)

N3 = ρ3 σ Yb3W0W1W2N0(NYb0τYb1)3 τ1τ2τ3 ∝ I470

(2.2)

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Here, all the parameters can be regarded as constants except τx. On the basis of eq 2, the theoretical description of the I800/I470 may be written as follows (eq 3): I470/I800 ∝ τYb1τ3

shown in Figure S2 and Figure S3, respectively. Quantum efficiencies of NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) with the emissions at 470 and 800 nm were measured under 980 excitation with the power density of 96 W/cm2, as shown in Figure 5. With the increase of Tm3+

(3)

Figure 4 shows typical decay profiles of F5/2 → F7/2 (980 nm) of Yb3+, and 1G4 → 3H6 (470 nm) of Tm3+ transitions in 2

2

Figure 5. Under the excitation of 980 nm, upconversion luminescence quantum efficiencies of NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) nanoparticles with the emission at 470 and 800 nm.

doping concentration, the quantum efficiency of nanoparticles with the emission at 470 nm decreases rapidly, while quantum efficiency of nanoparticles with the emission at 800 nm changes very slowly. When the doping concentration reaches 4%, the quantum efficiency of nanoparticles with the emission at 470 nm reduces to 7 × 10−7, which is much lower than the efficiency of 3.9 × 10−3 at 800 nm. In this case, the 800 nm emission can be treated as monochromatic and its efficiency is sufficient for fluorescence imaging. Fluorescence images of NaYF4: 20% Yb 3+ , 4% Tm 3+ nanoparticles with monochromatic emission of 800 nm and NaYF4: 20% Yb3+, 0.3% Tm3+ nanoparticles with mixed emissions of 800 and 470 nm were compared. The experimental system is depicted in Figure 6. The expanded

Figure 4. Typical time-resolved spectra of 1G4 → 3H6 (470 nm) of Tm3+ and 2F5/2 → 2F7/2 (980 nm) of Yb3+ transitions in NaYF4: 20% Yb3+, 0.3% Tm3+ and instrument response functions.

NaYF4: 20% Yb3+, 0.3% Tm3+ and the instrument response function. The decay times between relevant energy levels and theoretical results of I800/I470 in NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) are shown in Table 1. The decay Table 1. Decay Times of 1G4 → 3H6 (τ3) of Tm3+ and 2F5/2 → 2F7/2 (τYb1) of Yb3+ Transitions in NaY0.8−xF4: Yby, Tmx (y = 20%; x = 0.3%, 1%, 2%, 3%, 4%) Nanoparticles and Corresponding Theoretical and Experimental Results of I800/I470 I800/I470

lifetime 3+

concentration Tm 0.003 0.01 0.02 0.03 0.04

τYb1 (ms) 1.77 1.07 0.80 0.58 0.39

τ3 (ms) 0.95 0.47 0.35 0.26 0.16

theory 31 103 185 344 833

a

experiment 31 122 209 416 757

Theoretical values for I800/I470 can be described by 1/τYb1τ3. To compare with the experimental values of I800/I470, 1/τYb1τ3 were multiplied by a constant (52), which was calculated by the experimental I800/I470 divided by the theoretical I800/I470 at Tm3+ concentration of 0.3%. a

times of Yb3+ (2F5/2 → 2F7/2) and Tm3+ (1G4 → 3H6) decrease with the increase of Tm3+ doping concentration, which is consistent with the report by Prorok et al.21 The I800/I470 increases rapidly with the increase of Tm3+ doping concentration. Quantum efficiency as an important indicator for the quality of a fluorescent bioprobe should be measured. We employed the absolute method to conduct the measurement of quantum efficiency, as described by Mello et al.22 The measuring system was calibrated by a standard lamp to get the system response functions, which were used to calibrate the fluorescence spectrum. Method for the measurement of absolute quantum efficiency of nanoparticles and a typical complete spectrum are

Figure 6. Schematic of the fluorescence imaging system.

980 nm laser diode with a power of 60 mW as excitation illuminated the phantom from below. Two identical tissue phantoms were prepared with the thickness of 10 mm and the diameter of 5.5 cm, including intralipid 10% and water 90%. The optical parameters of tissue phantoms referred to the results reported by van Staveren et al.23 At the excitation wavelength, the tissue phantoms have a reduced scattering coefficient μs′ (980 nm) = 6.83 mm−1 and an absorption coefficient of μa (980 nm) = 0.26 mm−1. At the emission 2823

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Figure 7. (a) and (b): Comparing fluorescence imaging of nanoparticles with and without impurity band emissions. (c) and (d): Corresponding fluorescence intensity distribution of cross sections in (a) and (b).

superior to other UCNPs with multicolor fluorescence in terms of improving the contrast of fluorescence imaging.

wavelengths of interest, the reduced scattering coefficients μs′ (470 nm) = 16.42 mm−1, μs′ (800 nm) = 8.93 mm−1 and the absorption coefficients μa (470 nm) = 0.016 mm−1, μa (800 nm) = 0.070 mm−1. To mimic the tissue autofluorescence, 2 mL of 40 μM/L water-soluble rhodamine B was added into both phantoms, respectively. There were two of the same capillary tubes with inner diameter of 0.9 mm and outer diameter of 1.1 mm submerged into the liquid phantoms, respectively. The same submerged depth of the two capillary tubes was 4 mm to the upper surface of the liquid phantom. The nanoparticles with monochromatic emission of 800 nm and mixed emissions of 800 and 470 nm were dissolved into cyclohexane with the same concentration of 1 wt % and each was separately filled into a capillary tube. Complementary Metal Oxide Semiconductor (CMOS) with a series of lens and a short-pass filter were used to capture the images. A short-pass filter with the cutoff wavelength 900 nm was put in front of CMOS to filter the excitation of 980 nm. The optical density of the filter is no more than 2. Parts (a) and (b) in Figure 7 show fluorescence imaging of nanoparticles with and without impurity band emissions, respectively. As expected, the imaging with mixture fluorescence of 800 and 470 nm shows strong background due to scattering and autofluorescence (Figure 7a), while the imaging with monochromatic emission of 800 nm shows much higher contrast and better spatial resolution (Figure 7b). Parts (c) and (d) of Figure 7 show the fluorescence intensity distribution of cross-section profiles in parts (a) and (b), respectively, of Figure 7. The Full Wave at Half Maximum (FWHM) of fluorescence intensity distribution of nanoparticles with monochromatic emission of 800 nm is much smaller than that with mixed emissions of 470 and 800 nm, with an improved factor of 1.45. In fluorescence imaging, the contrast of imaging depends on fluorescence properties of bioprobes. Decreased scattering and autofluorescence in tissues would lead to higher contrast fluorescence imaging. Therefore, NaYF4: 20% Yb3+, 4% Tm3+ nanoparticles with monochromatic fluorescence at 800 nm are

4. CONCLUSIONS Monochromaticities of 800 nm emission in NaYF4: Yb3+, Tm3+ nanoparticles were investigated for different Tm3+ doping concentrations. With increasing of the Tm3+ doping concentration, monochromaticity of emission at 800 nm is improved. When Tm3+ doping concentration reaches 4%, nanoparticles show excellent monochromatic 800 nm emission with the I800/ I470 ratio of 757. At such doping level, the quantum efficiency of nanoparticles with the emission at 470 nm is much smaller than the 3.9 × 10−3 for the emission at 800 nm. The tissue imaging using monochromatic 800 nm emission exhibits much higher contrast compared with the imaging using mixed fluorescence of 800 and 470 nm due to the elimination of autofluorescence and reduced scattering. In this sense, our NaYF4: 20% Yb3+, 4% Tm3+ nanoparticles with monochromatic upconversion emission of 800 nm provide a promising agent for high-contrast fluorescence imaging.

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ASSOCIATED CONTENT

S Supporting Information *

TEM images of NaYF4: Yb, Tm nanoparticles with the Tm3+ doping concentration of (a) 0.3%, (b) 1%, (c) 2%, and (d) 3%; method to measurement of quantum efficiency and typical complete spectra for the measurement of absolute quantum efficiency of nanoparticles NaYF4: 20% Yb3+, 4% Tm3+. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 045188060760. *E-mail: [email protected]. Phone: 0451-86402639. *E-mail: [email protected]. Phone: 0451-86402639. Notes

The authors declare no competing financial interest. 2824

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(16) Prasad, P. N. Introduction to Biophotonics; John Wiley & Sons, Inc.: Hoboken, NJ, 2003. (17) Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. High Contrast in Vitro and in Vivo Photoluminescence Bioimaging Using Near Infrared to Near Infrared Up-Conversion in Tm3+ and Yb3+ Doped Fluoride Nanophosphors. Nano Lett. 2008, 8, 3834−3838. (18) Chen, G. Y.; Shen, J.; Ohulchanskyy, T. Y.; Patel, N. J.; Kutikov, A.; Li, Z. P.; Song, J.; Pandey, R. K.; Agren, H.; Prasad, P. N.; et al. (αNaYbF4:Tm3+)/CaF2 Core/Shell Nanoparticles with Efficient NearInfrared to Near-Infrared Upconversion for High-Contrast Deep Tissue Bioimaging. ACS Nano 2012, 6, 8280−8287. (19) Wang, F.; Liu, X. G. Upconversion Multicolor Fine-Tuing: Visible to Near-infrared Emission from Lanthanide Doped NaYF4 Nanoparticles. J. Am. Chem. Soc. 2008, 130, 5642−5643. (20) Xu, W.; Gao, X. Y.; Zheng, L. J.; Zhang, Z. G.; Cao, W. W. An Optical Temperature Sensor Based on the Upconversion Luminescence from Tm3+/Yb3+ Codoped Oxyfluoride Glass Ceramic. Sens. Actuators, B 2012, 173, 250−253. (21) Prorok, K.; Gnach, A.; Bednarkiewicz, A.; Strek, W. Energy Upconversion in Tb3+/Yb3+ Codoped Colloidal Alpha-NaYF4 Nanocrystals. J. Lumin. 2013, 140, 103−109. (22) Mello, J. C.; Wittmann, H. F.; Friend, R. H. An Improved Experimental Determination of External Photoluminescence Quantum Efficiency. Adv. Mater. 1997, 9, 230−232. (23) van Staveren, H. J.; Mode, C. J. M.; van Marle, J.; Prahl, S. A.; van Gemert, M. J. C. Light Scattering in Intralipid-10% in the Wavelength Range of 400−1100 nm. Appl. Opt. 1991, 30, 4507−4514.

ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program (973 Program) under contract number 2013CB632900.

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