Breast Cancer Res Treat (2007) 103:2328
DOI 10.1007/s10549-006-9347-0
PRECLINICAL STUDY
Simultaneous two-color spectral fluorescence lymphangiography
with near infrared quantum dots to map two lymphatic flows
from the breast and the upper extremity
Yukihiro Hama Yoshinori Koyama
Yasuteru Urano Peter L. Choyke
Hisataka Kobayashi
Received: 14 July 2006 / Accepted: 17 July 2006 / Published online: 21 September 2006

Springer Science+Business Media B.V. 2006
Abstract Due to their small size and poor access, the
lymphatic function has been difficult to study in vivo.
Especially difficult is the mapping of lymphatic drain-
age from two basins into the same node. Quantum dots
can be used to perform multicolor images with high
fluorescent intensity and are of a nano-size size suitable
for lymphatic imaging via direct interstitial injection.
Here we show simultaneous two-color in vivo wave-
length-resolved spectral fluorescence lymphangiogra-
phy using two near infrared quantum dots with
different emission spectra, which allow non-invasive
and simultaneous visualization of two separate lym-
phatic flows draining the breast and the upper
extremity and variations in the drainage patterns and
the water sheds within the axillary node. Two-color
spectral fluorescence lymphangiography can provide
insight into mechanisms of drainage from different
lymphatic basins that may lead to sentinel lymph nodes
detection of the breast cancer as well as prevention of
complications such as lymphedema of the arm.
Keywords Lymphatic drainage Imaging Breast
cancer Lymph node Lymphedema Nanotechnology
Yukihiro Hama and Yoshinori Koyama are contributed
equally to this work.
Y. Hama Y. Koyama P. L. Choyke H. Kobayashi (&)
Molecular Imaging Program, Center for Cancer for Cancer
Research, National Cancer Institute, NIH, Bldg. 10, Room
1B40, MSC 1088, Bethesda, MD 20892-1088, USA
e-mail: Kobayash@mail.nih.gov
Y. Urano
Graduate School of Pharmaceutical Sciences,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Quantum dots Spectral fluorescence imaging
Near infrared
Introduction
The lymphatic system is difficult to evaluate because its
channels are small and not directly accessible. More-
over, when assessing lymphatic drainage from two
separate drainage basins, techniques such as X-ray
lymphangiography, MR lymphangiography or radio-
nuclide radioscintigraphy are inadequate because it is
impossible to differentiate the contributions from each
lymphatic basin. Quantum dots (Qdots) are charac-
terized by sharply defined emission spectra and can be
synthesized to emit a variety of wavelengths including
in the near infrared (NIR) [1]. Qdots can vary in size
but some are appropriately sized to conduct lymphatic
studies based on prior studies using macromolecular
dendrimer particles on MRI, which demonstrate opti-
mal lymphatic uptake for particles 912 nm in diame-
ter [2, 3]. Kim et al. [4] reported remarkable in vivo
imaging of the lymphatics using a NIR Qdot in order to
detect the sentinel lymph node (SLN) arising from
breast tissue. However, few in vivo multicolor imaging
studies for visualizing two separate physiological
functions have been reported [5], although the multi-
color imaging with two Qdots has been introduced in in
vitro microscopic imaging as a possible application of
nanotechnology to the bio-imaging. Therefore, we
chose two NIR Cadmium-Tellurium (CdTe) Qdots
with different emission spectra (705 and 800 nm peak
emission), based on their appropriate physical nano-
size for lymphatic imaging compared with other kinds
of Qdots such as Cadmium Selenium (CdSe) series. We
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simultaneously injected the two Qdots, into the mam-
mary gland and the skin of the middle phalange in the
upper extremity in order to monitor the lymphatic
drainage functions from these two basins, which both
drain into the axillary lymph nodes.
Materials and methods
Chemicals
Carboxyl quantum dots, Qdot 705 ITKTM (peak
emission wavelength at 705 nm) and Qdot 800
ITKTM (peak emission wavelength at 800 nm), which
had CdTe core with an additional thin semiconductor
shell (zinc sulfide) of ~6 and ~12 nm in diameter,
respectively, coated by thin polymer (12 nm) con-
taining carboxyl groups, were purchased from Invitro-
gen Corporation (Carlsbad, CA, USA). A mixture of
800 nmol/l Qdot 705 ITKTM or Qdot 800 ITKTM
was prepared in PBS.
In vivo wavelength-resolved two-color spectral
fluorescence imaging
All in vivo procedures were carried out in compliance
with the Guide for the Care and Use of Laboratory
Animal Resources (1996), National Research Council,
and approved by the National Cancer Institute Animal
Care and Use Committee. Ten-week-old normal fe-
male athymic mice were anesthetized with intraperi-
toneal injection of 1.15 mg sodium pentobarbital
(Dainabot, Osaka, Japan). Then, ten consecutive mice
were given intracutaneous injections of 10 ll (8 pmol)
of Qdot 800 ITKTM into the middle phalange of the
left upper extremity and subcutaneous injections of
20 ll (16 pmol) of Qdot 705 ITKTM into the left
breast. Another five consecutive mice were adminis-
tered the Qdots in the opposite order (i.e. intracuta-
neous injections of 10 ll (8 pmol) of Qdot 705
ITKTM into the middle phalange of the left upper
extremity and subcutaneous injections of 20 ll (16
pmol) of Qdot 800 ITKTM into the left breast).
Immediately after injection of the Qdots, wavelength-
resolved spectral imaging was carried out using a
spectral imaging system (Maestro In-Vivo Imaging
System, CRI Inc., Woburn, MA, USA). Animals were
placed in the prone and in the right lateral decubitus
position under pentobarbital anesthesia. The excitation
band pass filter 575605 nm was used. The tunable
filter was automatically stepped in 10-nm increments
from 600 to 950 nm with the same exposure time for
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Breast Cancer Res Treat (2007) 103:2328
images captured at each wavelength. Collected images
were analyzed by the Maestro software, which uses
spectral unmixing algorithms to separate autofluores-
cence from quantum dot signals, and a composite im-
age consisting of Qdot 705 ITKTM and Qdot 800
ITKTM signals and autofluorescence was generated.
Unmixed composite images of in vivo lymphangiog-
raphy as well as ex vivo LN images for both Qdot 705
ITKTM and Qdot 800 ITKTM were compared inde-
pendently by two reviewers (Y.K. and H.K.). The
qualitative visibility of the axillary LNs on lymphan-
giograms obtained with each fluorescence signal (705
or 800 nm) was rated on a 5-point scale (, , +, ++,
and +++). Any discrepancy between the two reviewers
was resolved by discussion.
Two-color spectral fluorescence microscopic
imaging of the lymph node section
The mice were killed with intravenous injection of
sodium pentobarbital 1 h after Qdots injection. The
axillary, lateral thoracic and cervical LNs were dis-
sected and removed and frozen in Tissutek (Sakura,
Torrance, CA, USA) and stored at 80C. Frozen
samples were sectioned by a cryostat microtome
(CM1800, Leica, Bannockburn, IL, USA) at a thick-
ness of 30 lm. Slides were analyzed under an Olympus
BX61 microscope (Olympus America Inc., Melville,
NY, USA) equipped with the following filters: Cy5
cube set for Qdot 705 ITKTM; excitation wavelength
590650 nm, emission wavelength 665730 nm band
pass, Cy7 cube set for Qdot 800 ITKTM; excitation
wavelength 675745 nm, emission wavelength 770
850 nm band pass.
Results
In vivo wavelength-resolved two-color spectral
fluorescence lymphangiography of the axillary
region visualized the territory of the lymphatic
drainage from two different basins
Two-color NIR lymphangiography following injections
of Qdot 705 ITKTM in the breast and Qdot 800
ITKTM in the upper extremity successfully visualized
the lymphatic vessels as well as demonstrated the
lymphatic drainage territory in all ten mice using
spectral fluorescence imaging (Fig. 1). In the two-color
wavelength-resolved spectral fluorescence lymphangi-
ography, eight axillary lymph nodes (LNs) had mixed
contributions from both the breast Qdot (705 nm) and
Breast Cancer Res Treat (2007) 103:2328
Fig. 1 A schematic illustration of two-color NIR fluorescence
imaging with a graph of emission spectra of two NIR quantum
dots (Qdot705; red, and Qdot800; green) visualizing the bidirec-
tional axillary lymphatic flows from the mammary pad (Qdot705;
red) and the upper extremity (Qdot800; green). The axillary,
lateral thoracic and superficial neck lymph nodes were the
draining lymph nodes visualized in this experiment
the upper extremity Qdot (800 nm) (Fig. 2a). These
findings were clearly depicted in images at the surgery
(Fig. 2b) and validated by the fluorescence microscopic
images of frozen LN sections (Fig. 2c). Among these
eight animals, however, there were variable contribu-
tions from each basin indicating different flow rates in
each animal, a phenomenon that was illustrated by the
varying proportion of the two Qdots in the resected
axillary node (Table 1, Fig. 2a). Two axillary LNs re-
ceived only one color (800 nm) of Qdot indicating that
the lymphatic flow was exclusively arising from the
upper extremity (Fig. 3). Interestingly, the mice whose
axillary lymph nodes received lymphatic flow exclu-
sively from the upper extremities and not the breast
25
tissues had increased lymphatic flow from the breasts
to the cervical LNs indicating that the principal
drainage pattern of the breast had been altered in these
animals (Fig. 3, Table 1). Ex vivo fluorescence imaging
and fluorescence microscopy depicting the mixture of
Qdots within resected LNs validated the lymphatic
drainage found in the in vivo lymphangiography in all
ten mice (Table 1).
Inappropriate use of quantum dots failed to show
two-color lymphangiography
When the Qdot 800 ITKTM was injected in the breast
and the Qdot 705 ITKTM was injected in the upper
extremity, the lymphatic drainage from the breast was
only faintly visualized on in vivo spectral NIR fluo-
rescence images. In these five animals only the Qdot
705 ITKTM was consistently visualized as the Qdot
800 ITKTM signal was too weak (Fig. 4). Visualization
score of the lymphatic drainage from the breast with
Qdot 800 ITKTM was significantly less than that with
Qdot 705 ITKTM (P < 0.005). The in vivo findings
corresponded with the ex vivo fluorescence imaging
and fluorescence microscopy of the resected LNs
(Table 1).
Discussion
Based on prior results with Gadolinium labeled
dendrimer-based nano particles, 910 nm in diameter
is an ideal diameter for uptake by the lymphatic system
from either the mammary gland or the subcutaneous
space for identifying a sentinel LN, however a broad
range of diameters (530 nm) can still be used [6, 7].
Interestingly, for visualizing the lymphatics of the up-
per extremity by intradermal injection, a larger parti-
cle, of approximately 13 nm or larger in diameter was
optimal for visualizing the lymphatic flow [2]. There-
fore, in this experiment we matched the Qdot size to
the lymphatic basin. The smaller Carboxyl Qdot705
has a core diameter of ~6 nm and a thin coating of
12 nm making it ideal for imaging the lymphatic
drainage from the mammary gland, while the larger
Carboxyl Qdot800 has a core diameter of ~12 nm and
the same thin coating making it ideal for imaging the
lymphatic drainage from the upper extremity. With this
selection of Qdots, we were able to image the respec-
tive lymphatic drainage basins from both the mammary
gland and the upper extremity and determine the
trafficking of lymphatic flow in the axillary node in all
10 mice. However, when the Qdots were reversed and
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the larger Qdot800 was injected in the mammary tis-
sue, the lymphatic drainage from the mammary gland
was barely visualized due to its larger size. In mice, in
which the lymphatic flows from the mammary gland
were faintly visualized as shown in Fig. 4, fluorescence
signal of Qdot800 was too weak compared with that of
Qdot705 to display both colors on a single display.
m
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Breast Cancer Res Treat (2007) 103:2328
The practical implications of this finding are that it
is now possible to study the drainage patterns and
mixing of adjacent lymphatic basins in vivo using a
high resolution imaging in a laboratory environment.
There are a number of nodes in the lymphatic system
that receive afferent lymphatic vessels from more
than one body region especially in human yet there is
variability in these drainage patterns that is poorly
understood. The immunologic consequences of these
drainage patterns have not been studied. Two-color
spectral fluorescence imaging could assist in the
understanding of lymphatic flow patterns. This may
also aid in the understanding of the development of
lymphedema following resection of lymph nodes such
as the axillary or inguinal nodes. Lymphedema is a
major complication of radical breast cancer and mel-
anoma surgery [811] and a possible complication for
the SLN biopsy [914]. Since the procedure described
here is similar to that of lymphoscintigraphy without
radioactivity, it may be possible to visualize the ax-
illary LN both from the upper extremity and from the
breast cancer simultaneously and thereby predict the
likelihood of postoperative lymphedema and reduce
the morbidity associated with axillary lymph node
dissection by avoiding the disruption of lymphatic
flow from the upper extremity. However, to be clini-
cally relevant, Qdots composed of non-toxic materials
will need to be developed.
To our knowledge, this is the first demonstration of
simultaneous imaging of two different lymphatic
drainages in vivo and their trafficking to a LN. Since
Fig. 2 a In vivo and ex vivo spectral fluorescence imaging of a
mouse injected with Qdot800 (green) intracutaneously into the
middle digit of the left upper extremity and Qdot705 (red) into
the left mammary pad. The axillary lymph node received the two
different lymphatic flows simultaneously from the mammary pad
and the upper extremity. Lymph nodes as well as the lymphatic
vessels (arrows) were clearly visualized through the skin. b In
vivo spectral fluorescence imaging neck and axillary regions at
the surgery of the mouse shown in a. The lymphatic system
especially lymphatic draining ducts from both the breast (red)
and the extremity (green) were more clearly depicted after
removal of the skin and the fat tissue than before the surgery as
shown in a. c Differential interference contrast (DIC) and
fluorescence microscopy of extracted axillary lymph node in the
same mouse shown in a. The two-color fluorescence microscopy
more clearly and reliably demonstrated the intra-lymphatic
drainage territory as well as the water shed of the two different
lymphatic flows (interface between the green and red Qdots).
The fluorescence microscopy findings were compatible with the
in vivo optical lymphangiography in all mice. Eight of ten mice
examined showed similar findings to the mouse shown in figure
but in two mice drainage was exclusively from the upper
extremity (see Fig. 3). The filters used for excitation and
emission were 590650 and 665730 nm for Qdot705 and 675
745 and 770850 nm for Qdot800, respectively
Breast Cancer Res Treat (2007) 103:2328 27

Table 1 Summary of in vivo and ex vivo spectral fluorescence imaging of the axillary and the cervical LNs
Animal# Injected Qdot In vivo spectral Ex vivo spectral In vivo spectral
fluorescence imaging fluorescence imaging fluorescence imaging
of the axillary LN of the axillary LN of the cervical LN
Breast Hand 705 nm 800 nm 705 nm 800 nm 705 nm 800 nm

1 705 800 ++ +++ ++ +++ +++

2 705 800 +++ +++ +++ +++
3 705 800 +++ +++ +++
4 705 800 ++ ++ ++ +++ ++
5 705 800 +++ +++ +++ +++ +
6 705 800 +++ +++ +++ +++ ++
7 705 800 + +++ ++ +++ +++
8 705 800 +++ +++ ++
9 705 800 +++ ++ +++ ++ ++
10 705 800 ++ +++ ++ +++
11 800 705 +++ +++
12 800 705 +++ +++
13 800 705 +++ +++
14 800 705 +++ +++
15 800 705 +++ +++ +

Fig. 3 In vivo and ex vivo spectral fluorescence imaging of a
mouse injected with Qdot800 (green) intracutaneously into the
middle digit of the left upper extremity and Qdot705 (red) into
the left mammary pad. The axillary lymph node received
lymphatic flow exclusively from the upper extremity (green).
The lymphatic vessel from the upper extremity to the axillary
lymph node (green arrow) and that from the breast to the
superficial neck lymph node (red arrow) were clearly shown
Fig. 4 In vivo and ex vivo spectral fluorescence imaging of a
mouse injected with Qdot705 (red) intracutaneously into the
middle phalange of the left upper extremity and with Qdot800
(green) into the left breast (opposite injection). The lymphatic
drainage from the breast (green) was too weak to show up in two-
color NIR in vivo imaging. All five mice examined showed the
similar finding as the mouse shown in figure

the two colors of each Qdot were related to their size,
it was possible to match the Qdot to its most appro-
priate lymphatic basin, based on diameter thus, opti-
mizing imaging for both lymphatic tracts. Qdots hold
promise as an imaging method for the exploration of
the lymphatic system that may have implications for
the processing of the cancer metastasis and the im-
mune response to cancer cells or vaccines in the lymph
nodes.
Acknowledgment This research was supported by the Intra-
mural Research Program of the NIH, National Cancer Institute,
Center for Cancer Research.
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