Size and Shell Effects on the Photoacoustic and Luminescence

Subscriber access provided by TULANE UNIVERSITY

Article

Size and Shell Effects on the Photoacoustic and Luminescence Properties of Dual Modal Rare-Earth Doped Nanoparticles for Infrared Photoacoustic Imaging Yang Sheng, Lun-De Liao, Aishwarya Bandla, Yu-Hang Liu, Nitish V. Thakor, and Mei Chee Tan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00012 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on April 2, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Size and Shell Effects on the Photoacoustic and Luminescence Properties of Dual Modal Rare-Earth Doped Nanoparticles for Infrared Photoacoustic Imaging

Yang Sheng1, Lun-De Liao2,5, Aishwarya Bandla2,3, Yu-Hang Liu2,4, Nitish Thakor2,3,4, Mei Chee Tan1,*

1. Engineering Product Development, Singapore University of Technology and Design, Singapore, 8 Somapah Road, Singapore 487372 2. Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, 28 Medical Drive, #05-COR, Singapore 117456 3. Department of Biomedical Engineering, National University of Singapore, 21 Lower Kent Ridge Rd, Singapore 119077 4. Department of Electrical and Computer Engineering, National University of Singapore, 21 Lower Kent Ridge Rd, Singapore 119077 5. Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, 35 Keyan Rd., Zhunan Town, Miaoli County 35053, Taiwan, R.O.C.

*To whom correspondence should be addressed. Email: [email protected]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Infrared-emitting rare-earth (Ytterbium or Erbium) doped nanoparticles (REDNPs) have recently emerged as an excellent probe for both deep tissue luminescence and photoacoustic (PA) imaging with high resolutions and contrast. Here we report on the first study of the size and surface effects of the infrared PA imaging of dual modal REDNPs. We show that the PA signal amplitude generated by REDNPs is increased by increasing the size and coating the inorganic shell (undoped NaYF4 or silica). We have also discovered that the choice of the coating material is critical as undoped NaYF4 shell was able to enhance PA signal amplitude (by up to ~30%) and infrared emission (19 times) simultaneously. The simultaneous enhancement of PA signal amplitude and infrared emission was due to increased phonon modes and reduced surface effects. The in vivo PA images obtained demonstrated that in addition to being excellent luminescent probes, the REDNPs also performed as successful PA contrast agents to visualize rodent cortical blood vessels.

Key words: rare-earth, core/shell, nanoparticle, infrared, dual-modality, biomedical imaging, photoacoustic imaging.

2


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Imaging techniques have facilitated the visualization of biochemical pathways and the characterization of the structure-function relationships at the cellular and anatomical levels. However, any given individual imaging modality has its own intrinsic limitations such as low sensitivity or resolution, which make it difficult to obtain accurate and reliable information of the target tissue. To address the limitations, combination of several imaging modalities seek to simultaneously improve the resolution and signal-to-noise ratio. The combination of luminescence and photoacoustic (PA) imaging presents an inexpensive intraoperative system that can be easily adapted for surgical guidance and provide detailed anatomical information of deep tissue1-4. However, intrinsic optical contrast agents such as hemoglobin and melanin generates strong PA signals in the visible spectral region5, where light attenuation is higher than infrared region. In addition, some biological tissues such as lymphatic systems and bladders do not have intrinsic optical absorption contrast5-7. Therefore, exogenous contrast agents are needed to facilitate the visualization of the structural and functional states of tissues8, 9.

We have previously demonstrated that NaYF4 nanoparticles co-doped with ytterbium (Yb3+), erbium (Er3+) are excellent luminescent probes for deep tissue shortwave infrared (SWIR) imaging under the near infrared (NIR) excitation due to their low cytotoxicity, good biocompatibility and tunable optical properties4, 10-12. We further demonstrated that in addition to being excellent luminescence probes, the rare-earth doped micron-particles can also be used for PA imaging contrast enhancement13,14. 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Our results showed that the morphology of particles influenced the PA signal intensity and particles with high aspect ratio are preferred. However, the dimension of these micron-sized particles severely limits their application for in vivo biomedical and molecular imaging. Nanomaterials in the sub-100 nm range are needed to overcome the biological barriers such as reticuloendothelial system (RES) uptake, intracellular barrier and blood-brain barrier for effective bio-distribution15-17. To date, the size and surface effects of REDNPs on the PA signals remain unclear, and there is lack of experimental studies that demonstrate how to tailor the PA property of REDNPs.

Here, we report the first study of the size and shell effects of nano-sized NaYF4:Yb,Er REDNPs for enhancing PA signal amplitude. For the first time the luminescence intensity and PA signal amplitude have been increased simultaneously by tuning the REDNP particles size. Preliminary experiments also showed that the choice of shell material is important. For example, coating a biocompatible silica shell enhanced PA signal but reduced luminescence. In contrast, undoped NaYF4 shell was able to enhance the luminescence intensity and PA signal amplitude simultaneously. Our study demonstrates two useful strategies for increasing PA amplitude without compromising the luminescence intensity of REDNPs. In addition, in vivo PA imaging was conducted to evaluate the feasibility using core/shell REDNPs as a PA contrast agent. As PA contrast agents, our nano-sized REDNPs are able to differentiate the target cortical blood vessel from the surrounding tissue. The obvious signal enhancement from our REDNPs was demonstrated. 4


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Results Physical characterization of REDNPs. SEM images (Fig. 1a,b; Supplementary Fig. S1a,b) show the average size of the as-prepared REDNPs are 11.4 ± 3.1 nm (RE(11)), 18.4 ± 3.2 nm (RE(18)), 29.3 ± 3.8 nm (RE(29)) and 38.4 ± 3.9 nm (RE(38)). After coating with undoped NaYF4 shell, the average size of the REDNPs was increased. Figure 1c,d show there is a difference of 12 to 18 nm in average sizes between core and core/shell REDNPs, suggesting a shell of 6 to 9 nm was successfully grown on the REDNPs. As the shell thickness is uniform for all samples, the shell effects on both the luminescence emissions and PA amplitudes can be compared.

To determine the crystal structure of the as-prepared REDNPs, X-ray diffraction (XRD) was used for both core and core/shell REDNPs (Data for REDNPs sample RE(29) shown in Fig. 1e; data for other nanoparticles shown in Supplementary Fig. S2). The positions of XRD peaks correspond to the standard hexagonal NaYF4 phase, and no peak from other phases or impurities were observed. Hexagonal NaYF4 as a host material is advantageous due to its low phonon energy and multiple lattice sites for substitution with lanthanide ions10. The XRD profile shows obvious narrowing of diffraction peaks, indicating crystal size increase. Using the Scherrer equation18, the calculated grain size for the uncoated REDNPs are 12.4 ± 0.8 nm, 16.4 ± 0.4 nm, 21.9 ± 0.8 nm, and 32.5 ± 1.1 nm. The estimated crystal size is similar to the SEM 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

observation, indicating that single crystal REDNPs were synthesized.

Luminescence emissions and decay time characterization upon excitation at 975 nm. To demonstrate that the synthesized REDNPs exhibited the expected characteristic luminescence emissions upon excitation at 975 nm, we measured the steady state luminescence spectra (Fig. 2a; Supplementary Fig. S3). Both the SWIR (1530 nm) and visible (540 and 650 nm) emissions increased with core size. The integrated SWIR emission intensity of the largest core REDNPs is ~5-fold of that of the smallest (Fig. 2b). The enhancement was largely attributed to the reduced surface-to-volume ratio and increased phonon-assisted energy transfer of large nanoparticles. The growth of an inorganic NaYF4 shell also significantly enhanced the visible upconversion and SWIR downshifting luminescence of REDNPs (Fig. 2a,b). For example, a 19-fold enhancement in SWIR emission and 32-fold increase in visible emission was observed when comparing the core and core/shell systems for the REDNPs with a core size of 29 nm. The integrated emission intensities of all the core/shell REDNPs are at least 8.75 times higher than the largest core REDNPs as shown in Table 1 . Our findings suggest that coating with an undoped NaYF4 shell, the luminescence emissions are enhanced more significantly (increased by 46 times) than increasing particles size (increased by only 5 times). This is because the coating of an undoped shell effectively reduced the surface quenching effects which dominates as particle size decreases due to increasing surface-to-volume ratios. The mechanism for luminescence enhancement is discussed in further detail in the later 6


Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sections.

To understand the luminescence enhancement characteristics affected by the size and undoped shell of the rare-earth host material, the luminescence decay time was measured. Visible up-conversion emissions occur via nonlinear optical process which makes it difficult to analyze the effects on the decay time due to the interdependence of the multiple pathways19. In contrast, the down-shifting SWIR emission is a linear optical process where the emission intensity is dependent on one pathway. Therefore, the luminescence decay time of the 4I13/2 → 4I15/2 down-shifting SWIR transition was measured to study the effects of particle sizes. Figure 2c shows the measurement of time-resolved luminescence spectra of SWIR emission. To quantify the decay constants, all decay curves were fitted to a double exponential equation,

I (t ) =

 t A1 × exp  − τ1 

  + 

 t A2 × exp  − τ2 

  

(1)

where I(t) is the decaying intensity at the maximum of the emission band 1530 nm, A1 and A2 are the exponential pre-factors and τ1 and τ2 are the fitted decay times. The average decay time constant τave can be determined using the following equation20,

τ ave =

A1 τ 12 + A2 τ 22 A1 τ 1 + A2 τ 2

(2)

The fitting of all spectra were completed using Origin 8, and the fitted SWIR luminescence decay times and errors were plotted in Figure 2d and summarized in Table 2.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Decay time characterizes the contribution of non-radiative relaxation processes from a selected excited state. Generally, a long decay time indicates low non-radiative losses and strong luminescence emission, while short decay time indicates weak luminescence emission and high non-radiative losses. Therefore, the decay time can be used as an indicator for estimating the PA amplitude. As shown in Figure 2d and Table 2, the decay time was increased from 0.19±0.04 ms to 6.84±0.03 ms as the particle size increased from 11 to 38nm for uncoated REDNPs. Significant increase in decay time for the largest REDNPs (38nm) compared to that of the smallest REDNPs (11nm) suggests that particle sizes of uncoated REDNPS are critical to the PA performance. As PA signal is considered as one form of the non-radiative energy, the smaller uncoated REDNPs are expected to exhibit higher PA signal amplitude. After coating with the undoped NaYF4 shell, the luminescence decay time of all core/shell REDNPs showed a significant increase to between 9.75±0.06 and 11.11±0.51 ms. It should be noted that only an incremental increase in decay time of ~10% was observed upon comparing the smallest and largest core/shell REDNPs. The smaller increase in decay time can be attributed to effective minimization of non-radiative losses from surface quenchers with the coating of a shell. The increase in luminescence efficiency (i.e. reduced non-radiative losses) as indicated by the longer decay time would have a significant impact on the PA signal amplitude, where the shell coating could lead to a decrease in PA signal intensities.

PA signals from REDNPs upon excitation at 975 nm. Our previous study shows 8


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


isotropic REDMPs with short decay time generally have high PA signal amplitude14. However, we also found that the particle morphologies affected the PA signal amplitude. A strong PA signal amplitude can be obtained from anisotropic particles which also show a long decay time and strong luminescence emission14. In this work, we studied the size effect by measuring the PA response of core REDNPs of four different sizes using pulsed 975 nm excitation source. The PA amplitude increased from 0.14 to 0.32 as the particle size increased from 11 to 38 nm for REDNPs (Fig. 3a). In contrast to our previous expectation, the results show that by increasing the particle size of uncoated REDNPs, both luminescence emissions and PA amplitude were simultaneously enhanced. After coating an undoped shell of the rare-earth host material, the PA signal amplitude was increased by 10% to 30% when compared to uncoated REDNPs (Fig.3a). Figure 3b shows the PA images of core/shell REDNPs of different sizes with measured PA signal amplitudes from 0.16 to 0.37. Coating an undoped NaYF4 shell improved the PA response of REDNPs in addition to enhancing the luminescence. Our results demonstrate that inorganic shell coating can simultaneous improve both luminescence and PA signals. The PA intensity data for both core and core/shell REDNPs are summarized in Table 1.

Influence of shell materials on the luminescence and PA properties of REDNPs. Preliminary experiments were conducted to study the influence of the shell material on the luminescence and PA properties. Amorphous silica was coated on the core REDNPs as a comparison to the crystalline undoped NaYF4 shell21. Figure 4a shows 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the SEM image of the silica coated REDNPs (RE(29)) with an average size of ~150 nm. The steady state luminescence measurement (Fig. 4b) shows the luminescence intensity decreased by ~2 times for SWIR emission and by ~3 times for visible green emission after coating silica shell. The luminescence decay time was estimated to be 4.10±0.01 ms, which is shorter than the uncoated REDNPs (6.03±0.03 ms). Upon measuring the PA response, we found a significant enhancement of the PA signal amplitudes using silica-coated REDNPs compared to REDNPs that were coated with undoped shell. A PA amplitude of 0.34 was obtained using silica-coated REDNPs at lower concentration of 0.04 mmol/mL (~ 8mg/mL, based on the core REDNPs). A lower concentration of silica-coated REDNPs was used in our experiments due to the difficulty of injecting the particles at higher concentrations. The higher viscosity at high concentration of silica-coated REDNPs can be attributed to the stronger inter-particle interactions from hydrogen bonding as a result of the high density of hydroxyl bonds on the surfaces of the silica coating. Our results from the use of different surface coating material highlighted the importance of selecting the right shell material to enhance both the luminescence and PA signals simultaneously. A detailed systematic study on the effects of the surface coating on both the luminescence and PA signals is warranted, which is beyond the scope of this work.

In vivo animal PA imaging of REDNPs upon excitation at 975 nm. Our nano-sized REDNPs were used to demonstrate their effectiveness as in vivo PA contrast agents by evaluating their ability to image the fine cortical blood vessels in the rat’s brain. 10


Page 10 of 40

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Core/shell REDNPs with 38 nm core size and an undoped NaYF4 shell were injected via the tail vein. These REDNPs then circulated reaching the cortical blood vessel around the superior sagittal sinus (SSS) and were monitored by a PAM system upon excitation at 975 nm. The benefit of using the 975 nm excitation is that deeper penetration depths can be reached since tissue attenuation in the infrared region is low. The PA signal was monitored for ~30 min after administration of REDNPs (Fig. 5). The background signal from blood absorption was detected to be ~0.19 before the injection and blood vessel was barely recognized. The PA signal amplitude increased to ~0.28 at 15 min mark after injection, which was ~30% higher than the background signal. A good PA contrast of the blood vessel was obtained which could well differentiate it from the surrounding tissues in the PA image. The PA signal amplitude was observed to decrease slowly over the following 15 min, indicating successful elimination of REDNPs in the bloodstream. The optimum signal enhancement from our REDNPs was observed at the 15 min mark and enhanced visualization of the blood vessel from the cortical SSS lasted for ~10 min. The successful in vivo infrared PA imaging demonstrated for the first time that in addition to being SWIR luminescence probes, these REDNPs are excellent infrared PA contrast agents that can be tailored for dual-modal luminescence and PA imaging for deep tissue imaging and diagnosis.

Discussion 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Together with the development of innovative imaging techniques, complementary imaging probes are continually being co-developed to improve the resolutions and the signal-to-noise ratio (SNR)22-26. However, it remains challenging for any individual conventional imaging technique to achieve both high resolution and SNR simultaneously24-27. For Example, luminescence imaging is a fast and low cost imaging technique28, 29, while the spatial resolution and detection depth are limited due to the light scattering and absorption by tissue29,

30

. On the other hand, PA

imaging offers complementary features of high resolution imaging in deep tissues31. PA imaging converts laser energy at a specific wavelength into acoustic waves that can be detected using an ultrasound receiver. Only a few materials such as gold nanoparticles, QDs and carbon nanotubes have been previously evaluated for PA imaging, and none of them can be considered as ideal so far due to various issues including control of absorption behavior and cytotoxicity8, 9, 32-34. We are the first to explore the use of rare-earth doped material for luminescence/PA dual-modality possibilities14. To date, REDNPs doped with Yb and Er are the only rare-earth doped materials for dual-modal contrast agent for luminescence and PA imaging. Compared with gold nanoparticles, QDs and carbon nanotubes, our REDNPs have shown comparable capability in enhancing PA signal. In addition, our REDNPs are also capable of dual-modal imaging, which is lacking in previous PA enhancing agents.

Multi-modal imaging platform is an emergent technological development, which integrates several imaging modalities with compatible contrast agents to 12


Page 12 of 40

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


simultaneously capture visual information over many spatial scales28,

35, 36

. The

combination of complementary imaging techniques offers an opportunity for cooperative synergy of the strengths for individual modality. The data processing time can also be reduced with the co-registration of multiple signals instead of obtaining sequential images of independent modality. In addition, an all optical-based multi-modal imaging platform, such as luminescence and PA imaging, presents a non-invasive and inexpensive intraoperative system that can be easily adapted for surgical guidance1-4. However, there is still a lack of nano-sized contrast agents for multi-modal luminescence and PA imaging.

Luminescence imaging is based on the use of optical probes such as organic flurophores, quantum dots, and rare-earth doped nanoparticles (REDNPs) that emit light upon excitation at specific wavelengths. The emitted light is next detected to visualize biological tissues using an imaging system with either Si (for visible detection) or InGaAs (for IR detection) based camera. The high SNR gained from these optical probes enables the imaging of anatomical structures with enhanced sensitivity and contrast for effective and rapid diagnosis and analysis. However, most luminescence imaging systems that operate in the visible region face limited penetration depth due to significant tissue attenuation4. Recently, the use of the shortwave infrared (SWIR) tissue transparent window increased the imaging depth by up to ~1 cm due to lower tissue attenuation coefficient compared to the visible window10, 23. Despite limited penetration depth and relatively low spatial resolutions, 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the benefits of luminescence imaging include excellent temporal response, high sensitivity and an extensive library of available fluorophores. On the other hand, PA imaging detects the acoustic wave which is non-radiative vibration generated from the interaction between light and absorbers such as tissues, chromophores, and nanoparticles31. The generation of PA signal is determined by multiple parameters of the absorbers, including optical, thermal, elastic and geometrical, and the generated acoustic wave is transmitted through the tissue37. Its advantage over optical transmission is that the attenuation length of acoustic waves is much longer than that of the infrared optical signals and can reach up to ~4 cm with high contrast and good spatial resolution38.

REDNPs utilizing the near infrared (NIR) excitation for SWIR imaging have shown the capability to enable rapid and convenient real-time scanning with high sensitivity3, 4

. These REDNPs have shown their potential as contrast agents for PA imaging,

enabling improved resolution at deeper tissue depths compared to luminescence imaging13, 14. Therefore, in addition to being excellent luminescence probes, REDNPs have emerged as excellent PA contrast agents by harnessing the non-radiative losses. In this work, we have prepared nano-sized dual modal contrast agent which utilizes the tissue penetrating IR window for luminescence and PA imaging. We have demonstrated two strategies to enhance both radiative luminescence emissions and non-radiative PA amplitude simultaneously by increasing particle size and coating an undoped shell, which have not been previously reported. Animal experiments 14


Page 14 of 40

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


demonstrate that the core/shell REDNPs can be used to enhance the PA contrast effectively in vivo.

Our study shows that both luminescence and PA signals increased for core REDNPs as particle size increased (Fig. 2b; Fig. 3a). To account for these observations, there are two mechanisms that must be considered, especially for nano-sized particles: (1) surface effects and (2) phonon density of states (PDOS). The first mechanism to be discussed, affecting both luminescence and PA properties, is the surface effects of REDNPs. As the particle size decreases, the surface-to-volume ratio of REDNPs increases and surface effects becomes more significant. Lanthanide ions in the surface layer experience different electronic and structural environment from the interior ions as surface atom disordering occurs and surface quenchers exist. The absorbed excitation energy of nanoparticles was easily dissipated to the surrounding due to the energy transfer from these surface ions to nearby surface quenchers such as defects, impurities and ligands39. Since the surface effect is less significant for larger REDNPs, energy losses due to surface effects were decreased, leading to the observed enhancements in both the luminescence and PA signals. The other mechanism is the phonon modes which are also dependent on the size of the REDNPs39. Phonon-assisted energy transfer between lanthanide ions of Yb3+ and Er3+ affects the luminescence emissions of REDNPs, while the acoustic phonon modes affect the PA signal amplitude. As the size increases, there is likely an increase in the acoustic phonon modes and enhanced energy transfer, leading to enhancement for both 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

luminescence and PA signal39.

The choice of the shell material was also found to influence the luminescence and PA properties of REDNPs. Undoped crystalline shells of the rare-earth host material (NaYF4) and amorphous silica shells were used in this study because of the absence of lattice mismatch with the core REDNPs. By coating undoped NaYF4 shell, both luminescence emission and PA signal were found to be improved simultaneously. The surface effects were removed due to the increased separation of lanthanide ions from surface quenchers40. Consequently, the absorbed excitation energy was effectively transformed as radiative luminescence emissions and non-radiative PA signals. On the other hand, the undoped NaYF4 shell also increased the size of the REDNPs. As the host of the core and the shell were the same material of NaYF4, the phonon modes and the rate of phonon-assisted energy transfer increased41. Therefore, both luminescence and PA signals were enhanced. In contrast, coating a silica shell only enhanced the PA signal amplitude while the luminescence intensity was reduced. The enhancement of the PA signal amplitude was due to the high phonon energy of amorphous silica compared to NaYF442, 43. With higher phonon energy, multiphonon non-radiative losses are likely to increase, resulting in higher PA signal amplitude. However, this would also lead to a decrease in luminescence emissions, where the higher phonon energy of silica and the hydroxyl groups (–OH) associated with the silica shell increases the non-radiative losses significantly21. Surface groups such as – OH has been shown to significantly quench the infrared luminescence emissions19. 16


Page 16 of 40

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusion In summary, we have successfully synthesized hexagonal phase core NaYF4:Yb,Er REDNPs with different sizes and core/shell REDNPs using undoped NaYF4 and silica. Our findings show that by increasing nanoparticles size, the PA signal was enhanced due to reduced surface effect and increased phonon modes. Surface effects result in energy loss from surface lanthanide ions to surface quenchers such as defects, impurities and ligands, while high phonon modes enhance the phonon-assisted energy transfer. Our result also shows that the choice of the coating material influences the luminescence and PA properties. The silica shell only enhanced PA signal amplitude, while the undoped NaYF4 shell enhanced both luminescence and PA signals. The core/shell REDNPs with NaYF4 shell demonstrated the effectiveness of our probes for in vivo PA imaging. This work provides a new insight into the understanding of how REDNPs can be tailored to control the luminescence and PA properties. By further controlling the shape and surface modification of REDNPs, another dimension is to be added to optimize the contrast of our dual-modal SWIR luminescence and PA imaging system for deep tissue diagnosis.

Methods Materials. All chemicals were purchased from manufacturers and used as obtained without any further purification. Lanthanide oxides (99.99%, Y2O3, Yb2O3, Er2O3), sodium trifluoroacetate (98.0%, CF3COONa), 1-octandecene (97%), oleylamine 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

(70%), oleic acid (90%), cyclohexane (99.5%), tetraethyl orthosilicate (98%) Igepal CO-520, 1-hexanol (98%), and ammonium hydroxide (28%) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). Lanthanide trifluoroacetates ((CF3COO)3Ln) were prepared by dissolving the respective lanthanide oxides in trifluoroacetic acid (CF3COOH) in a glass bottle in an oven (Memmert Gmbh, Schwabach, Germany) at 80 °C overnight until all solids were dissolved and clear solution was obtained.

Synthesis of β-NaYF4:Yb,Er Core/Shell β-NaYF4:Yb,Er/NaYF4 Nanoparticles. Typically, to synthesize 11 nm β-NaYF4:Yb,Er nanoparticles, a mixture of CF3COONa (2 mmol, 272 mg), (CF3COO)3Y (0.78 mmol, 334 mg), (CF3COO)3Yb (0.2 mmol, 102 mg), and (CF3COO)3Er (0.02 mmol, 10 mg) was dissolved in ODE (10 mmol) with OA (8 mmol) and OM (5 mmol) at 140 °C for 1 h until a clear solution was formed. The mixture was heated to 340 °C, stirred for 1 h, and then cooled to room temperature. Nanocrystals in the solution were precipitated by addition of excess ethanol (∼ 40 mL), followed by centrifugation, redispersion, and washing (yield: ~120 mg). To change the size of the as-synthesized REDNPs, the amount of sodium trifluoroacetate (CF3COONa) precursor added was varied.

For

synthesizing 18, 29 and 38 nm NaYF4:Yb,Er nanoparticles, the amount of CF3COONa was reduced from 2 mmol to 1.8, 1.6 and 1.5 mmol, respectively. Concentration for all other precursors and solvent amounts were kept constant when the amount of CF3COONa was varied. Undoped NaYF4 shell was coated by adding a 18


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mixture containing CF3COONa (2 mmol), (CF3COO)3Y (1 mmol), ODE (10 mmol) with OA (8 mmol) and OM (5 mmol) to the reaction solution after keeping at 340 °C for 1 h. The reaction was maintained at 340 °C for another 30 min before cooling to room temperature (yield: ~260 mg).

Synthesis of Silica Coated β-NaYF4:Yb,Er Nanoparticles. Silica shell was coated by adopting previous reported method with some modification21. In general, surfactants of Igepal CO-520 (18 mL) and 1-hexanol (10 mL) were dispersed in cyclohexane (70 mL) by stirring. A solution of NaYF4:Yb,Er nanoparticles in cyclohexane (20mg, 5mL) was added. The resulting mixture was stirred, and ammonium hydroxide (0.5 mL, 28%) was added to form a transparent reverse microemulsion. Finally, TEOS (300 µL) was added, and the reaction was continued for 24 h. The NaYF4:Yb,Er/silica core/shell nanoparticles were collected by centrifuging and washed with ethanol.

Materials Characterization. The size and morphology of the synthesized NaYF4:Yb,Er particles were characterized by field emission scanning electron microscopy (JSM-7600F, JEOL Ltd., JP). Images for core REDNPs were collected using an accelerating voltage of 10 kV and an working distance 4.7 mm under SEM mode. The images for core/shell REDNPs were obtained at an accelerating voltage of 1 kV and working distance of 3.5 mm under GB_High Mode. Powder XRD patterns were measured using the D8 ADVANCE ECO powder diffractometer (Bruker AXS 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Inc., Madison, WI) with CuKα (λ=1.541 Ǻ) source operating at 40 V and 25 mA with a step size of 0.02° and duration of 0.5 s. The sample was prepared by pressing the dry powder of REDNPs on glass slide and was then placed on the holder. Steady state and time-resolved spectra were measured using a Edinburgh Instruments spectrometer (FLS980, Edinburgh Instruments, Livingston, U.K.) equipped with PMT (Hamamatsu R928P, Hamamatsu Photonics K.K., Japan) and NIR-PMT (Hamamatsu H1033A-75, Hamamatsu Photonics K.K., Japan) upon excitation with a 975 nm continuous wave laser (CNI MDL-III-975, Changchun New Industries Optoelectronics Tech. Co. Ltd, China). To measure steady state spectra, dry powder samples were packed in demountable Spectrosil far UV quartz Type 20 cells (Starna Cells, Inc., Atascadero, CA) with 0.5 mm path lengths for emission collection. The power of the 975 nm laser was set to ~ 10 mW. The optical path for all photoluminescence measurements was kept constant. All luminescence measurements were measured three times and the average curves were shown in this study. To measure the time-resolved luminescence spectrum, the excitation source was modulated using an electronic pulse modulator to obtain excitation pule at a pulse duration of 20 µs with a repetition rate of 10 Hz. The obtained spectra were fitted can calculated using Origin 8 with the equations shown in the Results section.

PA Measurements. Samples for in vitro PA imaging and signal measurements were obtained by dispersing the nanoparticles in the ethanol. Concentration of 0.2 mmol/mL (~40 mg/mL; concentration based on the nanoparticles obtained directly 20


Page 20 of 40

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


form synthesis) was obtained for core and core/shell REDNPs. Concentration of 0.04 mmol/mL (~0.8 mg/mL; concentration based on core NaYF4:Yb,Er nanoparticles) was used for NaYF4:Yb, Er/silica nanoparticles. To evaluate the PA properties of our REDNPs, PA signals of our REDNPs that were injected into a ~15 cm polyethylene tubings (0.38 × 1.09 mm, Scientific Commodities, Inc, Lake Havasu City, Arizona) were measured using a dark-field confocal photoacoustic microscopy system upon excitation at 975 nm. Afterwards, the tubing was positioned at a depth of the transducer’s focus, i.e., the depth of 9 mm with respect to the transducer in water tank. The system was maintained in a 25°C water bath throughout the experiment. The contrast changes were imaged using a 50-MHz dark field confocal PAM system with 32 × 61 µm resolution. An optical parametric oscillator pumped by a frequency-tripled Nd:YAG Q-switched laser was employed to provide ~4 ns laser pulses at a pulse repetition rate of 10 Hz. Laser energy was delivered by a 1 mm multimode fiber. The fiber tip was coaxially aligned with a convex lens, an axicon, a plexiglass mirror, and an ultrasonic transducer on an optical bench, forming dark field illumination that was confocal with the focal point of the ultrasonic transducer. Laser pulses at 975 nm were used for PA wave excitation. A large numerical-aperture, wideband 50-MHz ultrasonic transducer was employed to allow for the efficient collection of PA signals. The scanning step size was 10 µm for each B-scan.

The PA signals received by the ultrasonic transducer were pre-amplified by a low-noise amplifier (noise figure 1.2 dB, gain 55 dB, AU-3A-0110, Miteq, USA), 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

cascaded to an ultrasonic receiver (5073 PR, Olympus, USA), then digitized and sampled by a computer-based 14-bit analog to digital (A/D) card (CompuScope 14200, GaGe, USA) at a 200-MHz sampling rate for data storage. Fluctuations in the laser energy were monitored by a photodiode (DET36A/M, Thorlabs, USA). Recorded photodiode signals were applied to compensate for PA signal variations caused by laser energy instability before any further signal processing.

In Vivo PA Cortical SSS Blood Vessel Imaging.

The experimental setup for brain

vasculature imaging using dark-field strong-focusing PAM is described in detail elsewhere44, 45. Six Male Sprague Dawley rats weighing 250 to 300 g (InVivos Pte Ltd, Singapore) each, were used for in vivo PA imaging. In this study, we used the B-scan of PA imaging to show the sectional view of the blood vessel.

The animals were

housed at a constant temperature and humidity with free access to food and water. Before the imaging experiment, the rats were fasted for 24 hours but given water ad libitum. All experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of the National University of Singapore. The animals were anesthetized with pentobarbital (50 mg/kg bolus and 15 mg/kg/h maintenance, intraperitoneal) throughout the experiments. The body temperature was measured via a rectal probe and maintained at 37 ± 0.5°C by a self-regulating thermal plate (TCAT-2 Temperature Controller, Physitemp Instruments, Inc., New Jersey, USA). The anesthetized rats were mounted on a custom built acrylic stereotaxic head holder, and the skin and muscle were cut off to expose the bregma landmark. The 22


Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


anteroposterior distance between the bregma and the interaural line was surveyed directly. Furthermore, the craniotomy was performed for each animal and the bilateral cranial window with approximately 5 (horizontal) × 3 (vertical) mm size was fashioned with a high-speed drill. The interaural and bregma references were then used to position the animal’s head in the PAM system without additional surgery in following experiments. The designed particles were administered via tail vein infusion as an aqueous solution (150 µL) of REDNPs (40 mg/mL). None of the animals suffered any ill effects as a result of nanoparticle injection.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References 1.

Kaijzel, E. L.; van der Pluijm, G.; Löwik, C. W. G. M. Whole-body optical imaging in animal models

to assess cancer development and progression. Clin. Cancer Res. 2007, 13, 3490-3497. 2.

Wang, F.; Liu, X. Recent advances in the chemistry of lanthanide-doped upconversion

nanocrystals. Chem. Soc. Rev. 2009, 38, 976-989. 3.

Tan, M. C.; Al-Baroudi, L.; Riman, R. E. Surfactant effects on efficiency enhancement of

infrared-to-vsible upconversion emissions of NaYF4:Yb-Er. ACS Appl. Mater. Inter. 2011, 3, 3910-3915. 4.

Naczynski, D. J.; Tan, M. C.; Zevon, M.; Wall, B.; Kohl, J.; Kulesa, A.; Chen, S.; Roth, C. M.; Riman, R.

E.; Moghe, P. V. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 2013, 4, 2199. 5.

Kim, C.; Favazza, C.; Wang, L. V. In vivo photoacoustic tomography of chemicals: high-resolution

functional and molecular optical imaging at new depths. Chem. Rev. 2010, 110, 2756-2782. 6.

Xie, Z.; Roberts, W.; Carson, P.; Liu, X.; Tao, C.; Wang, X. Evaluation of bladder microvasculature

with high-resolution photoacoustic imaging. Opt. Lett. 2011, 36, 4815-4817. 7.

Pan, D.; Cai, X.; Yalaz, C.; Senpan, A.; Omanakuttan, K.; Wickline, S. A.; Wang, L. V.; Lanza, G. M.

Photoacoustic sentinel lymph node imaging with self-assembled copper neodecanoate nanoparticles. ACS Nano 2012, 6, 1260-1267. 8.

De La Zerda, A.; Zavaleta, C.; Keren, S.; Vaithilingam, S.; Bodapati, S.; Liu, Z.; Levi, J.; Smith, B. R.;

Ma, T.-J.; Oralkan, O.; Cheng, Z.; Chen, X.; Dai, H.; Khuri-Yakub, B. T.; Gambhir, S. S. Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nat Nanotechnol. 2008, 3, 557-562. 9.

Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting

polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nat. Nano. 2014, 9, 233-239. 10. van Saders, B.; Al-Baroudi, L.; Tan, M. C.; Riman, R. E. Rare-earth doped particles with tunable infrared emissions for biomedical imaging. Opt. Mater. Express 2013, 3, 566-573. 11. Xiong, L.; Yang, T.; Yang, Y.; Xu, C.; Li, F. Long-term in vivo biodistribution imaging and toxicity of polyacrylic acid-coated upconversion nanophosphors. Biomaterials 2010, 31, 7078-7085. 12. Hemmer, E.; Venkatachalam, N.; Hyodo, H.; Hattori, A.; Ebina, Y.; Kishimoto, H.; Soga, K. Upconverting and NIR emitting rare earth based nanostructures for NIR-bioimaging. Nanoscale 2013, 5, 11339-11361. 13. Maji, S. K.; Sreejith, S.; Joseph, J.; Lin, M.; He, T.; Tong, Y.; Sun, H.; Yu, S. W.-K.; Zhao, Y. Upconversion nanoparticles as a contrast agent for photoacoustic imaging in live mice. Adv. Mater. 2014, 26, 5633-5638. 14. Sheng, Y.; Liao, L.-D.; Thakor, N.; Tan, M. C. Rare-earth doped particles as dual-modality contrast agent for minimally-invasive luminescence and dual-wavelength photoacoustic imaging. Sci. Rep. 2014, 4, 6562. 15. Garnett, M. C.; Kallinteri, P. Nanomedicines and nanotoxicology: some physiological principles. Occup. Med. 2006, 56, 307-311. 16. Li, W.; Szoka, F., Jr. Lipid-based nanoparticles for nucleic acid delivery. Pharm. Res. 2007, 24, 438-449. 17. Georgieva, J. V.; Kalicharan, D.; Couraud, P.-O.; Romero, I. A.; Weksler, B.; Hoekstra, D.; Zuhorn, I. S. Surface characteristics of nanoparticles determine their intracellular fate in and processing by human blood-brain barrier endothelial cells in vitro. Mol. Ther. 2011, 19, 318-325. 18. Patterson, A. L. The Scherrer Formula for X-Ray particle size determination. Phys. Rev. 1939, 56,

24


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


978-982. 19. Yuan, D.; Tan, M. C.; Riman, R. E.; Chow, G. M. Comprehensive study on the size effects of the optical properties of NaYF4:Yb,Er nanocrystals. J. Phys. Chem. C 2013, 117, 13297-13304. 20. Tan, M. C.; Kumar, G. A.; Riman, R. E.; Brik, M. G.; Brown, E.; Hommerich, U. Synthesis and optical properties of infrared-emitting YF3:Nd nanoparticles. J. Appl. Phys. 2009, 106, 063118. 21. Qian, L. P.; Yuan, D.; Shun Yi, G.; Chow, G. M. Critical shell thickness and emission enhancement of NaYF4:Yb,Er/NaYF4/silica core/shell/shell nanoparticles. J. Mater. Res. 2009, 24, 3559-3568. 22. Wang, L. V. Multiscale photoacoustic microscopy and computed tomography. Nat. Photon. 2009, 3, 503-509. 23. Hong, G.; Diao, S.; Chang, J.; Antaris, A. L.; Chen, C.; Zhang, B.; Zhao, S.; Atochin, D. N.; Huang, P. L.; Andreasson, K. I.; Kuo, C. J.; Dai, H. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photon. 2014, 8, 723-730. 24. Nahrendorf, M.; Keliher, E.; Marinelli, B.; Waterman, P.; Feruglio, P. F.; Fexon, L.; Pivovarov, M.; Swirski, F. K.; Pittet, M. J.; Vinegoni, C.; Weissleder, R. Hybrid PET-optical imaging using targeted probes. P. Nat. Acad. Sci. 2010, 107, 7910-7915. 25. Kircher, M. F.; de la Zerda, A.; Jokerst, J. V.; Zavaleta, C. L.; Kempen, P. J.; Mittra, E.; Pitter, K.; Huang, R.; Campos, C.; Habte, F.; Sinclair, R.; Brennan, C. W.; Mellinghoff, I. K.; Holland, E. C.; Gambhir, S. S. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat. Med. 2012, 18, 829-834. 26. Kircher, M. F.; Mahmood, U.; King, R. S.; Weissleder, R.; Josephson, L. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res. 2003, 63, 8122-8125. 27. Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat. Meth. 2010, 7, 603-614. 28. Sheng, Y.; De Liao, L.; Thakor, N. V.; Tan, M. C. Nanoparticles for molecular imaging. J. Biomed. Nanotechnol. 2014, 10, 2641-2676. 29. Hong, G.; Zou, Y.; Antaris, A. L.; Diao, S.; Wu, D.; Cheng, K.; Zhang, X.; Chen, C.; Liu, B.; He, Y.; Wu, J. Z.; Yuan, J.; Zhang, B.; Tao, Z.; Fukunaga, C.; Dai, H. Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window. Nat. Commun. 2014, 5, 4206. 30. Gu, L.; Hall, D. J.; Qin, Z.; Anglin, E.; Joo, J.; Mooney, D. J.; Howell, S. B.; Sailor, M. J. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles. Nat. Commun. 2013, 4, 2326. 31. Zhang, H. F.; Maslov, K.; Stoica, G.; Wang, L. V. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nat. Biotech. 2006, 24, 848-851. 32. Song, K. H.; Kim, C.; Cobley, C. M.; Xia, Y.; Wang, L. V. Near-infrared gold nanocages as a new class of tracers for photoacoustic sentinel lymph node mapping on a rat model. Nano Lett. 2009, 9, 183-188. 33. Shashkov, E. V.; Everts, M.; Galanzha, E. I.; Zharov, V. P. Quantum dots as multimodal photoacoustic and photothermal contrast agents. Nano Lett. 2008, 8, 3953-3958. 34. Liang, X.; Deng, Z.; Jing, L.; Li, X.; Dai, Z.; Li, C.; Huang, M. Prussian blue nanoparticles operate as a contrast agent for enhanced photoacoustic imaging. Chem. Commun. 2013, 49, 11029-11031. 35. Naczynski, D. J.; Tan, M. C.; Riman, R. E.; Moghe, P. V. Rare earth nanoprobes for functional biomolecular imaging and theranostics. J. Mater. Chem. B 2014, 2, 2958-2973. 36. Kim, J.; Piao, Y.; Hyeon, T. Multifunctional nanostructured materials for multimodal imaging, and

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

simultaneous imaging and therapy. Chem. Soc. Rev. 2009, 38, 372-390. 37. Todorović, D. M.; Nikolić, P. M.; Ristovski, Z. D., Quantitative determination of absorption using coupled amplitude and phase photoacoustic spectra. Springer US: New York, 1991; pp 641-646. 38. Xu, M.; Wang, L. V. Photoacoustic imaging in biomedicine. Rev. Sci. Instrum. 2006, 77, 041101. 39. Liu, G. Advances in the theoretical understanding of photon upconversion in rare-earth activated nanophosphors. Chem. Soc. Rev. 2015, 44, 1635-1652. 40. Lu, H.; Gillin, W. P.; Hernandez, I. Concentration dependence of the up- and down-conversion 3+

emission colours of Er -doped Y2O3: a time-resolved spectroscopy analysis. Phys. Chem. Chem. Phys. 2014, 16, 20957-20963. 41. Wang, F.; Deng, R.; Wang, J.; Wang, Q.; Han, Y.; Zhu, H.; Chen, X.; Liu, X. Tuning upconversion through energy migration in core–shell nanoparticles. Nat. Mater. 2011, 10, 968-973. 42. Zhao, J.; Lu, Z.; Yin, Y.; McRae, C.; Piper, J. A.; Dawes, J. M.; Jin, D.; Goldys, E. M. Upconversion luminescence with tunable lifetime in NaYF4:Yb,Er nanocrystals: role of nanocrystal size. Nanoscale 2013, 5, 944-952. 43. Schietinger, S.; Menezes, L. d. S.; Lauritzen, B.; Benson, O. Observation of size dependence in multicolor upconversion in single Yb3+, Er3+ codoped NaYF4 nanocrystals. Nano Lett. 2009, 9, 2477-2481. 44. Liao, L. D.; Lin, C. T.; Shih, Y. Y. I.; Lai, H. Y.; Zhao, W. T.; Duong, T. Q.; Chang, J. Y.; Chen, Y. Y.; Li, M. L. Investigation of the cerebral hemodynamic response function in single blood vessels by functional photoacoustic microscopy. J. Biomed. Opt. 2012, 17, 061210. 45. Liao, L. D.; Li, M. L.; Lai, H. Y.; Shih, Y. Y. I.; Lo, Y. C.; Tsang, S. N.; Chao, P. C. P.; Lin, C. T.; Jaw, F. S.; Chen, Y. Y. Imaging brain hemodynamic changes during rat forepaw electrical stimulation using functional photoacoustic microscopy. Neuroimage 2010, 52, 562-570.

Acknowledgements MC Tan and Y Sheng would like to gratefully acknowledge the funding support from the Singapore University of Technology and Design (SUTD) Start-up Research Grant and the SUTD-MIT International Design Center. MC Tan and Y Sheng would like to thank Dr. Du Yuan for the guidance on the synthesis of rare-earth doped nanoparticles. L-D Liao, A Bandla, Y-H Liu and N Thakor also would like to thank the funding support from the National University of Singapore, A*STAR and the Ministry of Defense to the Singapore Institute for Neurotechnology (SINAPSE). 26


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Author contribution Y.S., M.C.T., A.B., Y.H.L., L.D.L. and N.T. conceived the study and designed the experiments. Y. S. performed the experiments to synthesize and characterize the materials. L. D. L., A.B., Y.H.L. performed the PA signal measurements and imaging. Y.S., M.C.T. and L.D.L. discussed and analyzed the data. Y.S., M.C.T., L.D.L. and N.T. wrote and revised the manuscript.

Competing financial interests The authors declare no competing financial interests.

Supporting information This material is available free of charge via the Internet at http://pubs.acs.org

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Legends of Figures

Figure 1 SEM images of core REDNPs of (a) RE(29) and (b) RE(38) and corresponding core/shell REDNPs of (c) RE(29)_NaYF4 and (d) RE(38)_ NaYF4. All scale bars are 100 nm. The corresponding sizes of as-prepared REDNPs are 29.3 ± 3.8 nm and 38.4 ± 3.9 nm, while after coating the sizes increased to 40.5 ± 5.8 nm and 50.3 ± 5.6 nm. Insets: histogram for the size distribution of corresponding REDNPs. The histograms were obtained by counting at least 80 nanoparticles for (a) to (d). (e) XRD comparison for core REDNPs and core/shell REDNPs. RE(29) and RE(29)_NaYF4 REDNPs are chosen for direct comparison. The XRD pattern 28


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


indicates that the crystal size was increased after undoped NaYF4 shell coating. All samples are hexagonal phase (PDF no. 16-0334). (f) Illustration of core/shell REDNPs with varied sizes of core REDNPs and similar shell thickness.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2 (a) Steady state luminescence emission spectra of RE(29) and RE(29)_ NaYF4. The spectra were measured using continuous wave 975 nm laser at an output power of 10 mW for both samples. (b) Integrated intensity of SWIR emission intensity of core REDNPs and core/shell REDNPs. The data was measured at 10 mW for all samples. (c) Time-resolved luminescence spectra of RE(29) and RE(29)_ NaYF4 corresponding to the 4I13/2-4I15/2 transition of Er3+ at 1530 nm emission. (d) Fitted decay time of core REDNPs and core/shell REDNPs with different core sizes. The values and errors of decay time τave of both core and core/shell REDNPs obtained from equation 2 were summarized in Table 2. The error bars are given by fitting using Origin, which represents the deviation of the fitted value from the measured data.

30


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3 (a) Size dependent PA signals for core REDNPs (black dot and dot line) and core/shell REDNPs (red dot and dot line). (b) Corresponding PA images of core/shell REDNPs. The PA amplitudes of the core REDNPs are RE(11): 0.14, RE(18): 0.24, RE(29): 0.28, and RE(38): 0.32.The PA amplitudes of the core/shell REDNPs are RE(11)_NaYF4:

0.16,

RE(18)_NaYF4:

0.26,

RE(29)_NaYF4:

0.34,

and

RE(38)_NaYF4: 0.37. All measurements were performed using a 50-MHz dark field confocal PAM system with ~4 ns laser pulses (975 nm) at a pulse repetition rate of 10 Hz. The concentration of all samples was 0.2 mmol/mL. (Scale bar: 100 µm)

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4 (a) SEM image of NaYF4:Yb,Er/silica nanoparticles. (b) Steady state luminescence emission spectrum of corresponding particles compared with core REDNPs (RE(29)). (c) Time-resolved luminescence spectra corresponding to the 4

I13/2-4I15/2 transition of Er3+ at 1530 nm emission of RE(29) and RE(29)_SiO2. (d) PA

image of corresponding nanoparticle. Measurement was performed using a 50-MHz dark field confocal PAM system with ~4 ns laser pulses (975 nm) at a pulse repetition rate of 10 Hz. The concentration of all samples was 0.04 mmol/mL. (Scale bar: 100 µm)

32


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5 The plot shows the PA signal amplitude of RE(38)_NaYF4 around the cortical superior sagittal sinus (SSS) blood vessel with respect to time. The PA images show the visualization of SSS before and 10, 15, 30 min after injection of our REDNPs. (scale bar: 0.5 mm). The blood vessel was hardly recognized without the contrast agent, as shown in the image before injection of REDNPs. However, the differentiation between the blood vessel and surrounding tissues became obvious and the position of the blood vessel was determined with the assistance of our REDNPs as contrast agents.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

Table 1 Summary of IR and PA intensity for core and core/shell REDNPs Sample Integrated IR intensity (a.u.) PA intensity (a.u.) 6 RE(11) 4.6×10 0.14 7 RE(18) 1.1×10 0.24 RE(29) 1.5×107 0.28 7 RE(38) 2.4×10 0.32 8 RE(11)_NaYF4 2.1×10 0.16 8 RE(18)_ NaYF4 2.3×10 0.26 RE(29)_ NaYF4 2.9×108 0.34 8 RE(38)_ NaYF4 3.1×10 0.37

Table 2 Summary of SWIR luminescence decay time at 1530 emission for core and core/shell REDNPs Core

Decay time (ms)

Core/shell

Decay time (ms)

RE(11)

0.19±0.04

RE(11)_ NaYF4

9.75±0.06

RE(18)

4.71±0.05

RE(18)_ NaYF4

10.30±0.24

RE(29)

6.03±0.03

RE(29)_ NaYF4

11.82±0.19

RE(38)

6.84±0.03

RE(38)_ NaYF4

11.11±0.51

34


Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only

In addition to being excellent luminescent probes, REDNPs also served as successful PA contrast agents with enhanced PA signal by controlling the size and surface shell. Under the laser excitation, PA signal from the REDNPs was detected to image the vasculature of the brain vessels around the superior sagittal sinus of in vivo rat model.

Manuscript title: Size and Shell Effects on the Photoacoustic and Luminescence Properties of Dual Modal Rare-Earth Doped Nanoparticles for Infrared Photoacoustic Imaging Authors: Yang Sheng, Lun-De Liao, Aishwarya Bandla, Yu-Hang Liu, Nitish Thakor, Mei Chee Tan1

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 40

Figure 2 (a) Steady state luminescence emission spectra of RE(29) and RE(29)_ NaYF4. The spectra were measured using continuous wave 975 nm laser at an output power of 10 mW for both samples. (b) Integrated intensity of SWIR emission intensity of core REDNPs and core/shell REDNPs. The data was measured at 10 mW for all samples. (c) Time-resolved luminescence spectra of RE(29) and RE(29)_ NaYF4 corresponding to the 4I13/2-4I15/2 transition of Er3+ at 1530 nm emission. (d) Fitted decay time of core REDNPs and core/shell REDNPs with different core sizes. The values and errors of decay time τave of both core and core/shell REDNPs obtained from equation 2 were summarized in Table 2. The error bars are given by fitting using Origin, which represents the deviation of the fitted value from the measured data. 256x183mm (150 x 150 DPI)


Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3 (a) Size dependent PA signals for core REDNPs (black dot and dot line) and core/shell REDNPs (red dot and dot line). (b) Corresponding PA images of core/shell REDNPs. The PA amplitudes of the core REDNPs are RE(11): 0.14, RE(18): 0.24, RE(29): 0.28, and RE(38): 0.32.The PA amplitudes of the core/shell REDNPs are RE(11)_NaYF4: 0.16, RE(18)_NaYF4: 0.26, RE(29)_NaYF4: 0.34, and RE(38)_NaYF4: 0.37. All measurements were performed using a 50-MHz dark field confocal PAM system with ~4 ns laser pulses (975 nm) at a pulse repetition rate of 10 Hz. The concentration of all samples was 0.2 mmol/mL. (Scale bar: 100 µm) 248x98mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4 (a) SEM image of NaYF4:Yb,Er/silica nanoparticles. (b) Steady state luminescence emission spectrum of corresponding particles compared with core REDNPs (RE(29)). (c) Time-resolved luminescence spectra corresponding to the 4I13/2-4I15/2 transition of Er3+ at 1530 nm emission of RE(29) and RE(29)_SiO2. (d) PA image of corresponding nanoparticle. Measurement was performed using a 50-MHz dark field confocal PAM system with ~4 ns laser pulses (975 nm) at a pulse repetition rate of 10 Hz. The concentration of all samples was 0.04 mmol/mL. (Scale bar: 100 µm) 204x147mm (150 x 150 DPI)


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5 The plot shows the PA signal amplitude of RE(38)_NaYF4 around the cortical superior sagittal sinus (SSS) blood vessel with respect to time. The PA images show the visualization of SSS before and 10, 15, 30 min after injection of our REDNPs. (scale bar: 0.5 mm). The blood vessel was hardly recognized without the contrast agent, as shown in the image before injection of REDNPs. However, the differentiation between the blood vessel and surrounding tissues became obvious and the position of the blood vessel was determined with the assistance of our REDNPs as contrast agents. 183x90mm (150 x 150 DPI)


Size and Shell Effects on the Photoacoustic and Luminescence

Recommend Documents