Manganese doped fluorescent paramagnetic nanocrystals for dual-modal imaging

Article abstract of DOI:10.1002/smll.201401143

In this work, dual-modal (fluorescence and magnetic resonance) imaging capabilities of water-soluble, low-toxicity, monodisperse Mn-doped ZnSe nanocrystals (NCs) with a size (6.5 nm) below the optimum kidney cutoff limit (10 nm) are reported. Synthesizing Mn-doped ZnSe NCs with varying Mn²⁺ concentrations, a systematic investigation of the optical properties of these NCs by using photoluminescence (PL) and time resolved fluorescence are demonstrated. The elemental properties of these NCs using X-ray photoelectron spectroscopy and inductive coupled plasma-mass spectroscopy confirming Mn²⁺ doping is confined to the core of these NCs are also presented. It is observed that with increasing Mn²⁺ concentration the PL intensity first increases, reaching a maximum at Mn²⁺ concentration of 3.2 at% (achieving a PL quantum yield (QY) of 37%), after which it starts to decrease. Here, this high-efficiency sample is demonstrated for applications in dual-modal imaging. These NCs are further made water-soluble by ligand exchange using 3-mercaptopropionic acid, preserving their PL QY as high as 18%. At the same time, these NCs exhibit high relaxivity (≈2.95 mM^-1 s^-1) to obtain MR contrast at 25°C, 3 T. Therefore, the Mn²⁺ doping in these water-soluble Cd-free NCs are sufficient to produce contrast for both fluorescence and magnetic resonance imaging techniques.

Full text of DOI:10.1002/smll.201401143

Quantum Dots
Manganese Doped Fluorescent Paramagnetic
Nanocrystals for Dual-Modal Imaging
Vijay Kumar Sharma, Sayim Gokyar, Yusuf Kelestemur, Talha Erdem, Emre Unal,
and Hilmi Volkan Demir*
I n this work, dual-modal (fluorescence and magnetic resonance) imaging capabilities
of water-soluble, low-toxicity, monodisperse Mn-doped ZnSe nanocrystals (NCs) with a
size (6.5 nm) below the optimum kidney cutoff limit (10 nm) are reported. Synthesizing
Mn-doped ZnSe NCs with varying Mn2+ concentrations, a systematic investigation of
the optical properties of these NCs by using photoluminescence (PL) and time resolved
fluorescence are demonstrated. The elemental properties of these NCs using X-ray
photoelectron spectroscopy and inductive coupled plasma-mass spectroscopy confirming
Mn2+ doping is confined to the core of these NCs are also presented. It is observed that
with increasing Mn2+ concentration the PL intensity first increases, reaching a maximum
at Mn2+ concentration of 3.2 at% (achieving a PL quantum yield (QY) of 37%),
after which it starts to decrease. Here, this high-efficiency sample is demonstrated for
applications in dual-modal imaging.These NCs are further made water-soluble by ligand
exchange using 3-mercaptopropionic acid, preserving their PL QY as high as 18%. At
the same time, these NCs exhibit high relaxivity (≈2.95 mM−1 s−1) to obtain MR contrast
at 25 °C, 3 T.Therefore, the Mn2+ doping in these water-soluble Cd-free NCs are sufficient
to produce contrast for both fluorescence and magnetic resonance imaging techniques.
1. Introduction
because of their exceptional optical properties. These include
Colloidal semiconductor quantum dots (QDs), also known high quantum yield (QY), broad absorption with narrow
as nanocrystals (NCs) make an important class of inorganic photoluminescence (PL) spectra, and a high resistance to
fluorophores, which are gaining widespread recognition photobleaching. This greatly enhances their potential of flu-
orescence-based imaging (FI).[1,2] However, in bio-imaging,
FI cannot provide three-dimensional (3D) anatomical infor-
mation. In contrast, magnetic resonance imaging (MRI)
Dr. V. K. Sharma, S. Gokyar, Y. Kelestemur,
T. Erdem, E. Unal, Prof. H. V. Demir
is an important diagnostic tool with its ability to generate
3D images of opaque and soft tissues with sufficient spatial
resolution and tissue contrast.[3,4] Nevertheless, despite its
imaging capability, the inherent low sensitivity of the MRI
technique demands the synthesis of high relaxivity contrast
enhancement agents. Contrast agents are currently applied
in 30 to 40% of clinical MRI scans. Most of the commercial
MR (T1 weighted) contrast agents contain the paramagnetic
Gd3+ ion, which has seven unpaired electrons and a long elec-
tronic relaxation time.[5,6] These contrast agents are intrave-
nously administered to patients, reducing the relaxation time
of water protons in the tissue of interest and increasing signal
intensity. Recently, there have been efforts reported to com-
bine different imaging techniques so that more information
UNAM-Institute of Materials
Science and Nanotechnology
Department of Electrical and Electronics Engineering
Department of Physics
Bilkent University
Ankara 06800, Turkey
E-mail: volkan@bilkent.edu.tr
Prof. H. V. Demir
Luminous! Center of Excellence for Semiconductor
Lighting and Displays
School of Electrical and Electronic Engineering
School of Mathematical and Physical Sciences
Nanyang Technological University
Singapore 639798, Singapore
DOI: 10.1002/smll.201401143
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V. K. Sharma et al.
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can be obtained from the sample under study.[7–9] With two
functionalities integrated into a single type of NCs, a sensitive
contrast agent for two very powerful and highly complemen-
tary imaging techniques can be obtained. Therefore, multi-
modal imaging has stimulated intense interest for accurate
medical diagnosis.
There are few reports in the literature on multimodal
imaging based on the conjugation of QDs and magnetic
nanoparticles (NPs).[10–16] Most of these use cadmium (Cd)
based QDs, for example, CdSe/ZnS,[10–14] CdTe/CdS,[15]
CdSeTe/CdS[16] for FI. For MRI, Gd3+[10,11,16] ion is com-
[12–15]
monly used for T1 weighted imaging and Fe3O4
NPs for
T2 weighted imaging in these previous reports. Cadmium is
a toxic element, which limits its potential applications, espe-
cially related to human health.[17] Although gadolinium (Gd)
has been the most popular choice among the paramagnetic
metals, it has been recently linked to a medical condition
known as nephrogenic systemic fibrosis (NSF).[18] NSF is a
rare but potentially harmful side effect observed in some
patients with severe renal disease or following liver trans-
plant. For obvious reasons, this has led to concerns over the
safety of Gd-based T1 contrast enhancement agents in MRI
applications.[19,20]
Figure 1. PL spectra of Mn-doped ZnSe NCs for different Mn2+
concentrations. The excitation wavelength used is 300 nm.
with their small size <<10 nm (below kidney cutoff limit)
Recently, Mn-doped NCs have been regarded as a prom- and Mn2+ doping confinement within the core, they offer
ising new class of nanophosphors, owing to their superior high PL QY for FI and high relaxivity for MRI, while being
luminescent properties and potential applications in opto- water-soluble.
electronics[21] and bio-imaging.[22] They exhibit a broad emis-
sion peak at 585 nm, with a large stoke`s shift of 160 nm,
avoiding the issue of self-absorption. The Mn2+ (S = 5/2) is
also used as a paramagnetic probe in several solids with a
2. Results and Discussion
magnetic moment of 5 µB. Manganese is considered to be safe Mn-doped ZnSe NCs were synthesized using nucleation
for use in MRI contrast agents with no relation to NSF. The doping strategy[21] which is explained in detail in the experi-
only issue is with the overexposure to free Mn ions, which mental section. The influence of Mn2+ doped concentration
must be avoided not to risk neurode-generative disorder.[18]
on the PL properties of Mn-doped ZnSe NCs was studied
There are only two known reports[23,24] of Mn-doped NCs and presented in Figure 1. The injected Mn2+ concentration
used for dual-modal imaging. Wang et al.[23] reported high was varied from 1.6 to 10.8 at% to investigate the effect of
QY (≈21% in water) and high relaxivity for CdSe/Zn1−xMnxS Mn2+ on the optical properties of these NCs while keeping
QDs. However, the problem with these QDs is the intrinsic the other experimental conditions fixed (with a constant
toxicity of Cd, which limits its widespread applications. molar ratio (Zn(St)2/TBPSe/SA = 3.16/2.4/1.4). The dopant
Another issue is that, Mn is present in the shell, which is PL peak position was found to be independent of the Mn2+
again potentially toxic if released from the shell.[18] Recently, concentration (with PL emission peaking at 585 nm and full-
there is another report on dual imaging contrast agent by width-half-maximum (FWHM) ≈ 54 nm). It was observed
Gaceur et al.[24] using Mn-doped ZnS NPs. This work reports that increasing the Mn2+ concentration from 1.6 to 4.8 at%,
blue-green emission in Zn0.9Mn0.1S NPs with a high relaxivity increases the PL emission intensity, which indicates the suc-
≈20 mM−1 s−1, but the QY of these NPs has not been studied cessful incorporation of Mn2+ into ZnSe NCs. The PL QY
in the paper. This report used Mn2+ doping for the MRI gradually increases from 14 to 37% by increasing the Mn2+
imaging, but not for the FI. Different than the previous lit- concentration from 1.6 to 4.8 at%.
erature, here we are reporting smaller, water-soluble and low
Further increasing the Mn2+ concentration (above
toxicity Mn-doped ZnSe NCs exhibiting high PL QY for FI 4.8 at%) leads to a decrease in the PL QY of these NCs.
and high relaxivity for MRI. These NCs are Cd-free and have When the concentration of Mn2+ is higher than this threshold,
Mn2+ doping mostly localized in the core of the NCs, which is the nonradiative energy transfer between neighboring Mn2+
confirmed by elemental characterization. Thus, our NCs can dopant ions suppresses the fluorescence due to the concen-
be considered to be less toxic with a sufficient Mn2+ concen- tration quenching effect.[26,27] Under our synthesis conditions,
tration in the core region enabling both high QY and high the formation of such pairs of Mn2+ dopant ions is observed
contrast in MRI. Moreover, these NCs also meets another when the amount of Mn2+ ions in QDs is higher than 4.8 at%.
important criterion related to their size. They are 6.5 nm in It is considered that the number of the luminescent Mn2+ ions
size, well below the optimum kidney cutoff limit, which is increases within nanocrystals (MnSe core and/or MnSe/ZnSe
10 nm. Therefore, these NCs can be used in most parts of the interface) in the low concentration region. On the other hand,
human body.[25] Here, these NCs are unique in that, together heavy doping with Mn2+ increases the nonradiative processes
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Manganese Doped Fluorescent Paramagnetic Nanocrystals for Dual-Modal Imaging
components measured in ms due to the Mn2+ ions. For the
sample with Mn2+ concentration of 1.6 at%, the decay pro-
file of the Mn2+ PL is found to be a single exponential with a
lifetime of 0.1 ms. As the Mn2+ concentration increases above
4.8 at%, a fast decay component appears in addition to the
0.1 ms lifetime component. The PL decay of isolated Mn2+
4
ions is slow because the T1–6A1 transition of single Mn2+
ions is parity- and spin-forbidden and is only weakly allowed
through the odd parity crystal field and the spin-orbit interac-
tion. It is known that the PL lifetime of Mn2+ pairs is shorter
than that of single Mn2+ ions[28,29] because in the exchange
coupled Mn2+ pairs the spin selection rule is relaxed and
the electric dipole transition is allowed. Therefore, we con-
clude that the faster decay PL component is attributed to the
Mn-Mn pairs in highly doped nanocrystals. This result is also
supported by the observation of PL intensity decreasing with
the increasing Mn2+ concentration above 4.8 at%.
We also performed an elemental analysis of these NCs
using X-ray photoelectron spectroscopy (XPS) and inductive
coupled plasma—mass spectroscopy (ICP-MS) to determine
the concentration of Mn2+ in these NCs. ICP-MS is one of
the most sensitive and robust technique for measuring the
elemental concentrations and XPS is selected for studying
the core/shell nature of these NCs since it is a surface sensi-
tive technique.[30] The ratio of Mn2+ to Zn2+ in the Mn-doped
ZnSe NCs determined by ICP-MS and XPS, compared with
the corresponding injected values, are presented in Table 1.
Since ICP-MS measurements provide the overall ratio of
the two cations in a sample, the observed Mn2+ to Zn2+ ratio
should reflect the real value. The Mn-to-Zn ratio of the NCs
determined by XPS shows lower values, which is consistent
with the targeted core/shell structure and suggests that Mn2+
ions are localized in the core. As a result, more Zn appears
in the recording of XPS for the core/shell NCs. A core/shell
structure with a diffused layer between the core and shell
is also possible in our case as reported by Pradhan et al.[21]
for high synthesis temperature. From the above results, we
observe that the sample with Mn2+ concentration ≈4.8 at%
(injected Mn2+ concentration) reaches the maximum PL QY
≈37%. The actual value of Mn2+ concentration as confirmed
by ICP-MS is 3.2 at%.
We investigated Mn-doped ZnSe NCs with Mn2+ con-
centration ≈4.8 at% having maximum PL QY as a potential
contrast agent for FI and MRI. These NCs were rendered
water-soluble by ligand exchange using 3-mercaptopropi-
onic acid (MPA) for bio-medical applications. We studied
and compared the optical and structural properties of these
Figure 2. Time-resolved fluorescence decay profiles measured at
585 nm of Mn-doped ZnSe NCs at different Mn2+ concentrations under
excitation of 310 nm.
Table 1. Mn2+ concentration (Mn/Zn molar ratio) obtained by ICP-MS
and XPS for the Mn-doped ZnSe NCs.
due to the formation of pairs of Mn2+ dopant ions, which in
turn decreases the PL QY.
Injected
[at%]
ICP-MS
[at%]
XPS
[at%]
To obtain a better understanding of the effect of Mn2+
concentration on the emission kinetics, we performed time-
resolved fluorescence (TRF) measurements on these NCs.
Figure 2 shows the PL decay profiles of these NCs with var-
ying Mn2+ concentrations excited at 310 nm and monitored
at 585 nm. The PL decay profiles exhibit an initial sharp
spike due to the surface defects followed by the longer decay
1.6
3.2
4.8
6.4
9.6
1.1
1.8
3.2
4.4
5.9
0.8
1.3
2.5
3.6
4.5
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Figure 4. a) TEM and b) SAED of Mn-doped ZnSe NCs with Mn2+
concentration (4.8 at%). In the inset, HR-TEM of Mn-doped ZnSe NCs is
shown. Scale Bar is 10 nm for (a).
resolution-TEM) HR-TEM image of Mn-doped ZnSe NCs.
SAED pattern shows three continuous rings with the inter-
planar spacing corresponding to the zinc blende structure of
ZnSe. The d spacing of the rings from the center are; d1 ≈
3.31 Å, d2 ≈ 2.03 Å, and d3 ≈ 1.73 Å which can be indexed
to (111), (220), and (311) planes of the zinc blende structure
of ZnSe. HR-TEM image corresponds to d spacing ≈ 3.33 Å
Figure 3. Photoluminescence and absorption (inset) spectra of toluene
and water-soluble Mn-doped ZnSe NCs with Mn2+ concentration
(4.8 at%). The excitation wavelength used is 300 nm.
NCs is toluene and water. Figure 3 shows the PL spectra of indexed to the most prominent (111) zinc blende phase of
the Mn-doped ZnSe (Mn2+ concentration ≈4.8 at%) NCs dis- ZnSe (JCPDS 80–0021). It is also observed that, the average
solved in toluene and water. The PL emission peak position size and size distribution of the NCs remained same after the
is close in the two different solvents with similar FWHMs. ligand exchange (not shown here).
Quantum yield for the water-soluble NCs (18%) is reduced
The utility of the water-soluble Mn-doped ZnSe NCs as
to almost half in comparison to toluene (37%). The reduction dual-mode imaging contrast agents was investigated in solu-
of QY during the transfer from organic phase to water results tion. NCs were studied by MRI and fluorescence microscopy
from a combination of different effects. First, isolation of the to confirm that Mn2+ content was sufficient to produce con-
NCs decreased the QY by ≈10–20% due to the loss of ligand trast in MR image and also the QY is enough to produce
molecules on the surface of the NCs and the further decrease fluorescence contrast in FI.
of QY in water can be attributed to the dipole effect. These
QY levels in water are comparable to the CdSe/Zn1−xMnxS 0.01, 0.02, 0.045, and 0.065 mM Mn2+ with DI water as a ref-
QDs.[23]
erence sample. T1-weighted images for increasing concentra-
Particles were dissolved in water at concentrations of
In the UV–Vis absorption spectroscopy (shown in the tion of Mn2+ with DI water as reference is shown in Figure 5.
inset of Figure 3) we observe the first excitonic peak at 425 nm, We can clearly observe the increase in the image contrast
which corresponds to the band edge absorption of the ZnSe with the increase in Mn2+ concentration. Relaxation time T1
NCs. While considering the Mn-dopant emission at 585 nm, was measured at 3 T, 25 °C using a Spin Echo sequence. From
the Stoke`s shift of ≈160 nm is observed as compared to the the slope of (1/T1 – 1/T0) vs Mn2+ concentration as shown in
first excitonic absorption peak of the ZnSe NCs. Photolumi- the Figure S3 of Supporting Information, we obtain the relax-
nescence excitation (PLE) spectra (Figure S1 of Supporting ivity (r1) value ≈2.95 mM−1 s−1, where T0 is relaxation time of
Information) has similar features as the absorption spectra DI water. Comparable relaxivity values were reported for Gd
(excitonic peak ≈425 nm) and the PL spectra (almost 50%
drop in efficiency at 300 nm). The PL decay profile (Figure S2
of Supporting Information) of the orange emission is found
to be single exponential with an average lifetime of 0.1 ms for
both toluene and water-soluble Mn-doped ZnSe NCs, which
means the concentration of Mn2+ remains similar in both
cases. This further confirms that the Mn2+ ions are only pre-
sent in the core and not in the shell. Such slow decay further
confirmed the assignment of this emission band to the spin
forbidden doped Mn2+ 4T1 to 6A1 transition.[31]
Transmission electron microscopy (TEM) was performed
to understand the morphology and structure of these NCs.
Figure 4a indicates that the size distribution of the Mn-doped
Figure 5. MRI of Mn-doped ZnSe NCs; a) from the Left (DI Water) to
ZnSe NCs was reasonably uniform with nearly spherical
shape with an average diameter ∼6.5 nm. Figure 4b shows
selected area electron diffraction (SAED) pattern and (high
right (higher Mn2+ concentration). b) Integrated intensity of the same
samples for clear understanding. The black line represents the average
of these signals.
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Manganese Doped Fluorescent Paramagnetic Nanocrystals for Dual-Modal Imaging
4. Experimental Section
Materials: Zinc stearate (ZnSt2, purum 10–12% Zn basis), octa-
decylamine (ODA, 97%), 1-octadecene (ODE, technical grade 90%),
tributyl phosphine (TBP, ≥ 93.5%), stearic acid (SA, ≥ 98.5%) and
manganese chloride (MnCl2, ≥ 99%), were purchased from Sigma-
Aldrich. Selenium powder (Se, ≥ 99.5%), tetramethylammonium
hydroxide (TMAH, 25 wt% in methanol), 3-mercaptopropionic acid
(MPA, ≥ 99%) and methanol (anhydrous, 99.8%) was purchased
from Alfa Aesar. All chemicals were used without further purification.
Synthesis of Manganese Stearate (MnSt2) : In a typical syn-
thesis, SA (2.25 g) was dissolved in 15 mL of anhydrous methanol
and heated to 75 °C until it became a clear solution. The solution of
TMAH was prepared by taking 0.7 mL in 5 mL anhydrous methanol
and mixed with SA solution. The mixture was stirred for 15 min in a
beaker. To this solution, MnCl2 solution of 0.5 g in 5 mL anhydrous
methanol was added drop wise with vigorous stirring and a white
precipitate of MnSt2 slowlyflocculated. The synthesis is carried out
inside glove box. The precipitates were washed repeatedly with
hot methanol. Then the white precipitant was dried under vacuum
for two days. The synthesized MnSt2 was stored inside glove box.
Preparation of Stock Solutions: TBPSe stock solution was
prepared in a glove box by adding 1.9 g of Se into 10 mL of TBP
by stirring at room temperature. The zinc stock solution was pre-
pared by dissolving ZnSt2 (1.8 g) and 0.4 g of SA in 10 mL of ODE.
The selenium precursor solution was prepared by mixing 1 mL
of TBPSe stock solution and 1 g of ODA in a vial heated at 75 °C
inside the glove box until it turns clear. Amine precursor solution
was prepared by mixing 0.5 g of ODA and 0.63 mL of ODE in a vial
at 75 °C inside glove box.
Synthesis of Mn-doped ZnSe NCs: In a typical reaction, variable
amount of MnSt2 (0.05, 0.10, 0.15, 0.20 and 0.30 g) and 25 mL
of ODE was loaded in 50 mL three neck flask and degassed under
argon for 20 min at 100 °C. Then, the temperature of clear brownish
manganese precursor solution was increased to 280 °C. At this tem-
perature the solution became transparent; the selenium precursor
(≈2 mL) was quickly injected into the main reaction vessel. After the
injection, the color of the solution turned faint yellow indicating for-
mation of MnSe nanoclusters. The temperature of the main reaction
was dropped to 260 °C and was held there for 1 h. At the same
time, the zinc stock solution was heated at 125 °C under the argon
gas flow until a clear solution was formed. The reaction tempera-
ture of main reaction was set to 290 °C for the injection of zinc pre-
cursor. Once it hit the target temperature, 3 mL of the zinc precursor
solution was injected quickly. Immediately after the Zinc precursor
injection, the solution glow yellow under UV-light, showing the
growth of ZnSe over MnSe NCs. The temperature of the main reac-
tion was decreased to 260 °C and held there for 20 min. Amine pre-
cursor solution was added to the main reaction. The same injection
strategy was performed three times with 20 min intervals for the
completion of the reaction and all the zinc precursor was injected
into the main reaction. The growth process was monitored through
successive UV–Vis and PL measurements. Finally, the reaction was
cooled to room temperature. We used a new method for purifica-
tion of these NCs. For purification, the NCs were warmed and cen-
trifuged. The lower part was taken and added methanol, then the
solution was mixed well and warmed again. After taking the lower
part and adding toluene, iso-propanol and methanol the mixture is
precipitated. Finally, toluene was added and centrifuged. Finally,
Figure 6. Fluorescence Image of a drop cast Mn-doped ZnSe NCs on
quartz substrate.
based MR contrast agents and it is observed that the relax-
ivity increases with the increase in the values of magnetic
field.[32] Thus, these NCs offer a potential alternative to the
Gd based MR contrast agents. In addition, our NCs possess
high fluorescent QY which can be used as an additional tool
for medical diagnosis along with MRI.
To demonstrate that these NCs possesses ample
luminescence for optical imaging, samples of the same con-
centrations of NCs employed in the MR studies were imaged
using fluorescence microscopy. NCs at a concentration that
produced high signal enhancement for MRI were dropped
on quartz and imaged by fluorescence microscopy with
405 nm diode laser excitation, 560 nm long pass filter at the
detector. The high intensity of emission observed under these
weak excitation conditions indicates the high QY of these
NCs (Figure 6).
From the PLE spectra of Mn-doped emission (≈585 nm)
(Figure S1, Supporting Information), we observe that PL
emission is maximum for the excitation wavelength of 300 nm,
whereas the emission is only 50% of the maximum value at
an exciation wavelength of 405 nm. Mn2+ doped NCs also
have a potential of multiphoton excitation which improves
tissue penetration depth and resolution of in vivo.[22] Thus, in
our case we are reporting low toxic (Cd-free) NCs with high
QY for fluorescence imaging with Mn atoms too localized in
the core and not in the shell. Therefore, the doped Mn2+ con-
centration in these NCs is sufficient for both high QY for FI
and high contrast in MRI.
3. Conclusion
In this work, we have demonstrated that Mn-doped ZnSe
NCs are potential candidates for dual-modal imaging. They
possess high QY (18% in water) for fluorescence imaging
and high relaxivity (≈2.95 mM−1 s−1) for magnetic reso-
nance imaging. These NCs are small in size (≈6.5 nm) and
less toxic for use in the human body. Thus, these NCs hold
a great promise for improved diagnosis using dual mode
imaging.
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obtained with a fluorescence spectrophotometer (Varian – Cary
Eclipse). The quantum yield of the NCs was measured using Horiba
Jobin Yvon Time resolved fluorescence setup using an integrating
sphere F-3018. Transmission electron microscopy (TEM – Tecnai
G2 F30) images were obtained using a high resolution transmis-
sion electron microscope (HRTEM) operating at 300 kV. Elemental
analysis is done using energy dispersive x-ray spectroscopy (EDS),
X-ray photoelectron spectroscopy (XPS – Thermo K-Alpha) and
inductive coupled plasma mass spectroscopy (ICPMS – Thermo X
Series II). Fluorescence Imaging of NCs drop casted on the quartz
were obtained using Carl Zeiss Axio Scope Fluorescence Micro-
scope. An excitation wavelength of 405 nm (LED) (power less than
5 mW) was used with a 560–600 nm emission pass filter. Magnetic
Resonance imaging experiments were performed at room temper-
ature on a 3 T Siemens TrioTim MRI scanner. MRIs were acquired
using a spin echo image sequence (slice thickness = 3 mm,
flip angle = 90°, acquisition matrix = 256 pixels × 256 pixels,
FoV = 90 mm × 90 mm, TE = 13 ms, TR from 100 to 10 000 ms),
where T1 is extracted by fitting an exponential function to these
curves by using least squares curve fitting algorithm. The longitu-
dinal (r1) relaxivity was determined from the following equation:[9]
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This work is supported by EU-FP7 Nanophotonics4Energy NoE, and
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Received: April 25, 2014
Revised: June 13, 2014
Published online: August 8, 2014
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