COMMUNICATION www.rsc.org/materials | Journal of Materials Chemistry

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In vivo multiple color lymphatic imaging using upconverting nanocrystals†

Hisataka Kobayashi,*a Nobuyuki Kosaka,a Mikako Ogawa,a Nicole Y. Morgan,b Paul D. Smith,b

Christopher B. Murray,c Xingchen Ye,c Josh Collins,c G. Ajith Kumar,d Howard Belld and Peter L. Choykea

Received 28th May 2009, Accepted 21st July 2009

First published as an Advance Article on the web 30th July 2009

DOI: 10.1039/b910512c

Upconverting nanocrystals are unique nano-sized particles that emit

light at shorter wavelengths (visible and near infrared) after exci-

tation in the near infrared that dramatically reduces background

autofluorescence in in vivo two color lymphatic imaging for depicting

the lymphatic channels and nodes.

In vivo fluorescence imaging has become an important technique in

biomedical imaging1 because of its high sensitivity, low cost and

feasibility for clinical translation. However, normal tissue auto-

fluorescence interferes with conventional in vivo fluorescence imaging

by lowering the target to background ratio. Currently, there are two

strategies for overcoming autofluorescence: employing ultrabright

fluorophores such as quantum dots2,3 or fluorescent proteins,4 or

acquiring spectral images and then post-processing the imaging data

to remove the background signal.5 Ultrabright fluorophores mostly

consist of solid state quantum dots, which contain cadmium and

selenium and are therefore potentially toxic. Spectral imaging is

relatively slow; the acoustoptic tunable filters used in commercial

systems have switching times of approximately 100 ms, leading to

a minimum time of several seconds to acquire a full image cube. Real

time post-processing of the data for the spectral unmixing is also

problematic.

Upconverting nanocrystals (UCNPs) represent a new strategy for

eliminating background signal. They are excited by infrared or NIR

light yet emit in the visible-NIR. As a result, there is minimal

excitation of autofluorophores, and no spectral overlap between the

upconverter emission and any autofluorescent background, effec-

tively eliminating background signal6 (Fig. 1). This enables near real-

time luminescence imaging with high target to background ratios but

without complex and time consuming image processing. Moreover,

by changing the composition of the doping metals within UCNPs, it

is possible to alter their emission wavelength.7 Although these

materials have been known for nearly 20 years, only recently have

they been grown as nanocrystals in small sizes suitable for in vivo

imaging.8,9 In this study, we demonstrate the feasibility of using

UCNPs for in vivo lymphatic imaging, which is a promising clinical

application of nano-materials, using two UCNPs with different

emission spectra.

aMolecular Imaging Program, Center for Cancer Research, NationalCancer Institute, NIH, Bldg. 10, Room 1B40, MSC 1088, Bethesda, MD20892-1088, USA. E-mail: Kobayash@mail.nih.govbNational Institute of Biomedical Imaging and Bioengineering, NIH,Bethesda, MD 20892, USAcUniversity of Pennsylvania, Philadelphia, PA 19104, USAdSunstone BioSciences, Inc., Philadelphia, PA 19104, USA

† Electronic supplementary information (ESI) available: Fig. S1–S3. SeeDOI: 10.1039/b910512c

This journal is ª The Royal Society of Chemistry 2009

Two upconverting NaYF4 nanocrystals doped with rare earth

activating pairs, Yb, Tm, or Yb, Er were synthesized for near-

infrared (800 nm) and green (540 nm) emissions, respectively, Both

nanocrystals, approximately 50 nm in diameter, �20 nm core

measured by DLS (Delsa� Nano C system, Beckman Coulter,

Fullerton, CA) and 10–15 nm thick surface PEG coatings, were

employed for all in vivo experiments (see ESI Fig. S1†). The following

chemicals were used as received without any further purification:

Na(CF3COO) (GFS Chemical, Inc., Powell, OH), Y(CF3COO)3

(GFS Chemical, Inc.), Yb(CF3COO)3 (GFS Chemical, Inc.),

Tm(CF3COO)3 (GFS Chemical, Inc.), Er(CF3COO)3 (GFS Chem-

ical, Inc.), oleic acid (technical grade, 90%, Sigma-Aldrich, St. Louise,

MO) and 1-octadecene (ODE, Sigma-Aldrich, technical grade, 90%).

The synthesis was based on the methods reported by Mai et al. and

Shan et al.10,11 The methods reported by Mai and Shan have been

modified slightly to increase heating efficiency and to obtain higher

heating temperatures as close to the boiling point of oleic acid as

possible. These modifications allow for a single step method by which

pure b phase nanocrystals can be achieved. The ability to obtain

b phase nanocrystals will ensure that we are utilizing nanocrystals

with the highest quantum efficiency thus giving the greatest signal to

Fig. 1 Near infrared excitation does not produce autofluorescence from

the mouse abdomen. (a) A scheme of the excitation/emission profiles of

quantum dots and UPNCs. (b) A comparison of autofluorescence

emission spectra from the mouse abdomen excited with 468 nm blue (blue

line) and 980 nm near infrared (red line). Near infrared light produces no

detectable background from the body in the visible or near infrared

ranges. (c) A scheme of the in vitro and in vivo macroscopic/animal

imaging set-up.

J. Mater. Chem., 2009, 19, 6481–6484 | 6481

Fig. 2 Luminescence signals from two different upconverting nano-

crystals can be resolved with both spectral and single shot imaging. (a)

Luminescence images of two different UPNCs (UC-NIR and UC-green)

obtained with either spectral or single shot imaging at the respective

emission peaks (550 nm for green and 800 nm for NIR) are shown. The

two UPNCs can be distinguished even without spectral analysis. (b) The

emission spectra of the two UPNCs (UC-NIR and UC-green) ranging

from 500 nm to 900 nm detected with the Maestro In Vivo Imaging

System under excitation with 980 nm light. (c) TEM images of two

upconverting nanocrystals. UC-Green: NaYF4:Yb/Er (left) and UC-

NIR: NaYF4:Yb/Tm (right) nanocrystals show that cores of both

particles are �20 nm in diameter.

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noise ratio. Additionally, it is widely accepted in the community of

upconverting nanophosphors that the hexagonal NaYF4 (b phase) is

the most efficient host for green and blue up-conversion, which shows

visible emission upon infrared excitation (980 nm) when doped with

Yb3+/Er3+ or Yb3+/Tm3+.

One pot synthesis can produce purely b phase NaYF4, as can be

seen from the Table S2 of the Mai et al. paper. More importantly, we

demonstrate the XRD pattern of our as-synthesized nanocrystals

after the incorporation of dopants Yb3+/Er3+ (ESI Fig. S2b†), to be

consistent with Fig. 11a of the Mai et al. paper.

Briefly, for UC-green, 1.8 mmol Na(CF3COO), 1 mmol

Y(CF3COO)3, 0.18 mmol Yb(CF3COO)3 and 0.02 mmol

Er(CF3COO)3 (for UC-NIR, 0.2 mmol Yb(CF3COO)3 and

0.0025 mmol Tm(CF3COO)3 were used) were loaded to a 50 mL

reaction flask. After adding 20 mmol oleic acid and 20 mmol

1-octadecene (ODE), the mixture was heated to 110 �C under

vacuum for 30 minutes to remove oxygen and moisture, forming

a transparent solution. The solution was then heated up to 330 �C

and kept at 330 �C for 30 minutes under N2 atmosphere. After

cooling to room temperature, ethanol was added to the crude

solution to precipitate nanoparticles and remove reaction by-prod-

ucts. The as-prepared nanocrystals were washed several times with

ethanol before being redispersed in hexane.

Surface modification of doped b-NaYF4 nanocrystals was per-

formed as shown below. 15 mL hexane solution of nanocrystals was

mixed with 15 mL of 0.5 M aqueous hexadecyltrimethylammonium

bromide (CTAB) solution and the resulting solution was stirred

vigorously for 12 h at 45 �C. The mixture was then heated up to 70 �C

to evaporate the hexane. The nanocrystals were further washed with

ethanol twice before being redispersed in 10 mL water. Then, we

added a PBS solution of PL-PEG (Avanti Polar Lipids, 1.5 mg ml�1)

to the aqueous nanocrystal solution and sonicated them for 1 h. The

coated nanocrystals were then centrifuged at 7500 rpm for 5 minutes

to remove any agglomerated nanocrystals, ensuring a monodisperse

solution. The as-synthesized samples are hydrophobic. After surface

modification of the UCNP to render them hydrophilic, they are

stable in PBS buffer for up to 2 weeks, mainly due to the high ionic

strength of the buffer solution. As stated, both nanocrystal prepa-

rations using CTAB and PEG have exhibited excellent short-term

stability for up to 2 weeks, retaining their optical properties while still

resisting agglomeration and settling. The stability of the nanocrystals

for longer durations does reduce but only minimally and without

affecting the upconversion processes. Some aggregation will occur

after long-term storage but the nanocrystals can easily be re-dispersed

by sonication for 3 minutes.

For analyzing structural characterization of NaYF4 nanocrystals,

TEM images were taken on a JEM-1400 Transmission Electron

Microscope (JEOL Ltd, Tokyo, Japan) operating at 120 kV. Powder

X-ray diffraction (XRD) of the nanocrystals were recorded on

Rigaku Smartlab system (Rigaku America, Woodlands, TX) at

a scanning rate of 0.2� min�1 using Cu Ka radiation (l ¼ 1.5406 A)

(ESI Fig. S2b†).

Both UCNPs can be excited with a 980 nm laser. One particle,

NaYF4; + Yb and Tm (UC-NIR), has two narrow emission peaks at

470 nm and 800 nm (ESI Fig. S2a†).7 Another particle, NaYF4; + Yb

and Er (UC-Green), has two narrow emission peaks at 550 and 670

nm (Fig. 2b, also see ESI Fig. S2b†). Both UC-NIR and UC-Green

could be detected both with spectral imaging and directly with single-

shot imaging at the respective peak emission wavelengths using an

6482 | J. Mater. Chem., 2009, 19, 6481–6484

in vivo spectral imaging system (Maestro In Vivo Imaging System,

CRI Inc., Woburn, MA, USA) (Fig. 2a).

All in vivo procedures were approved by the National Cancer

Institute Animal Care and Use Committee. Initially, the auto-

fluorescence emission spectral profile of the mouse abdomen was

determined with blue light and NIR excitation. Blue light resulted in

a broad and high amplitude autofluorescence spectrum which

extended up to 750 nm. NIR light at 980 nm, in contrast, produced

no detectable background in the spectral window of interest (Fig. 1b).

Since both UC-NIR and UC-Green are excited at 980 nm, in theory,

there should be no contribution from the tissue background at their

emission peaks (Fig. 2b).

To perform in vivo luminescence lymphatic imaging, animals were

placed in the supine position whilst under pentobarbital anesthesia.

Lymphatic imaging was performed 5 to 30 min after injection of 200

mg of either UC-NIR or UC-Green (n¼ 4 in each group) or quantum

dot 545 (Invitrogen, Co., Carlsbad, CA) as a control. Additionally,

UC-NIR and UC-Green (200 mg each) were serially injected in

another group of mice within a 30 min interval to achieve 2-color

lymphatic imaging in different lymphatic drainage basins. The

nanocrystals were injected intradermally into the lower lip with the

expected drainage pattern to nodes within the neck. Spectral and

single shot, real time luminescence imaging was performed with

a 980 nm diode laser excitation (continuous wave, power density:

250 mW cm�2, BWF-5A, B&W TEK Inc., Newark, DE) (Fig. 1c).

The injection sites were masked with a non-fluorescent black tape.

After obtaining in vivo images, the draining lymph nodes were

removed and evaluated ex vivo with spectral luminescence imaging.

For the in vivo spectral imaging (Maestro), the tunable filter was

automatically stepped in 10 nm increments from 500 to 950 nm.

This journal is ª The Royal Society of Chemistry 2009

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Collected images were analyzed by the Maestro software (Nuance

Ver 2.4, CRi), which uses spectral un-mixing algorithms based on

user-selected regions of interest to separate the autofluorescent

background from the signal of interest. Single shot luminescence

images were obtained at the peak emission for each upconverting

nanocrystal.

Injections with UC-NIR clearly depicted the draining lymph nodes

both in vivo and in situ during surgery in all 4 mice. Due to the

minimal background, single shot luminescence images obtained at

800 nm were comparable to images obtained with the spectral camera

(Maestro) using the unmixing algorithm to remove autofluorescence

and requiring approximately 30 seconds for acquisition (Fig. 3a). In

contrast, injections with UC-Green enabled visualization of the

draining lymph nodes only when the skin was removed during

surgery because of poor tissue penetration of green light (Fig. 3b).

Serial injections of both nanocrystals revealed enhancement of the

same draining lymph nodes at two wavelengths, however, the

UC-NIR, which was injected earlier, moved further than the UC-

Green which was injected later (Fig. 4). As before only the UC-NIR

was visible before surgery but both agents could be used to visualize

the draining node during surgery and ex vivo after the overlying tissue

was removed (Fig. 4).

Current in vivo fluorescence imaging results in excitation of

autofluorescence and this background signal must be overcome

before the target is detected. Bioluminescence imaging (BLI) has

the advantage of not requiring excitation light, and therefore has

greatly reduced autofluorescence signal. However, BLI requires

the transfection of an enzyme produced by another species such as

luciferase, which is normally produced by the firefly.12 The light

Fig. 3 Luminescence lymphatic imaging with upconverting nanocrystals ca

depicted the draining lymph nodes during in vivo and in situ imaging in all 4 mice

at 800 nm were comparable to the spectrally unmixed images which were post pr

to depict the draining lymph nodes only in situ during surgery probably becau

This journal is ª The Royal Society of Chemistry 2009

emitted during BLI is weak and so far the emission wavelength is

limited to spectral regions with poor tissue penetration, necessi-

tating long exposure times and obscuring deep objects.13 Trans-

lation of BLI to humans is very unlikely. In contrast, the UC-NIR

can be effectively excited with a 980 nm near infrared laser, which

is well above the excitation wavelengths of endogenous fluo-

rophores. Moreover, its emission at 800 nm allows optimal tissue

penetration.9 Unlike BLI, translation of UCNPs to humans is

feasible although extensive toxicity testing must be completed.

Another technology, which can obtain an emission light of shorter

wavelength than the excitation light, is the two-photon imaging.

However, conventional two-photon imaging is not technically

easy to apply to macroscopic in vivo imaging because the emission

light is in the blue or green range that cannot penetrate well in the

tissue. Therefore, almost all in vivo two-photon imaging has been

performed on the exposed surface of the living animal with

a microscope or an optical fiber.14 As our data show, imaging with

UC-NIR depicts the lymphatics with no background signal during

in vivo, in situ (during surgery) and ex vivo imaging without the

need for post processing. Results from Shan and Collins et al.11

indicate very limited to no toxicity of carboxyl and amino func-

tionalized nanocrystals when incubated up to 9 days with human

osteosarcoma cells. There was a slight concentration effect

observed when we compared nanocrystal concentrations of

0.25 mg/ml and 0.0625 mg/ml, 93% and 96.3% survival was

observed. This minimal difference suggests that the observed

concentration effects are negligible under the assay conditions.

Upconverting nanocrystals can be designed to emit various colors

of light with a single excitation similar to quantum dots. Therefore,

n be obtained with both spectral and single shot imaging. The NC-NIR

. Due to the minimal background, single shot luminescence images obtained

ocessed to remove autofluorescence (a). In contrast, the NC-green was able

se of poor penetration of green emission light through tissue (b).

J. Mater. Chem., 2009, 19, 6481–6484 | 6483

Fig. 4 Two color lymphatic imaging with serial injection of two upconverting nanocrystals. With serial injection of NC-NIR and NC-green,

enhancement at both wavelengths was seen within the draining node. The NC-NIR, which was injected earlier, moved further than the green particle,

which was injected 30 min later. Images were obtained 5 min after injection of the NC-green nanocrystal.

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luminescence imaging using UCNPs could permit multi-color imaging

as shown in Fig. 4 with the advantage over quantum dots of not

producing interfering background autofluorescence (ESI Fig. S3†).

This dramatic reduction in background should permit the use of lower

doses of contrast agents, somewhat mitigating toxicity concerns.

Despite recent reductions in the size of UCNPs, their current size

(�20 nm for core;�50 nm with coating) is still larger than the 10–20

nm size that is optimal for in vivo imaging.3,15 Therefore, in their

current form, UCNPs have suboptimal uptake by the lymphatics.

However, smaller and brighter nanocrystals are under development

and could be ideally suited to lymphatic imaging in comparison to

existing agents.16

In conclusion, we demonstrate that lymphatic imaging with

UCNPs is feasible and results in images without autofluorescence.

This enables the direct, real time capture of lymphatic images without

extensive post processing of the images. Upconverting nanocrystals

have the potential to be a robust component nanostructure for

developing future molecular imaging probes.

Acknowledgements

This research was supported by the Intramural Research Program of

the NIH, National Cancer Institute, Center for Cancer Research, and

supported by the Ben Franklin Technology Development Authority

through the Ben Franklin Technology Partners of Southeastern

Pennsylvania and The Nanotechnology Institute.

6484 | J. Mater. Chem., 2009, 19, 6481–6484

Notes and references

1 R. Weissleder and M. J. Pittet, Nature, 2008, 452, 580–589.2 S. Kim, Y. T. Lim, E. G. Soltesz, A. M. De Grand, J. Lee,

A. Nakayama, J. A. Parker, T. Mihaljevic, R. G. Laurence,D. M. Dor, L. H. Cohn, M. G. Bawendi and J. V. Frangioni, Nat.Biotechnol., 2004, 22, 93–97.

3 H. Kobayashi, Y. Hama, Y. Koyama, T. Barrett, C. A. Regino,Y. Urano and P. L. Choyke, Nano Lett., 2007, 7, 1711–1716.

4 R. M. Hoffman, Nat. Rev. Cancer, 2005, 5, 796–806.5 R. M. Levenson and J. R. Mansfield, Cytometry A, 2006, 69, 748–758.6 H. J. Zijlmans, J. Bonnet, J. Burton, K. Kardos, T. Vail,

R. S. Niedbala and H. J. Tanke, Anal Biochem., 1999, 267, 30–36.7 F. Wang and X. Liu, J. Am. Chem. Soc., 2008, 130, 5642–5643.8 D. K. Chatterjee, A. J. Rufaihah and Y. Zhang, Biomaterials, 2008,

29, 937–943.9 M. Nyk, R. Kumar, T. Y. Ohulchanskyy, E. J. Bergey and

P. N. Prasad, Nano Lett., 2008, 8, 3834–3838.10 H. X. Mai, Y. W. Zhang, R. Si, Z. G. Yan, L. D. Sun, L. P. You and

C. H. Yan, J. Am. Chem. Soc., 2006, 128, 6426–6436.11 J. Shan, J. Chen, J. Meng, J. Collins, W. Soboyejo, J. S. Friedberg and

Y. Ju, J. Appl. Phys., 2008, 104, 094308–094307.12 C. S. Levin, Eur. J. Nucl. Med. Mol. Imaging, 2005, 32(Suppl. 2),

S325–345.13 C. P. Klerk, R. M. Overmeer, T. M. Niers, H. H. Versteeg,

D. J. Richel, T. Buckle, C. J. Van Noorden and O. van Tellingen,Biotechniques, 2007, 43(7–13), 30.

14 O. Garaschuk, R. I. Milos and A. Konnerth, Nat. Protoc., 2006, 1,380–386.

15 H. Kobayashi and M. W. Brechbiel, Adv. Drug Deliv. Rev., 2005, 57,2271–2286.

16 T. Barrett, P. L. Choyke and H. Kobayashi, Contrast Media Mol.Imaging, 2006, 1, 230–245.

This journal is ª The Royal Society of Chemistry 2009