2. The recent emergence of fluorescent nanodiamond (FND)
has sparked a new era in cell labeling, imaging, and tracking
with nanoparticles.5−7
This is attributed to the inherent
biocompatibility and unique optical properties of this sp3
-
carbon-based nanomaterial. However, unlike other fluorescent
nanoparticles that can be synthesized with wet chemistry
methods, FND can be fabricated only by physicochemical
means under extreme conditions.7
Although the fabrication is
technically demanding, it has benefited enormously from
physicists who have carried out detailed characterization of
the crystallographic defects in diamond both experimentally
and theoretically since the 1950s.8,9
Researchers in the field are
now working toward using surface-functionalized nanodia-
monds for bioimaging, quantum sensing, and drug delivery.10
It
has been reported that FNDs containing nitrogen-vacancy
defect centers can emit bright, stable, and tissue-penetrating red
photons. Additionally, the surface of FNDs can be conveniently
derivatized with functional groups for protein and nucleic acid
immobilization.11,12
FNDs let researchers visualize, track, and
quantify molecules as well as cells with high spatial resolution,
which is necessary should one wish to fully understand complex
biological systems both in vitro and in vivo. As technological
demands in life sciences increase, the surface-functionalized
FNDs are expected to become one of the most favorable optical
nanoprobes for biomedical imaging, diagnostics, and treatment.
In the last 10 years, FNDs have attracted much attention
from biologists, chemists, physicists, and material scientists
because of their excellent mechanical, optical, chemical, and
biomedical properties. Recently, many more innovative
applications of FNDs are emerging. This Account touches on
three new and significant areas, including long-term cell
tracking, super-resolution imaging, and nanoscale temperature
sensing, where FNDs are shown to be a versatile and powerful
tool.
2. THE NITROGEN−VACANCY CENTER
Nitrogen is the most common impurity in diamond. It is
responsible for the vast majority of impurity-related color
formation.13
Man-made diamonds synthesized by high-
pressure−high-temperature methods usually contain 100 ppm
of atomically dispersed nitrogen, classified as type Ib diamond.
The submicrometer powders of these diamonds are convenient
sources for FND fabrication. Vacancies in diamond are typically
produced as a result of radiation damage under bombardment
with high-energy particles such as electrons, neutrons, and
ions.9
These lattice vacancies are structurally unstable and yet
immobile at room temperature. They become mobile if the
radiation-damaged sample is subjected to annealing at 600 °C
or above to re-establish its crystalline structure. When one
encounters a nitrogen atom in the next lattice position, it binds
with that atom to form a stable NV complex (Figure 1a).
The NV centers in diamond can exist in two different forms:
NV0
and NV−
. The latter deserves special attention because of
its remarkable magneto-optical properties.14
First, the NV−
center absorbs light strongly at 550 nm for the electronic
transition 3
A → 3
E (Figure 1b), and its emission band peaks at
685 nm with a high fluorescence quantum yield. Second, the
fluorescence emission is perfectly stable, undergoing neither
photobleaching nor blinking, allowing for the detection of
single NV−
centers. Third, the electron spins in the ground
state can be optically polarized, provided by the intersystem
crossing in the excited state (Figure 1b), enabling optical
readout of their spin states (ms = 0 and ±1) at the single
molecule level by using optically detected magnetic resonance
(ODMR) techniques at room temperature.15
Fourth, nearly
70% of the emitted photons lie in the near-infrared window
(670−890 nm) of biological tissue (Figure 1c), and they can
penetrate into tissue like skin for more than 2 mm.16
Fifth, the
fluorescence lifetime of the emission is significantly longer than
those of cell and tissue autofluorescence (∼20 ns versus ∼3
ns),17
which permits fluorescence imaging of single FNDs in
cells and organisms by time gating (Figure 1d).18,19
Finally,
background-free detection of the color centers can be
accomplished by microwave and magnetic modulation of the
NV−
fluorescence.20−23
Conventionally, high-energy (typically 2 MeV) electrons
from a van de Graaff accelerator are used as the damaging
agents for vacancy creation.8
However, because the accelerator
is difficult to access in both availability and cost, FNDs cannot
be routinely produced. In response to this difficulty and the
request for a larger quantity of the nanomaterials for biological
applications, our group has explored the practicality of using a
40 keV He+
beam to construct high-density ensembles of NV−
in type Ib diamonds.7,24
The advantage of this method is that
the ion beam is high in flux but low in energy, allowing for
fabrication of FNDs on a daily basis in a chemistry laboratory
(Figure 2). To scale up the production, Boudou et al.25
have
developed a high-yield fabrication method based on electron
irradiation using a 10 MeV Rhodotron accelerator, followed by
high-energy ball milling to reduce the particle size down to 10
nm. Cigler and co-workers alternatively employed a 15.5 MeV
H+
beam to facilitate the process.26
Table 1 lists the
experimental approaches that have been reported in liter-
ature.5,7,24−29
Having the capability of fabricating FNDs in large
Figure 1. (a) Structure and (b) energy level diagram of the NV−
center in diamond. The red sphere, blue dashed circle, and black
spheres in (a) denote nitrogen, vacancy, and carbon atoms,
respectively. The green, red, blue sinusoidal, and black dashed arrows
in panel b denote optical excitation, fluorescence emission, microwave
excitation, and intersystem crossing relaxation, respectively. (c)
Comparison between the fluorescence spectrum of FNDs excited
with a 532 nm laser and the near-infrared (NIR) window of biological
tissue. (d) Comparison between the fluorescence lifetimes of FNDs in
water and endogenous fluorophores in cells. Time gating at 10 ns is
indicated for background-free detection.
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3. quantity, we have devoted our efforts to the development of
NV−
into bioimaging contrast agents and nanoscale quantum
sensors since 2008.7
Three salient applications of the FNDs are
illustrated in subsequent sections.
3. LONG-TERM CELL TRACKING
An early work proposed that FNDs could be useful as single-
particle biomarkers.6
However, due to the large size of the
nanomaterials (35−100 nm), FNDs are more suitable for use as
cell trackers than molecular tags. A number of studies, including
our own, have found that bare FNDs after acid wash can be
spontaneously taken up by adherent cells in culture.30−33
Close
examination of the uptake mechanism indicates that FNDs
enter cells through energy-dependent, clathrin-mediated
endocytosis and are subsequently trapped in endosomes and
lysosomes.30−32
Notably, the internalization of FNDs does not
cause any obvious cytotoxic and detrimental effects on the
proliferation of human cells like HeLa cervical cancer cells.
Figure 3a displays snap-shots of the real-time tracking of some
internalized FNDs through the cell cycle of a FND-labeled
HeLa cell by bright-field/epifluorescence microscopy.33
No
significant exocytosis of FNDs is found even during the cell
proliferation. This unique characteristic permits tracking of
these cells continuously over a week (Figure 3b), suggesting
that the FND can serve as a useful nanoprobe for long-term
labeling and tracking of cell division, proliferation, and
differentiation, particularly in stem cell research.
Stem cells are a group of undifferentiated biological cells that
have the ability to self-renew and differentiate into various
specialized cells. They are delicate and fragile, and their
properties such as growth rate and differentiation capacity are
prone to be affected by fluorescent labeling and gene
transfection. To validate the aforementioned suggestion, we
have applied the FND labeling technique to track the
engraftment and regenerative capability of transplanted FND-
labeled lung stem cells (LSCs) in mouse models.34
In vitro
experiments first verified that the FND labeling does not
eliminate the cells’ properties of self-renewal and differentiation
into type I and type II pneumocytes. The FND-labeled LSCs
were then injected into lung-injured mice via intravenous
administration. Mice were sacrificed at different time points and
organs were collected to search for the injected LSCs by
fluorescence microscopy. As commonly encountered in tissue
section imaging, the high autofluorescence background level
prevented clear identification of these cells in the lungs. Thanks
to the distinct fluorescence lifetime of the NV−
center, this
undesirable effect could be largely removed by use of
fluorescence time imaging microscopy (FLIM). Figure 4
shows FLIM images of the lung tissue collected on days 1
and 7.34
The injected cells can be clearly discerned, even when
the cells are stained with hematoxylin and eosin (H&E) to aid
Figure 2. Schematic diagram (top view) and photograph (side view)
of a 40-keV He+
ion beam facility for routine production of FNDs. A
thin diamond film is prepared on a copper ribbon, which rolls in
vacuum to allow continuous exposure of the nanoparticles to the ion
beam for irradiation.
Table 1. Methods of Creating Vacancies in Nanodiamonds
by Radiation Damage
damaging agents energy ranges references
e−
10 MeV 25
e−
13.9 MeV 27
H+
3 MeV 5
H+
15.5 MeV 26
He+
20 keV 28
H+
, He+
40 keV 7, 24
H+
, He+
, Li+
, N+
20−250 keV 29
Figure 3. (a) Time-lapse images of a FND-labeled HeLa cell
undergoing division, acquired by differential interference contrast
and epifluorescence microscopy. The cell nuclei are stained in blue
with Hoechst 33342, while the FNDs appear as red spots. Scale bars:
10 μm. (b) Long-term tracking of FND-labeled HeLa cells over 8 days
by flow cytometry. The fluorescence intensity of each cell decreases
exponentially with time due to cell proliferation. Reproduced with
permission from ref 33. Copyright 2011 Wiley.
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4. histological examination. The FND-labeled LSCs preferentially
reside at the terminal bronchioles of the lungs on day 7 after
intravenous administration, as confirmed by time-gated
fluorescence (TGF) imaging of the tissue sections of
naphthalene-injured mice with single-cell resolution. Addition-
ally, the damaged lung cells can rapidly recover after
transplantation of the FND-labeled LSCs into the mice. The
method offers new insights into the components that limit the
acceptance of the transplanted stem cells as well as the
mechanism of their regeneration within a host. It holds great
potential for monitoring the homing of different kinds of stem
cells (such as mesenchymal stem cells) in larger animal models
(such as miniature pigs) for preclinical experimentation.
The extraordinary long-term tracking ability of FNDs led us
to apply this methodology to find slow-proliferating and
quiescent cancer stem cells (CSCs).35
These cells have long
been considered to be a source of tumor initiation; however,
there have been no effective tools for their identification and
isolation even in vitro. FNDs are well suited for the purpose
because they are both chemically and photophysically stable.
Prior to the cell tracking experiments, we first carried out
genotoxicity tests with comet and micronucleus assays for
human fibroblasts and breast cancer cells to confirm that FNDs
neither cause DNA damage nor impair cell growth. We then
employed AS-B145-1R breast cancer cells as the model cell line
for CSCs and compared in parallel the performance of FND
with the commonly used cell trackers, carboxyfluorescein
diacetate succinimidyl ester (CFSE) and 5-ethynyl-2′-deoxyur-
idine (EdU). Results indicate that our technique not only
enables quantitative analysis of the FND-labeled cells by flow
cytometry but also outperforms CFSE and EdU in regards to its
long-term tracking capability (Figure 5). It is anticipated that
further integration of the FND-based cell tracking platform
with the functional assays of protein markers will greatly
enhance our understanding of the CSCs both in vitro and in
vivo.
4. SUPER-RESOLUTION IMAGING
Fluorescence microscopy is a noninvasive, sensitive, and
quantitative technique that has profoundly advanced our
knowledge of cell biology. However, the revelation of the
detailed structure of cellular organelles is limited by the
diffraction of light. In the past decade, super-resolution
fluorescence imaging has overcome this diffraction limit and
substantially improved our ability to comprehend subcellular
processes.36
Stimulated emission depletion (STED) micros-
copy is one of the techniques.37
In STED microscopy, two laser
beams at different wavelengths are colinearly aligned with
nanometric precision. The main laser beam brings the
fluorophore of interest to its excited state, while the second
laser beam, having a doughnut shape at its focus, depletes the
fluorescence from all molecules except those in the middle of
the excitation volume. Consequently, the resultant “fluorescent
volume” becomes smaller than the diffraction limit. The higher
the excitation power is, the smaller is the fluorescent volume
and thus the higher is the image resolution. Being an
exceptionally photostable fluorophore, the NV−
centers are
ideal to achieve the highest possible spatial resolution with
STED.
The STED microscopy was first employed by Hell and co-
workers38,39
to detect single NV−
centers in bulk diamond with
a remarkable resolution of ∼6 nm. Using the same technique,
our group has effectively procured high-resolution images of 35
nm FNDs spin-coated on a glass slide.40
We obtained a
resolution of ∼40 nm, essentially limited by the size of the
particles. A later study by Arroyo-Camejo et al.41
demonstrated
that STED can resolve single NV−
centers in 40−250 nm sized
NDs with an improved resolution of ∼10 nm. There was no
photobleaching even under intensive STED laser illumination
(>100 MW/cm2
). Leveraging this fact, we have applied STED
to image FNDs taken up by HeLa cells. To prevent particle
agglomeration in cell medium, we first coated FNDs non-
covalently with bovine serum albumin (BSA) and delivered
them into the cells by endocytosis (Figure 6a). Using green
light for the excitation and a doughnut-shaped 740 nm laser
beam for the depletion, an improvement of the spatial
resolution from ∼240 to ∼40 nm has been reached for the
BSA-coated FNDs in HeLa cells (Figure 6b,c). We isolated the
Figure 4. Tracking the engraftment and regenerative capability of
transplanted lung stem cells using FNDs in a lung-injured mouse
model. Lung tissue sections are examined on days 1 and 7 after
intravenous injection of the FND-labeled lung stem cells. Arrows
indicate the identified cells. Scale bars: 50 μm. Reproduced with
permission from ref 34. Copyright 2013 Nature Publishing Group.
Figure 5. Comparison of the long-term tracking capability of EdU,
CFSE, and FND. The assays are conducted for the mammospheres
generated from AS-B145-1R cells labeled separately with EdU, CFSE,
and FND and then dissociated for flow cytometric analysis with a 4-
day period for 20 days. Reproduced with permission from ref 35.
Copyright 2015 Wiley.
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403
5. individual FNDs in fixed cells and distinguished them from
FND aggregates trapped in the endosomes.
Despite the remarkable resolution that has been achieved
with STED for single NV−
centers, the high power of the
STED beam is detrimental to super-resolution imaging of
FNDs in living cells. To reduce the photodamage effect,
converting NV−
to NV0
by using a low power laser is a possible
alternative. Chen et al.42
have recently explored this possibility
and demonstrated that the charge-state depletion microscopy
can resolve the NV−
centers in bulk diamond with a resolution
of ∼4 nm at a STED laser power of ∼20 mW. Further decrease
of the laser power by one more order of magnitude will make
the technique better suitable for live cell imaging. It should be
noted that other researchers in the field have also developed
approaches to achieve subdiffraction imaging of single NV−
centers on a parallel course. These include (1) deterministic
emitter switch microscopy (DESM) with NV−
manipulated by
microwave radiation,43
(2) single-spin stochastic optical
reconstruction microscopy (STORM) with the charge state
of NV switching between −1 and 0,44
and (3) tip-enhanced
fluorescence with FLIM in conjunction with atomic force
microscopy.45,46
Taken together, the combination of the unique
optical properties of FND with the subdiffraction imaging
capability of STED microscopy and its variations has opened up
exciting new opportunities to probe intracellular interactions
and dynamics, not only with single-particle sensitivity but also
with nanometric resolution and precision.
5. NANOSCALE TEMPERATURE SENSING
Thermogenesis is defined as a process in which the body of an
organism generates heat. Visualizing thermal changes at
subcellular resolution can reveal in depth the heat production
processes and how they are correlated with biological activities.
Recent advances in luminescence nanothermometry have made
it practical to measure temperature changes in intracellular
environment with high precision.47
The first application of
FNDs as tiny thermometers in living cells was made in 2013
and has since aroused considerable interest in the scientific
community.48
Although there are several notable achievements
afterward,49−51
the field of the NV-based nanoscale temper-
ature sensing is still in its infancy and possesses many
obstructions to real-world applications.
The NV−
center in diamond has a triplet ground state with a
total spin of 1. The ground state exhibits a crystal field splitting
Figure 6. (a) Confocal fluorescence image of a HeLa cell labeled with
BSA-coated FNDs by endocytosis. The corresponding image of the
entire cell is given in the inset (white box). (b) STED image of single
BSA-coated FNDs enclosed within the green box in panel a. (c)
Comparison of confocal and STED fluorescence intensity profiles of
the particle indicated in panel b with a blue line. Solid curves are best
fits to one-dimensional Gaussian (confocal) or Lorentzian (STED)
functions, with the corresponding full widths at half-maximum (fwhm)
given in parentheses. Reproduced with permission from ref 40.
Copyright 2011 Wiley.
Figure 7. (a) Covalent conjugation of polyarginine with carboxylated FNDs through carboxyl-to-amine cross-linking by carbodiimide chemistry for
subsequent physical adsorption of bare GNRs. (b) TEM image of polyarginine-coated FNDs decorated with multiple 10 nm × 41 nm GNR particles.
(c) Temperature rise of the FND in a GNR−FND hybrid heated by an 808 nm laser. Inset shows a typical fluorescence image of the spin-coated
particle (denoted by the yellow arrow) used for the measurement. Reproduced with permission from ref 54. Copyright 2015 Springer.
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6. of D = 2.87 GHz, which separates the ms = 0 and ±1 sublevels
and can be measured by ODMR.49−51
Since a change in
environmental temperature (T) can induce crystal strain, which
in turn shifts the magnetic resonance, a precise measurement of
the thermal shift (ΔD/ΔT), which varies from −75 kHz/K at
300 K to −150 kHz/K at 600 K,52,53
can expose the
temperature change. Kucsko et al.48
have measured the electron
spin resonance spectra of 100 nm FNDs in human embryonic
fibroblasts with a continuous-wave laser and deduced the local
temperature change with a precision of 0.1 K and a spatial
resolution of 200 nm. They also introduced gold nanoparticles
into the same cells as the heat source and demonstrated high-
precision temperature control as well as thermal mapping at the
subcellular level. To expand the versatility of this nano-
thermometry, we conjugated FNDs with gold nanorods
(GNRs) using polyarginine as the interface to form dual-
functional nanoparticles (Figure 7a).54
More than one GNR
(10 nm in diameter and 41 nm in length) can be attached to a
single 100 nm FND (Figure 7b), making the nanohybrid an
effective energy absorber and hence an efficient laser-activated
nanoheater. The availability of these dual-functional nano-
particles has enabled us to achieve highly localized heating with
a near-infrared laser (808 nm) and simultaneously probe the
corresponding temperature changes in situ with ODMR for the
individual gold/diamond nanohybrids (Figure 7c). The utility
of these hybrid nanomaterials for hyper-localized hyperthermia
applications is demonstrated with the endocytosed particles in
HeLa cells.
While measuring temperature at the nanoscale is important
in many areas of science, much of the information about the
underlying phenomena is contained in the dynamics. The FND
provides a powerful new tool to disclose such information.
Enlightened by the fact that the widths of the ODMR peaks of
NV−
in FND are insensitive to temperature change,52,53
we
have developed a three-point sampling method in conjunction
with pump−probe-type spectroscopy to map the temporal
profile of this physical parameter.51
In a proof-of-principle
experiment, we conducted time-resolved temperature measure-
ment for a GNR solution heated by a tightly focused 808 nm
laser along with a 100 nm FND particle as the temperature
sensor (Figure 8a). We then determined the ODMR peak shifts
by measuring the fluorescence intensities of the FND being
probed at three preselected microwave frequencies (f1, f 2, and
f 3 as indicated in Figure 8b), instead of scanning the whole
spectra. Results show that the method not only allows real-time
monitoring of the temperature change over ±100 K (Figure 8c)
but also permits detailed studies of the nanoscale heat transfer
at a temporal resolution of better than 10 μs (Figure 8d).51
6. CONCLUSION
The application of nanodiamonds in biomedicine is a
conceptual breakthrough. While diamond has been prevailing
as gems and industrial grinding materials for centuries,
considerably less attention has been paid to its potential use
in life sciences. It was first demonstrated in 2005 that FNDs
containing high density ensembles of NV centers are brightly
fluorescent and highly biocompatible,5
especially promising for
biomedical applications. Since then, this sp3
-bonded nano-
carbon material has developed into a key element that is
capable of bridging the information gap between physics and
biology, placing the emerging new technologies including
subdiffraction optical imaging and nanoscale quantum sensing
within a subcellular context.
Figure 8. (a) Experimental scheme for the time-resolved temperature measurement with a 100 nm FND particle submerged in aqueous solution
containing 10 nm × 41 nm GNRs (black rods) heated by an 808 nm laser (red hyperboloid). (b) Time sequences of the laser, microwave, and
detection pulses (all in μs) used in the time-resolved nanothermometry with a three-point method. The typical number of cycles is n = 70 000. (c)
ODMR spectra of the GNR solution heated by the near-infrared laser with its power varying from 0 to 10 mW. (d) Time evolution of the heat
dissipation of the GNR solution at the radial positions of r = 1.0 and 1.5 μm, as indicated in panel a.
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7. However, FNDs are not without their limitations. For
instance, in order to meet some demanding bioimaging
applications and to integrate it with the existing advanced
protein labeling technologies such as HaloTag,55
there is a need
to reduce the particle size down to at least 10 nm. Also, in vivo
tracking of cells with good image contrast is an area still not
well-developed, as researchers are struggling to adequately
perform deep tissue imaging of FNDs at sufficient levels of
sensitivity and resolution. Any of these improvements, if
successful, will undoubtedly invite more talents to the field and
lead to new advances in bioimaging and nanoscale sensing with
unprecedented sensitivity, resolution, and precision.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: hchang@gate.sinica.edu.tw.
Notes
The authors declare no competing financial interest.
Biographies
Wesley W.-W. Hsiao graduated from the Department of Chemical
Engineering at the University of Waterloo in Canada. He obtained his
Ph.D. degree in Chemical Biology and Molecular Biophysics from the
Taiwan International Graduate Program at Academia Sinica, the
national academy of Taiwan. In 2013, he had been awarded top 100
Ph.D. On-the-Job Training Postdoctoral Fellowship, granted by the
Taiwan government. He is presently working as a Postdoctoral
Research Fellow on fluorescent nanodiamond research at the Institute
of Atomic and Molecular Sciences of Academia Sinica.
Yuen Yung Hui received his B.S. degree in Physics from the Chinese
University of Hong Kong in 1996 and his Ph.D. degree in Physics from
the same university in 2003. He worked at the Hong Kong Science and
Technology Park on material characterization in the semiconductor
industry from 2004 to 2005. Now he is working as a Postdoctoral
Research Fellow at the Institute of Atomic and Molecular Sciences of
Academia Sinica. His current research interest is focused on
bioimaging and quantum sensing with fluorescent nanodiamonds.
Pei-Chang Tsai graduated from the Department of Physics at Chung
Yuan Christian University in 1996 and obtained his Ph.D. degree in
Physics from National Taiwan Normal University in 2009. His
expertise lies in lasers and optoelectronics. He has been working as a
Postdoctoral Research Fellow at the Institute of Atomic and Molecular
Sciences of Academia Sinica on optical and biomedical applications of
fluorescent nanodiamonds since 2013.
Huan-Cheng Chang received his B.S. degree in Agricultural
Chemistry from National Taiwan University in 1981 and his Ph.D.
degree in Physical Chemistry from Indiana University at Bloomington
in 1990. He joined the Institute of Atomic and Molecular Sciences of
Academia Sinica in 1994 and is now a Distinguished Research Fellow.
His research interests are focused on the development of new
methods, tools, and technologies based on physical chemistry and
applying them to solve problems of biological and medicinal
significance. He pioneered the development of single bioparticle
mass spectrometry and nanodiamond-based optical bioimaging. His
current research activities are devoted to the development and
applications of surface-functionalized fluorescent nanodiamonds as
diagnostic, imaging, sensing, and therapeutic tools.
■ ACKNOWLEDGMENTS
The authors gratefully acknowledge the kind support and
assistance from our collaborators, C.-C. Han, J.-H. Hsu, J. Yu,
A. L. Yu, M.-S. Chang, and Y. T. Lee. We also thank Y.-K.
Tzeng, V. Vaijayanthimala, C.-Y. Fang, B.-M. Chang, Y. Kuo,
and H.-H. Lin for their important contributions. This work was
supported by Academia Sinica and the Ministry of Science and
Technology of Taiwan.
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