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Anti-Stokes shift luminescent materials for

bio-applications
Cite this: DOI: 10.1039/c6cs00415f
Xingjun Zhu, Qianqian Su, Wei Feng* and Fuyou Li*

Anti-Stokes shift luminescence is a special optical process, which converts long-wavelength excitation
to short-wavelength emission. This unique ability is especially helpful for bio-applications, because the
longer-wavelength light source, usually referring to near infrared light, has a larger penetration depth
offering a longer working distance for in vivo applications. The anti-Stokes shift luminescence signal can
also be distinguished from the auto-fluorescence of biological tissues, thus reducing background
interference during bioimaging. Herein, we summarize recent advances in anti-Stokes shift luminescent
Received 31st May 2016 materials, including lanthanide and triplet–triplet-annihilation-based upconversion nanomaterials, and
DOI: 10.1039/c6cs00415f newly improved hot-band absorption-based luminescent materials. We focus on the synthetic strategies,
optical optimization and biological applications as well as present comparative discussions on the
www.rsc.org/chemsocrev luminescence mechanisms and characteristics of these three types of luminescent materials.

Key learning points

(1) Anti-Stokes shift luminescence mechanism.
(2) The strategies for the synthesis and modification of anti-Stokes luminescent materials.
(3) Preparation and setup for anti-Stokes shift luminescence bioimaging.
(4) Application models for anti-Stokes shift luminescence bioimaging.
(5) Typical designs of wavelength convertors using anti-Stokes luminescent materials.

1. Introduction
Stokes shift is a common definition used in the luminescence
process to describe the energy difference between the maxima
of the excitation and emission spectra peaks. As shown in
Scheme 1, in a typical luminescence process, the luminescence
centre absorbs one excitation photon with energy hn1 or
wavelength l1, and jumps to an excited state 1. After a rapid
non-radiative relaxation process to the excited state 2 with a
loss of a part of the energy, the following emissive transition
from the excited state 2 back to the ground state will generate
photon emission with energy hn2 or wavelength l2. Because of the
restriction of the law of energy conservation, the energy of the
emitted photon must be less than that of the excitation one.1,2
In recent years, anti-Stokes shift luminescent materials have Scheme 1 Schematic illustration of typical Stokes shift (left) and anti-Stokes
attracted much attention because of their unique ability to shift luminescence processes (right).
convert long wavelength (low energy) excitation photons to

short wavelength (high energy) ones (Scheme 1). This special

Institutes of Biomedical Sciences & Department of Chemistry & State Key
Laboratory of Molecular Engineering of Polymers, Fudan University,
wavelength conversion ability originates from the utilization of
220 Handan Road, Shanghai 200433, China. E-mail: [email protected], additional energy, from heat or another excitation photon, in
[email protected] the luminescence process. This feature is especially important

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in bio-applications as the longer wavelength light (i.e. near-

infrared light, NIR) irradiation could penetrate deeper into
biological samples and the unusual anti-Stokes shift luminescence
avoid the background fluorescence interference.3–5
This tutorial review will introduce the recent advances in
anti-Stokes luminescent materials and their applications based
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on different luminescence mechanisms. We hope that this review

will provide valuable guidance to the students or researchers who
want to enter this field or use anti-Stokes shift luminescence
technique for their own works.
Scheme 2 Schematic illustration of typical anti-Stokes shift luminescence
processes.
2. Mechanisms of different anti-Stokes
luminescence processes 2.1 Two-photon absorption process
Anti-Stokes luminescent materials used in bio-applications The first theoretical analysis of two-photon absorption (TPA)
are typically divided into three classes based on their different mechanism was proposed in 1930s. As shown in Scheme 2, the
mechanisms. TPA process involves simultaneous absorption of two photons

Xingjun Zhu was born in Shanghai, Qianqian Su was born in Anhui,

China, in 1990. He received his BS China. She obtained her BE
degree from Fudan University in degree in Materials Science
2012. Since then he has studied and Engineering from Shandong
as a PhD candidate under the University. She received her MS
guidance of Prof. Fuyou Li at degree in Polymer Chemistry and
Fudan University. His present Physics from Guangzhou Institute
research interests focus on the of Chemistry, Chinese Academy
development of multifunctional of Sciences and completed her
upconversion nanomaterials for PhD degree in the group of
bioimaging and cancer therapy. Professor Liu Xiaogang at the
National University of Singapore.
Xingjun Zhu Qianqian Su She currently holds a postdoctoral
research position with Professor
Fuyou Li at Fudan University. Her research interest focuses on the
development of luminescent materials for bioapplications.

Wei Feng was born in 1982 in Fuyou Li was born in Zhejiang,

Wuhan, China, and received his China, in 1973. He received his
BS degree in 2004 and PhD BS degree in 1995 and PhD
degree in 2009, both in chemistry degree in 2000 from Beijing
from Peking University. He sub- Normal University. He worked
sequently worked as a postdoctoral as a postdoctoral researcher at
researcher at Peking University for Peking University from 2000 to
two years, and joined Fudan 2002. He was then an associate
University in 2011. He is currently professor at Peking University
an associate professor in the from 2002 to 2003 and Fudan
Department of Chemistry. His University from 2003 to 2006.
research interests lie in the Since 2006, he has been working
Wei Feng design and synthesis of luminescent Fuyou Li as a full Professor at Fudan
nanomaterials with focus on their University. His current research
biological applications. interests involve anti-Stokes luminescent materials and
luminescent chemodosimeters for sensing and bioimaging. To
date, he has 200 scientific publications which have received more
than 10 000 citations.

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to excite a molecule from one state, e.g. ground state (0), to a luminescence process occurs at higher vibrational levels of the
higher energy level (1) which then jumps back to the ground singlet ground state, it may prefer molecules with rich vibrational
state by generating one photon with higher energy than either energy levels and is strongly temperature dependent due to the
of the absorbed photons. During the TPA process, a non- fact that the populations of higher vibration energy levels (0 0 )
stationary (virtual) state will emerge (illustrated by the dashed are determined by the Boltzmann distribution. Additionally,
line in Scheme 2) due to the interaction of photons and the populations of higher vibrational energy levels are very
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molecules. However, this virtual state does not exist indeed, limited so that effective anti-Stokes luminescence is usually
and thus the probability of occurrence of TPA is low. This observed in dye molecules with high quantum yield and molar
phenomenon results in an ultra-low cross section of TPA, which extinction coefficient. It is also worth noting that hot band
means that the realization of two-photon excitation lumines- absorption lies in the long wavelength tail of the whole absorption
cence needs extremely high excitation energy to ensure a large band. Dye molecules with small Stokes shifts will exhibit an
number of excitation photons. The experimental proof of optical absorption feature that more portions of the absorption
TPA was demonstrated in 1961, largely attributed to the invention spectrum extend to the long wavelength region of the emission
of lasers. Femto-second pulse lasers are commonly used in the band, and thus will have greater chances of generating hot band
commercial imaging systems for realizing luminescence signals absorption anti-Stokes luminescence.
of two-photon absorption.6 Until the establishment of a two-
photon fluorescence microscope in 1990, the application of this 2.3 Upconversion process
anti-Stokes luminescence process in the biological field just really
In the 1960s, the upconversion process was discovered and then
started. During the development of devices for TPA fluorescence
was quickly established to generate anti-Stokes shift luminescence.
imaging, the investigations of highly efficient TPA dyes were in
In the upconversion process (Scheme 2, right), the luminescence
progress at the same time. These dominated TPA dyes share some
centres can absorb energies from two or more photons sub-
common features in their molecular structure, such as conforma-
sequently (e.g. 0 - 2 - 1), and then generate one emission
tional rigidity and strong p conjugation. These features can
photon with higher energy than the one of each individual
significantly increase the TPA cross-sections. On the other hand,
excitation photon (1 - 0). Because of the existence of a real
the addition of an electron donor (D) and acceptor (A) on the
excited intermediate state (2), each process of photon absorp-
terminals of a p conjugated structure to build a D–p–A system is
tion is like a second-order elementary reaction, which has a
also helpful to boost TPA efficiency. Considering that a one-sided
higher degree of probability than the case of absorption of two
D–p–A structure results in only one direction of polarization
photons simultaneously.9
which reduces the TPA capacity, a centrosymmetry molecular
In fact, the upconversion process is generally divided into
structure like D–p–A–p–D is preferred.6 Although efforts have
two typical mechanisms. One typical upconversion mechanism
been made to optimize the molecular structure to enhance the
exists in the lanthanide (Ln)-based upconversion system using
TPA fluorescence, the exciting power density is still extremely high
lanthanide ions as emissive centres (Scheme 3, left). Lanthanide
which is a tough problem to face. Hence, several alternative anti-
ions with long-lived intermediate energy levels could be relatively
Stokes luminescence mechanisms gradually came into view.
stable in excited states. When the ions in excited states (2)
2.2 Hot band absorption process receive excessive energy from photons or other excited ions
(sensitizers), they will reach higher excited states (1).2,9 At this
The phenomenon of anti-Stokes luminescence induced by hot
moment, anti-Stokes luminescence might occur via radiative
band absorption has long been observed, which can be traced
transitions to make the ions come back to the ground states.
back to 1928.7 In this type of anti-Stokes process, additional
In the lanthanide-based upconversion process, the selection of
energy is provided by the heat originated from the original
sensitizers and emitters is very important. Sensitizers should
Boltzmann distribution of the fluorescent dye molecules
have a large absorption cross section and relatively simple
(Scheme 2, middle). While the excitation light, which irradiates
energy level structures to absorb photon energy as much as
the dye molecules, has a longer wavelength (lower energy) than
possible and avoid energy loss by relaxation. As for emitters,
that of the maximum emission, molecules located at a higher
they usually have multiple energy levels, which may generate
vibration energy level (0 0 ), also termed as ‘hot band’, can absorb
these photons and reach the excited state (1). Finally, the
excited state can decay back to the ground state and generate
the common fluorescence emission (1 - 0) whose wavelength
is shorter than that of the excitation light.8 Despite that
the phenomena of hot-band absorption induced anti-Stokes
luminescence have already been confirmed, studies in this field
are still rare for the time being, mainly because the detailed
rules to design efficient dye molecules with this anti-Stokes
luminescence remain unclear. According to the photophysical
characteristics during the hot band absorption process, some
possible principles can still be summarized. Since this anti-Stokes Scheme 3 Schematic illustration of two types of upconversion processes.

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emissions varying from the near infrared region to the ultraviolet In the case of the hot-band absorption process, there is no such
region. Sometimes emitters can function as sensitizers to donate mechanism in the system so that the lifetimes under anti-
the energy of excited states to energy transfer media or to other Stokes and Stokes excitations are nearly the same.11 Besides the
emitters to extend the diversity of the spectral lines. Considering luminescence lifetime, the relationship between the excitation
that sensitizers and emitters are usually metal ions, a host lattice power density and emission intensity also reflects the mechanism.
is needed to carry these ions. Crystalline materials with low In the upconversion process, the curve of the emission intensity as
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phonon energies are good candidate as host lattices because a function of the pump power density is usually nonlinear,
they can reduce phonon assisted nonradiative energy loss and indicating occurrence of a multiple-photon process. In contrast,
enhance radiative emission. Besides, the crystal symmetry of a linear curve will be obtained during the hot-band absorption
the host lattice will affect the energy transfer efficiency and the process, demonstrating the presence of a single-photon process.
splitting of emission spectra.
Another upconversion process is called triplet–triplet
annihilation (TTA)-based upconversion, which employs the 3. Composition and spectral properties
triplet state of dye molecules as an intermediate excited state of anti-Stokes luminescent materials
(Scheme 3, right). This system usually contains two types of
molecular dyes with matched triplet states. The sensitizer Full understanding of the mechanisms of anti-Stokes lumines-
molecules absorb energy from the excitation light and transfer cence processes paves the way for further investigations in this
the energy to the emitter molecules, resulting in a long-lived field. However, in practical works, it is also important to choose
excited triplet state (3T*). When two emitter molecules at anti-Stokes luminescent materials with desirable components
the excited states encounter in the system, the triplet–triplet and optical performances. This section will introduce the composi-
annihilation process with highly efficient fluorescence occurs, tion and spectral properties of these materials and discuss their
driving one emitter molecule to an excited singlet state (1S*) similarities and diversities. As to similarities in composition, all
through consuming two triplet excited states.2 Similar to the three types of anti-Stokes shift luminescent materials have emissive
Ln-based upconversion system, sensitizers for the TTA-based components with abundant and stable intermediate energy levels.
upconversion process should also have a large absorption cross- As to lanthanide and TTA-based upconversion materials, they share
section at the excitation wavelength. In addition, sensitizers more commonalities. For example, sensitizers and emitters are
should exhibit high intersystem crossing efficiency to generate necessary in both kinds of materials to accomplish the upconver-
a plenty of triplet states and their phosphorescence decay rate sion process effectively. Nonetheless, there still exist quite a lot
should be slow enough to allow triplet–triplet energy transfer differences among these materials due to the extensive sources of
(TTET) to emitters. To realize TTET, the emitter used in the TTA ingredients, from inorganic to organic.
process should have a triplet state with energy comparable to The optical performances of luminescent materials play a
that of the sensitizer and the lifetime of its triplet state should be decisive role in discovering new phenomena and extending the
long enough to ensure the occurrence of the energy transfer. scope of applications. Comparisons of some optical properties
Moreover, the fluorescence quantum yield of the emitter should of these materials are summarized in Table 1 in accordance
be high to obtain efficient upconversion emission.2 with the reported studies, from which we can see that these
materials have remarkable differences in some performances,
2.4 Differences among anti-Stokes shift luminescence such as the full width at half-maximum (FWHM) of the emission
processes peak, anti-Stokes shift, luminescence lifetime, etc. Researchers,
These three types of anti-Stokes shift luminescence processes especially novices, can choose desirable materials for their own
based on different luminescence mechanisms can efficiently works by referring to this table.
generate short-wavelength emission under long-wavelength
excitation, and therefore are all potentially useful in biological 3.1 Two-photon absorption materials
applications. However, for the beginners in this field, it may be TPA dye molecules comprise an electron donor and acceptor
a little bit challenging to distinguish these anti-Stokes shift and a p-conjugated bridge, and usually exhibit relatively high
luminescence processes just by the phenomena of wavelength molecular symmetry. According to the different features in
conversion in experiments. Thanks to the distinctive features of molecular configuration, TPA dyes can be generally divided
their luminescence mechanisms, we can differentiate these into two types with linear and planar geometries, respectively.
processes by investigating some photophysical properties. As to linear molecules, dialkyl and diaryl amino groups are
Firstly, the luminescence lifetimes of upconversion and hot-band widely used as electron donors and many p-deficient hetero-
absorption processes are quite different. In both lanthanide and cycles are designed to be acceptors. The p-Conjugated bridge
TTA-based upconversion systems, sensitizers and emitters are often takes vinylene or ethynylene. The molecular structures of
required simultaneously to accomplish the emission processes D–p–A–p–D with electron-deficient cores have more effective
(Scheme 3). Their luminescence lifetimes are consequently deter- TPA capacity than other types of structures. For planar mole-
mined by the rate of energy transfer (which is slow) rather than cules, porphyrins and expanded porphyrinoids can be adopted
that of emission (which is fast). As a result, much longer lifetimes as chromophores because of their large TPA cross-sections.
can be observed in the upconversion luminescence process.10 In addition, branched planar systems are also qualified to serve

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Table 1 General information of anti-Stokes shift luminescent materials for bioapplications

Hot-band Lanthanide-based TTA-based

absorption upconversion upconversion Ref.
Excitation range Vis, NIR NIR Vis, NIR 2, 3 and 11–15
Emission range Vis, NIR UV, vis, NIR UV, vis 2, 3 and 11–15
Full width at half-maximum (FWHM) of B50 nm B12 nm B70 nm 2, 3 and 9–15
emission peak
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Excitation power density for bioimaging o55 mW cm2 450 mW cm2 o20 mW cm2 2, 3 and 11–15
Quantum yield o2% o10% o30% 2, 3 and 11
Anti-Stokes shift o80 nm c100 nm 100–300 nm 2, 3 and 11–15
Luminescence lifetime Bns Bms Bms 2, 3 and 11
Photostability Good Excellent Medium 2, 3 and 10–15

Bioimaging level Cell, animal Cell, animal Cell, animal 2, 3, 11, 15 and 29
Coverage of bio-applications  Biodetection  Biodetection  Bioimaging 2, 3, 11, 15 and 29
 Bioimaging  Bioimaging  Tumor targeting
 Tumor targeting
 Cancer therapy
Metabolism rate Fast Slow Fair 2, 3, 11, 15, 16 and 39

as TPA dyes. For example, a trigonal branched molecule with

triazine as the core and electron-rich dimethylamino styryl as
arms exhibits remarkable TPA performance.6
TPA induced anti-Stokes emissions have several particular
spectral properties. The anti-Stokes shift of TPA emission is
quite large. If the frequency of the two absorbed photons is
the same, the anti-Stokes shift is approximately equal to the
wavelength of the excitation light. The wavelengths of the
excitation sources usually lie in the near infrared region, which
have a relatively large penetration depth in biological tissues.
However, to generate TPA emission, a pulsed laser with high
energy density is required, which may cause a relatively strong
photobleaching and heating effect in the focal region.6

3.2 Hot-band absorption materials Fig. 1 Chemical structures of the reported hot band absorption materials.

Typically, hot-band absorption anti-Stokes luminescent materials

are simple dye molecules, of which the synthetic strategies are
usually based on mature organic synthetic routes. It should be studied for their anti-Stokes luminescence properties and
noted that not all types of dyes possess the capability of hot-band developed to perform laser cooling.13,14 The design criteria of
absorption anti-Stokes emission. Hence, to obtain intense lumi- hot band absorption materials are not so comprehensive at
nescence, more attention is paid to the selection of fluorophores present, so the use of a reported fluorophore with hot band
and the design of the molecular structure. absorption capacity as the mother nucleus is a good choice. To
The reported dye molecules with hot band absorption further enhance the hot band absorption efficiency, in other
induced anti-Stokes luminescence are shown in Fig. 1. As we words to enrich vibrational energy levels, adding some rotatable
can see, most of the hot band absorption dyes in reported bonds like single bonds and some heteroatom like O, N, etc. may
works are commercial products with high luminescence effi- be helpful. To fit the cases in bio-applications such as achieving
ciency. The first observation of this anti-Stokes process is better imaging quality in the living body, the deliberate design
exactly based on the commercially available fluorescein.7 Early and modification of the dye molecules are required to shift the
investigations of hot-band absorption based anti-Stokes lumi- emission peaks to the near infrared range and to add some
nescence mainly focused on the photophysical process and the hydrophilic substituent groups on the structure. Here, we use a
applications in laser cooling and temperature sensing. Rhoda- reported work to illustrate the general process to design hot
mine derivates are frequently chosen due to their ultra-high band absorption materials. For example, Li and coworkers
quantum yield and easy availability. In a typical case, rhoda- synthesized a molecular fluorophore NRh-1 that based on
mine 101 excited by a 633 nm laser to generate emission at rhodamine nucleus.15 Such a rhodamine nucleus has been
600 nm can serve as an intracellular temperature sensing reported to have hot band absorption property. By expanding
probe.12 Other types of dyes with fairish quantum yield also the conjugated system in the rhodamine based structure, NRh-1
have the property of hot-band absorption anti-Stokes emission. shows a significant absorption band with a maximum at 700 nm,
For example, cyanine derivates and laser dye oxazine 1 were a less-pronounced shoulder peak at 670 nm and a long tail

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extended to about 800 nm. In the normal fluorescence process, rhodamine derivatives. More investigations on other types of
the optimized excitation wavelength is 670 nm or 700 nm. dyes are needed to supplement the alternatives for such anti-
Excitation at 808 nm occurs into the low-energy tail, namely Stokes emission. Theoretically, dyes with high Stokes lumines-
hot band, of the absorption spectrum. Finally, NRh-1 can generate cence quantum yields and broad long-wavelength absorption
anti-Stokes emission centred at 730 nm under 808 nm excitation. edges may be good candidates for hot-band absorption induced
The anti-Stokes emission profile has the same shape and location anti-Stokes luminescence.13 Hence, there is plenty of room for
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as the conventional Stokes emission spectrum. The normal Stokes improvement and advancement in this realm.
shift of NRh-1 is about 30 nm, while the anti-Stokes shift is
significantly expanded to about 80 nm.
The research of hot band absorption materials is still in the 3.3 Lanthanide-based upconversion materials
primary stage, so the spectral properties of this class of materials Lanthanide-based upconversion nanophosphors (Ln-UCNPs)
have to be summarized from only a few reported works. Detailed are generally composed of a sensitizer, emitter, and host lattice.
optical parameters and spectral profiles are shown in Table 1 Both sensitizer and emitter are lanthanide ions with similar
and Fig. 2, respectively. It can be found that the excitation and chemical properties, which makes the integration of these two
emission ranges of current hot band absorption materials components easy. The commonly used sensitizers are Yb3+ or
mainly cover the long-wavelength part of the visible region Nd3+, because of their relatively large absorption cross section
(Fig. 2). With modification of the molecular structure, the in the near infrared region. The emission colours of lanthanide-
emission wavelength can be expanded to the NIR region (e.g. based upconversion nanomaterials can be modulated by doping
NRh-1, ca. 730 nm).15 According to the results of some recently different types of emitter ions. To circumvent luminescence
published works,11,15 the excitation conditions of hot-band quenching resulted from cross-relaxation between emitter ions,
absorption materials for bioimaging are quite mild, which the doping amount of emitters is controlled at a rather low level
can reduce the excitation source induced thermal effect or (usually o2%). The emitter ions in lanthanide upconversion
photo-damage. The anti-Stokes shifts of hot band materials nanomaterials can be Er3+, Tm3+, and Ho3+. Recently, other
are not very large, but the present filters or other light splitting lanthanide ions, such as Tb3+, Eu3+, Sm3+ and Dy3+, are also
technologies are competent to exclude the interference of developed to serve as emitters through the energy migration
excitation in this case during bioimaging. The most intriguing process or cooperative upconversion mechanism.2,17 The selec-
thing is that hot-band absorption materials are a class of anti- tion of appropriate host materials is also an essential prerequisite
Stokes luminescent materials consisting of simple small mole- for desired optical properties. Lanthanide fluoride nanocrystals
cules. As is well known that small molecules have a much are the preferred upconversion host materials because of their
shorter clearance time than nanomaterials,16 which may reduce ultra-low phonon energy, which results in relatively high quantum
the possible adverse effect to the living body. Hot-band absorp- yield.18 Great efforts have been made in recent years to solve the
tion materials are very promising to serve as optical probes for problem of breaking down the size of lanthanide-based materials
diagnosis and detection in vivo. in the nanoscale for bio-applications. Thanks to the low solubility
One important problem to be addressed is the relatively low of lanthanide fluorides, nearly all wet chemical synthetic methods
luminescence efficiency of hot-band absorption materials. The can be successfully adopted in the synthesis of lanthanide
dyes involved in the reported works are mainly restricted to fluorides, such as co-precipitation, hydro(solvo)thermal synthesis,
thermal decomposition, and sol–gel route synthesis (Fig. 3).
To meet the needs of multifunctional applications and
improve optical performances, the establishment Ln-UCNPs
nanocomposites is quite essential. According to reported
works, the components combined with Ln-based upconversion
materials can be generally classified into metal ions, small
molecules, polymers, amorphous and crystalline inorganic
materials. The construction methods of these composites are

Fig. 2 Typical emission spectra of three types of anti-Stokes luminescent Fig. 3 General illustration of the synthesis of lanthanide-based upconversion
materials. nanoparticles.

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also different. The cation exchange method is often used to

combine metal ions with Ln-UCNPs. Due to the differences in
the solubility product (Ksp) of two types of crystals, a fast ion
exchange process can occur in the aqueous solution and finally
a layer of crystalline structure comprised of objective metal ions
will be formed on the surface of Ln-UCNPs.2,3 For integration
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with small molecules, specific modifications such as the intro-

duction of –OP(OH)2, –COOH and –SO3H groups on the mole-
cular structure is necessary to conjugate molecules with
lanthanide ions in Ln-UCNPs by coordination bonds. Several
classes of polymer materials have been used to coat on the
surface of Ln-UCNPs, of which amphiphilic polymers are very
representative, because this class of polymers can not only
Fig. 4 Illustration of the dye sensitized upconversion process and the
transform the as-synthesized hydrophobic Ln-UCNPs into corresponding energy transfer mechanism.
aqueous phase but also encapsulate some small molecules into
the hydrophobic interlayer for the construction of optical
sensing or drug delivery systems. The coating of amphiphilic The main drawback of lanthanide-based upconversion
polymers can be realized by dispersing polymers and Ln-UCNPs materials is the relatively low upconversion luminescence
in organic solvent and assembling together with solvent efficiency, which limits their development in bio-applications.
evaporation. Amorphous silica may be the most frequently The low upconversion luminescence efficiency is caused by the
used inorganic material to combine with Ln-UCNPs. The silica low absorption cross section of lanthanide sensitizers and
component can be fabricated into a mesoporous or yolk–shell the serious non-radiative decay of the excited states in nano-
structure, which can provide more loading capacity for drugs crystals. To conquer these problems, Hummelen and coworkers
or functional molecules. Moreover, the silica layer is also utilized near infrared dye molecules as antennae to absorb near
an excellent platform to conjugate other kinds of inorganic infrared excitation light over a broader range of wavelength of
materials. Well-established silica coating strategies such as 740–850 nm.20 The following energy transfer process can excite
reverse micro-emulsion and the Stöber method are all applicable Yb3+ in the upconversion nanocrystals. Recently, a similar
in the synthesis of the Ln-UCNPs@silica core–shell structure. dye-sensitized upconversion have also been realized in
Owing to the chemical inertness of Ln-UCNPs materials, few Nd-sensitized upconversion systems (Fig. 4).21,22 In this case, the
heterogeneous inorganic crystalline materials can be directly photon energy absorbed by dye molecules populate Nd3+ ions in
jointed. Hence, in most reported cases, the objective crystalline excited states, followed by energy transfer between Nd3+ and Yb3+
materials are analogues of Ln-UCNPs with nearly the same ions to stimulate Yb3+ ions into the 2F5/2 state. Subsequently,
crystal structure and elements. For example, Ln-UCNPs with a emitter ions such as Tm3+ are populated in an excited state like
1
host lattice of NaYF4 can be easily conjugated with NaLuF4, CaF2, G4 or 1D2, thereby giving rise to UCL emissions of Tm3+ at
etc. The integration method is based on the epitaxial growth 475 nm (1G4 - 3H5) or 450 nm (1D2 - 3F4) (Fig. 4). Another
principle, which uses a similar synthetic process of Ln-UCNPs.2,3 method to enhance the emission intensity is to directly grow an
Lanthanide-based upconversion materials possess several additional layer of host lattice without sensitizer and emitter ions
unique spectral properties, for instance, narrow-band emission, onto the luminescent core. An inert shell outside the luminescent
a large anti-Stokes shift and a long lifetime (Table 1). As shown in nanocrystal can fill up lattice defects and exclude the energy
the spectra of Er3+ and Tm3+ doped lanthanide-based upconver- transfer from the sensitizers to surface quenching species, like
sion nanoparticles (Fig. 2), the emission bands exhibit narrow ligands or solvent molecules. This strategy has already been used
peak widths, resulting in a collection of luminescence signals to fabricate various core–shell nanoparticles with nearly hundred
at different wavelengths without crosstalk, which is quite fold enhancement of upconversion emission.23
useful in ratiometric luminescence detection. A large anti-
Stokes shift can completely exclude the interference generated 3.4 TTA-based upconversion materials
from exciting light or the autofluorescence of biotissues in The sensitizers and emitters used in the TTA-based upconver-
bioimaging. Some lanthanide emitters, such as Tm3+, have a sion system are comprehensively reviewed in other works.2
long and tunable luminescent lifetime, and thus can be Generally speaking, sensitizers for TTA-based upconversion
utilized to develop a new-generation multiplexing technique have a large absorption cross-section at the excitation wave-
instead of fluorescent colour coding. For example, Jin and length and efficient intersystem crossing capacity to generate
coworkers prepared a series of upconversion nanocrystals with enough triplet states. Qualified molecules like platinum(II)
separate lifetime identities by altering the doping concen- tetraphenyl tetrabenzoporphyrin (PtTPBP), (2,3,7,8,12,13,17,18-
tration of Tm3+ and further used these nanocrystals to prove octaethylporphyrinato) palladium(II) (PdOEP) are often used as
the feasibility of lifetime encoding.19 This technology is sensitizers (Fig. 5).2 As to emitter molecules, high luminescence
expected to have a wide range of applications in life sciences, quantum yields and suitable lifetimes of triplet states are
medicine and data storage. required.2 Frequently used emitter molecules are shown in

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method to fabricate upconversion nanospheres.25 After that, Li

et al. fabricated TTA upconversion-based water-soluble nano-
capsules containing organic solvents using BSA-dextran as the
shell layer.10 Medical injectable soybean oil was used as solvent to
dissolve the TTA upconversion sensitizer and emitter. Soybean
oil not only serves as solvent in the nanocapsules, but also
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enhances the upconversion emission intensity. This is because

some reductants in soybean oil could consume the dissolved
oxygen, protecting the excited triplet states from oxygen-induced
upconversion luminescence quenching.
Fig. 5 Typical sensitizers of TTA-based upconversion materials.
The spectral features of TTA-based upconversion materials
largely depend on the sensitizer and emitter. Based on the
commonly used combinations of sensitizer and emitter, the
emission peaks of TTA-based materials are usually located in
the UV and visible region with relatively large peak widths
(Fig. 2). As optical probes for bio-applications, especially for
bioimaging, excitation and emission in the NIR region are
always preferred since the optical transparent windows of
biological tissues lie in this range of wavelengths. With the
investigation of some metal complexes and nanoparticles as
Fig. 6 Typical emitters of TTA-based upconversion materials.
sensitizers, the excitation wavelengths have been expanded to
the NIR region.26,27 For instance, Castellano et al. used the
supramolecular complex Pyr1RuPPZn2 as a sensitizer to realize
TTA upconversion emission under 780 nm excitation.27
Recently, Kimizuka et al. synthesized a lipophilic Os complex
as a sensitizer. By the energy transfer between the triplet states
of the sensitizer and emitter, TTA upconversion emission was
generated by rubrene under 938 nm irradiation.28 Unfortunately,
up to now, efficient emitters working at wavelengths greater than
650 nm have been scarce. If this problem can be solved, a new
dimension for applying TTA-based upconversion materials to
biological sciences will be opened up.
In addition, TTA-based upconversion materials have much
Fig. 7 Integration strategies of the TTA-based upconversion system.
higher luminescence efficiency, which means that strong
The TTA-based upconversion process can be achieved in (a) homogeneous
bulk system, (b) micro-emulsion micelles, (c) silica-based nanoparticles and upconversion emission can be obtained under relatively low
(d) BSA-dextran nanocapsules. excitation power density (Table 1). As indicated in the results of
Li et al., the upconversion luminescence quantum yield of their
TTA-based nanoparticles is 4.5% in pure water. The corres-
Fig. 6. Recent years have witnessed the efforts to find appro- ponding power density of the continuous wave laser used for
priate host materials to compact the sensitizer and emitter in vivo imaging is as low as 8.5 mW cm2.24 Such facile laser
(Fig. 7). One typical material to contain the sensitizer and irradiation effectively circumvents the heating effect and photo-
emitter and form an ultra-small particle size is amorphous damage induced by the excitation source.
silica. For example, PdOEP (sensitizer) and 9,10-diphenyl- One current issue with TTA-based upconversion materials is
anthracene (DPA, emitter) were dissolved in toluene, followed the relatively low photostabilities. Although the concerns of
by a silica coating process.24 After the removal of the solvent, photostabilities also exist in hot-band absorption materials,
TTA upconversion-based silica nanospheres were obtained. singlet oxygen generated by the energy transfer from the
However, the upconversion efficiency is lower than conven- sensitizer to O2 molecules has an extremely strong quenching
tional TTA system. This may be attributed to the fact that only a and damaging effect on the emitter. Some strategies have been
small amount of solvent remains in the silica spheres, and thus developed to deal with this problem lately. One strategy is to
the restriction of molecular movements may suppress the disperse the sensitizer and emitter into liquid oil capsules.10
intermolecular energy transfer between sensitizer and emitter. The oil capsules not only provide a hydrophilic atmosphere to
The majority of the effective sensitizers and emitters reduce the content of dissolved oxygen but also eliminate the
are hydrophobic dye molecules, which dissolve in non-polar singlet oxygen with help of some reductive species in oil that
solvents. Therefore, non-polar solvents as host materials can contains unsaturated double bonds. Another way is to modify
improve the mobility and reduce the quenching effect by the the sensitizer and emitter with alkyl chains to form liquid
matrix. Katharina and coworkers first employed a micro-emulsion molecular networks.2 In this system, chromophores are tightly

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linked with hydrophilic components, which effectively suppress

the diffusion of oxygen molecules.

4. Bio-applications of anti-Stokes
luminescent materials
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The bio-applications of the above-mentioned three kinds of

materials have different situations at present. Hot-band anti-Stokes
emission is an attractive alternative method for bio-applications,
but now is just in its infancy. TTA-based nanomaterials with higher
upconversion luminescence efficiency also attracted much atten-
tion, but their development is limited by the unstable assembly
system. Lanthanide-doped bio-probes have been well developed in
recent years because of the well-established synthetic routes and
their stable inorganic crystalline nature.

4.1 Anti-Stokes luminescent materials for bioimaging

One of the most important bio-applications of anti-Stokes
luminescent materials is bioimaging. The most distinctive
property of anti-Stokes luminescent materials is their wave-
length conversion process that converts low-energy excitation
into high-energy emission. This feature is very favourable for
Fig. 8 Cell imaging using anti-Stokes luminescent materials. (Reprinted
bioimaging because it generates nearly no autofluorescence with permission from ref. 23, 28 and 30. Copyright 2009, 2012, 2016,
and provides a higher signal to noise ratio. Moreover, low- American Chemical Society, Wiley-VCH Verlag GmbH & Co. KGaA.)
energy excitation can effectively reduce photo-damage to bio-
logical tissues.
Cells, as the basic units of the biological tissues and living For the use of anti-Stokes shift luminescent materials in
body, are very important objects in biological research. Optical bioimaging, the first problem encountered is the lack of
imaging is the most direct means to observe the physiological corresponding imaging instruments. Taking a small animal
behaviour of cells. To date, all three types of anti-Stokes imaging system as an example (Fig. 9), some key components
luminescent materials have successfully been applied in optical should be modified to make it applicable to anti-Stokes
cell imaging (Fig. 8). In hot band absorption materials, rhodamine luminescence bioimaging.31 At first, the excitation sources are
derivates are primarily adopted for cell imaging for the detection usually lasers with relatively higher power output than the
of intracellular temperature or hazardous metal ions.12,29 With commonly used Xeon lamp, and the lasers should have an
suitable modifications in the molecular structure, anti-Stokes appropriate wavelength which matches the requirement for
emission can be excited under NIR 808 nm irradiation, which excitation of the probe. Secondly, the dichroic mirrors should
reduces the photo-damage to cells. Due to the small molecule be short-pass or notch filters which can effectively block the
nature of hot band absorption materials, the distribution of this longer wavelength excitation light and allow the shorter wave-
class of materials in cells is more homogenous than upconversion length emission light to pass through. Moreover, the emission
nanomaterials, which is rather favourable to observe the overall filters should be carefully selected. Owning to the low upcon-
status of cells. Lanthanide upconversion nanocrystals serving as version luminescence efficiency as discussed in Section 3, the
luminescent probes are featured by their ultra-high photostability. intensity of the emission light is usually much lower than
For cell imaging, signals from commercial dyes, such as DAPI, are excitation light. This fact requires emission filters with high
quickly eliminated by photo-bleaching, while the upconversion efficiency and an appropriate cut-off wavelength to eliminate
signals from lanthanide nanocrystals remained nearly constant.30 the interference from the excitation laser light. Normally, short-
This superiority originates from the nature that the inorganic pass or notch filters do not have very steady transmission
lattice is hard to destroy by the excitation light. The unique anti- performance in a wide range of wavelengths. Transmission
Stokes shift upconversion luminescence also enables lanthanide data of filters are must-read parameters before purchasing,
probes to work with other fluorescent species. In addition, a for they can provide information about the cut-off edge and
lanthanide upconversion nanocrystal contains multiple lumines- transmission region. Besides the emission filters, other optical
cent centres. Therefore, the quality of imaging cannot be affected elements, including lens, need optimization for the near infra-
by the blinking phenomenon. Examples of cell imaging by using red light, because many optical elements are designed for the
TTA-based upconversion materials are very few. Li et al. utilized visible light, which will result in an obvious drop in the
TTA-based silica nanoparticles to achieve blue-light cell imaging performance for the near infrared light. Finally, the detection
under low power density of a 532 nm laser. unit of the instrument needs to be modified. Due to a rather

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experiment, Li and coworkers proved that 50 and 1000 cells

stained by NaLu(Gd)F4:Yb,Tm can be detected in bioimaging
via intravenous and subcutaneous injections, respectively.2,3
For the traditional lanthanide upconversion nanomaterials,
Yb3+ is employed as the sensitizer due to its relatively large
absorption cross section than the emitter ions. However, the
use of Yb3+ requires an excitation wavelength of about 980 nm,
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which has an obvious photon-induced heating effect on

biotissues.34–36 He and coworkers reported the use of a
915 nm laser as an alternative to the 980 nm laser.37 The
absorption of Yb3+ at 915 nm is much weaker than that of
Yb3+ at 980 nm, so the obtained upconversion signals were
also reduced. Recently, Sun, Yan and coworkers designed an
Fig. 9 Schematic diagram of the experimental setup for anti-Stokes shift
Nd3+-sensitized system to shift the excitation wavelength to
luminescence bioimaging.
808 nm, which will minimize the absorption from water, thus
reduce the heating effect.34 This principle was also demonstrated
low quantum yield of anti-Stokes luminescent materials, by Liu and coworkers in a well-designed core–shell structure with
EMCCD is adopted since it is more sensitive to weak lumines- only Nd3+ in the shell, which is able to further reduce the possible
cence than common CCD or CMOS detectors. After modifica- quenching from Yb3+ to the surface defects.35
tion, imaging instruments will be suitable in the experiments Recently, a series of bioimaging applications have been
using anti-Stokes shift luminescent nanomaterials as probes. explored by using these lanthanide-based upconversion nano-
For the hot-band absorption materials, there are only a few probes. Lymphatic imaging is one of the classic application
reports about their application in bioimaging. This is because models in this area. This model may be suitable for localizing
the vast majority of the related literature has been focused lymph nodes and determining the stage of cancer. It also plays
on commercial dyes with ultra-high fluorescence quantum an important role in sentinel lymph node dissection.3 Tumor
efficiency, while the emission wavelength locates in the visible imaging is another significant application of lanthanide-doped
range, which is not suitable for in vivo imaging. Recently, Li and upconversion nanoparticles (Fig. 10). Tumor targeting ability
coworkers utilized the synthesized near infrared dye molecules of upconversion nanoparticles usually originates from the
NRh-1 as the luminescent probe. Because of the anti-Stokes enhanced permeability and retention (EPR) effect of tumor
luminescence process and the use of the NIR-to-NIR technique, tissues. Nanosized upconversion probes are able to passively
this probe offers remarkably improved signal-to-noise ratios target the tumor site, and thus provide important information
and a higher tissue penetration depth than conventional fluor- about the tumor by an imaging technique. Targeting agents
escent dyes.15 These advantages make the molecular upconver- (i.e. folic acid, peptides, and antibodies) could also be conjugated
sion dyes particularly attractive for applications in whole body onto the surface of upconversion nanoprobes to endow them with
and lymph node imaging. In addition, this probe displayed active targeting ability to tumor sites.3,31
faster excretion when compared to inorganic probes, which Besides these applications that can also be achieved by
can minimize the possible side effects. For example, Li and using commercial fluorescent dyes, lanthanide upconversion
coworkers reported that the NRh-1 molecular probe can be nanoparticles have some specific imaging cases. For example,
easily cleared from the body within 72 h, while the strong
upconversion signal was still detectable after 6 days post-
injection in the lanthanide-doped inorganic nanoparticle treated
group. Nevertheless, this feature is unfavourable for passive
tumor targeting and imaging. It is should be noted that, there
is no EPR effect of small molecules in tumor tissues, so this type
of luminescent probe cannot be directly used in target imaging.
To achieve an active targeting function, further development
aims to conjugate targeting ligands to these NIR molecular dyes.
Owing to the relatively large penetration depth of the near
infrared working light, lanthanide-based upconversion nano-
probes are able to be applied for in vivo imaging.32 Up to now,
the most popular upconversion probes for in vivo imaging
use Tm3+ as the emitter, possessing 800 nm near infrared Fig. 10 Small animal bioimaging using anti-Stokes luminescent materials.
The bioimaging photos of the whole body, lymph node and tumor are
upconversion luminescence emission. For example, Han and
collected by using a hot band absorption material, TTA-based upconver-
coworkers obtained detectable upconversion signals with a sion material and lanthanide-based upconversion material, respectively.
large penetration depth of 1.5 cm by using NaYF4:Yb,Tm@CaF2 (Reprinted with permission from ref. 10, 23 and 30. Copyright 2009, 2012,
nanoparticles as probes.33 In addition, for the detection limit 2016, American Chemical Society, Wiley-VCH Verlag GmbH & Co. KGaA.)

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Weissleder and coworkers utilized Y2O3:Yb,Er upconversion emissive TTA upconversion system into a nanocapsule by the
nanoparticles as a probe for blood vessel imaging.38 Common micro-emulsion method using injectable bean oil as solvent.10
dyes have a shorter life-time in blood than lanthanide-doped The as-prepared nanocapsule has a high quantum yield up to
nanoparticles, causing the difficulty in performing vascular 4.8%. This kind of material which has a relatively fast meta-
imaging using dyes as a probe. Another important usage is bolism rate of about 24 h and rather low toxicity has also been
that these nanoparticles could be used to track the position of used for cell imaging and animal imaging.39 Because of the
labelled cells.3 Although commercial dyes have higher fluores-
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larger penetration depth of the green light than the blue light,
cence efficiency, they suffer from low stability in cell tracking lymph node imaging can be performed without the removal of
applications. The inorganic lattice makes the signals from skin tissue.
lanthanide-based upconversion nanoprobes ultra-stable, which Because of the higher upconversion luminescence efficiency,
is suitable for long-term observation. Liu and coworkers TTA-upconversion nanoprobes seem to have much more
reported that they can keep tracking cancer cells until they potential in biological applications than lanthanide-based
grow into a solid tumor. This work proved that upconversion upconversion nanoparticles. However, the self-assembled nano-
nanoprobes may not obviously influence the activity of cells.3 particles are susceptible to the surroundings, and the stability of
Anti-Stokes luminescence imaging has superior sensitivity, the developed TTA-based upconversion nanoparticles is yet to be
which is very useful for guiding lesion section during surgery. overcome. Thus, efforts to further enhance the stability of these
However, the penetration depth of the excitation and emission materials are much needed for practical applications.
light still does not meet the requirements of the application in In summary, all of these three types of probes with
clinical diagnosis. Other imaging modes, like magnetic reso- anti-Stokes luminescence can be used in bioimaging. Their
nance (MR) and X-ray computed tomography (CT), have a large common advantages lie in the resistance of background auto-
penetration depth in biotissues, which can compensate for this fluorescence because no anti-Stokes shift luminescence is
shortcoming. Integrating different imaging modes into one generated by biological tissues under identical excitation condi-
contrast agent will greatly improve the accuracy of lesion tions. The near infrared excitation light has a large penetration
localization and reduce the injuries and adverse effects to depth, which makes these probes suitable for in vivo imaging.
patients made by repeated administration of different contrast For the hot-band absorption induced anti-Stokes lumines-
agents. Owing to the abundant physical properties of lanthanide cence of small molecules, their bioimaging applications are
ions, lanthanide-based upconversion nanoparticles have been just in the initial stage. Because such anti-Stokes luminescence
developed for multi-modality imaging. For instance, a nano- emission is a common property of dye molecules under suita-
system serves as an MR contrast agent by introducing ble excitation conditions, there is a plenty of room to further
Gd3+-containing species into the system. For CT imaging, elements improve their performance and broaden the applications of
with larger atomic numbers are favourable. Therefore, Yb3+ and such probes.
Lu3+ are ideal ions to endow the nanoprobes with CT contrast For TTA-based upconversion nanomaterials, a general
ability.2,3 Recently, some radioactive imaging techniques such approach for the preparation of a stable nanosystem containing
as positron emission computed tomography (PET-CT) and both sensitizer and emitter with the ability of free movement is
single-photon emission computed tomography (SPECT) can highly needed at this point of time. If this method is estab-
also use lanthanide-based upconversion nanoprobes for quan- lished, the high upconversion efficiency of the TTA-based
titative imaging.2,3 Li and coworkers used 18F to modify the nanosystem will allow these materials to be used in many
as-prepared lanthanide-based upconversion nanoprobes.2,3 applications.
Similarly, upconversion nanoparticles modified with 153Sm3+ Benefiting from the in-depth investigation of lanthanide-
can serve as contrast agents in SPECT imaging.2,3 The radio- based upconversion nanomaterials in bio-applications, anti-
active imaging techniques can provide real time and quantita- Stokes luminescent probes seem ready to serve as an alternative
tive images of samples with very high spatial resolution. These of the commercial dyes for practical biomedicine where a high
technique could offer new possibilities for the research of signal-to-noise ratio is required. The main concern for the
upconversion nanoprobes and clinical translation. further development is the possible long-term toxicity of these
TTA-based nanomaterials also have been fabricated and probes.3 In addition, because of the low solubility of these
used for small animal bioimaging in recent years. Compared inorganic nanoparticles under biological conditions, they are
with lanthanide-based nanoparticles, these nanomaterials hard to be quickly excreted from living systems, which may
exhibit strong upconversion emission with higher luminescence cause some unwanted side effects and limit their applications.
efficiency, which can be excited with lower power-density lasers.
As mentioned previously, Li and coworkers developed TTA 4.2 Anti-Stokes luminescence materials for biodetection
upconversion nanoparticles with blue emission under green Hot-band absorption dyes whose molecular structures are
excitation by silica coating.24 Although the blue emission is not usually used to design chemodosimeters, can be easily modified
the optimal choice for in vivo applications, cell imaging and to suit the need for biodetection. For instance, the designed NRh
lymph node imaging can be realized under a lower excitation chemodosimeter combines a near-infrared (NIR) frequency
power-density than that in the case using lanthanide upconver- upconversion luminescent rhodamine derivative with a hydrazine
sion nanocrystals as probes (Fig. 10). Later, we integrated a green cyclization reaction site which provides a selective and sensitive

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Fig. 12 Upconversion detection principle.

lanthanide nanoparticles by a simple ligand exchange method.

Chemodosimeters with carboxyl groups can coordinate with
lanthanide ions on the surface of nanoparticles. This kind of
upconversion probes were used to detect and CN in solution
or living cells.2 However, because of the hydrophobicity of
chemodosimeters, the detection was carried out in semi-
Fig. 11 (a) Experimental design for Cu2+ detection by the hot-band absorp-
aqueous solution. Li and coworkers further developed a general
tion technique. (b) A schematic illustration of in vivo detection of Cu2+ and
diagnostic urine tests for Wilson disease patients. (Reprinted by permission method to fabricate nanocomposites containing upconversion
from ref. 10. Copyright 2016, Wiley-VCH Verlag GmbH & Co. KGaA.) nanoparticles and chemodosimeters to work in pure water.2
An amphiphilic PEG derivative was used to coat on the surface
of hydrophobic lanthanide upconversion nanoparticles to
turn-on response to copper(II) ions (Fig. 11).11 The sensing system make them hydrophilic and generate a hydrophobic layer to
showed an extremely low detection limit down to 3.2 ppb. load chemodosimeters. Using this method, the upconversion
Remarkably, the sensing strategy enables reliable detection of detection systems have been successfully fabricated for a series
Cu2+ in real biological samples for diagnosis of Wilson disease. It of target species in aqueous solutions.2 Li and coworkers used a
could be used for non-invasively monitoring the copper contents chemodosimeter with absorption transition in the near infra-
in the liver of a mouse model and urine of Wilson disease patients red range to construct an upconversion detection system and
with the assistance of the molecular upconversion probes. When explore its application in vivo. The near infrared emission of
we modified the NRh-1 molecule with a monothiospirolactone NaYF4:Yb,Tm nanoparticles at 800 nm was used to monitor
group, the detection platform exhibits high selectivity and the concentration of MeHg+. The in vivo imaging experiment
sensitivity toward both mercury(II) ions and methylmercury.29 proved that the detection system is also workable in vivo.41
Therefore, this strategy holds great promise for turn-on sensing Another general method is to use a silica shell to load
and imaging ions in biological samples. chemodosimeters. Zhang and coworkers coated a mesoporous
In recent years, lanthanide-based upconversion nanoprobes silica layer on lanthanide-based upconversion nanoparticles.
are widely used in detection systems to shift the excitation light Chemodosimeters were then loaded into the mesoporous silica
into the near infrared range. By conjugation with a certain shell and an upconversion detection system for Cu2+ was
chemodosimeter, upconversion nanoparticles will show a constructed.2 Li and coworkers synthesized a yolk–shell structure
particular emission colour determined by the energy transfer with a hollow silica shell outside upconversion nanoparticles. The
from the lanthanide ions to the chemodosimeter molecules. cavity provides the capability to load chemodosimeters, and the
After the reaction of the chemodosimeter with the analyte, the obtained nanocomposites were used for the detection of bioactive
structure of the chemodosimeter changes and the energy molecules or ions (i.e. cysteine, CN), offering a universal detec-
transfer process is broken. Thus, the upconversion emission tion platform in vitro.3
of lanthanide nanoparticles quenched by the chemodosimeter For the detection applications of lanthanide-based upcon-
will recover and finally another emission colour will be seen. version nanoparticles, there are still many problems to be
Based on this principle, detection of various kinds of species solved to further improve their performance. The first one is
based on lanthanide-based upconversion probes can be realized the efficiency of the energy transfer process. In current studies,
by integration with corresponding chemodosimeters (Fig. 12). energy transfer is usually accompanied by the inner filter effect
To date, several general methods have been reported for caused by the absorption of chemodosimeter molecules in the
the fabrication of the upconversion detection nanosystems. system. These two kinds of quenching of luminescent emission
Wolfbeis and coworkers reported a series of simple methods of upconversion nanoparticles can be distinguished by the life-
to integrate dye molecules together with upconversion nano- time analysis of the luminescent excited state. Unfortunately, few
particles, and the obtained nanocomposites exhibited upconver- research works realized this fact and no direct characterization
sion in response to pH, CO2, and ammonia with the selection of or analysis has been performed to properly distinguish the
the corresponding dye molecules.2,40 energy transfer process and inner filter effect currently.2
For biodetection of Hg2+ and CN in vitro, Li and coworkers The second key point is the effective energy transfer distance.
integrated chemodosimeter molecules onto the surface of For the well-recognized Förster resonance energy transfer (FRET)

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process, which gives a good description of the energy transfer therapeutic accuracy and mild treatment conditions at the
characteristics in the molecule-to-molecule system, the effective same time.43
energy transfer distance is often believed to be not longer than
10 nm. Nevertheless, this distance will be certainly enlarged for 4.4 Anti-Stokes luminescent materials for secondary
lanthanide-based nanomaterials, because of the relatively longer excitation
lifetime luminescence from lanthanide ions than small molecules.
Anti-Stokes luminescent materials have a unique ability to
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transfer long wavelength excitation to short wavelength emission,

4.3 Anti-Stokes luminescent materials as microscopic allowing them to serve as a secondary excitation source in some
temperature reporters light-driven biomedical devices. Generally, conventional devices
driven by visible or UV light can be integrated with anti-Stokes
The mechanism of anti-Stokes luminescence also brings some
luminescent materials to form a nanosystem. This nanosystem
interesting features that promote the application of anti-Stokes
can be used in vivo under long-wavelength excitation.
luminescent materials in broader areas.
Typically, the visible or ultra-violet light photocleavable units
One of the main characteristics of the anti-Stokes luminescence
can be integrated with upconversion materials to realize the near
process is the temperature-dependent emission intensity. In hot-
infrared light triggered dissociation (Fig. 14). Lanthanide-doped
band absorption molecules, the emission originates from the
upconversion nanomaterials were firstly used for this purpose.
vibrational–rotational states with higher energy than the ground
For example, Liu, Xing and coworkers utilized the ultraviolet
state, the population of these states, following the Boltzmann
upconversion luminescence of NaYF4:Yb,Tm nanoparticles to
distribution, is strongly temperature dependent (exponential
cut off the photosensitive connection between nanoparticles and
dependence). Based on this principle, Prasad and coworkers used
D-luciferin, achieving a pioneering demonstration of near infra-
anti-Stokes luminescence of rhodamine 101 to realize cell tem-
red light triggered drug release.44 Unfortunately, the successful
perature mapping under a confocal microscopy system (Fig. 10).12
realization of photocleavage process relying on lanthanide-based
Similarly, in the Er3+-doped lanthanide upconversion system, the
materials requires high excitation power. By virtue of the high
emissions derived from 2H11/2 - 4I15/2 (centred at 525 nm) and
upconversion efficiency of TTA-based materials, the excitation
2
S3/2 - 4I15/2 (centred at 545 nm) transitions were close in energy
conditions of the photocleavage system will be more moderate.
which also comply with a thermal equilibrium ruled by the
Kohane and coworkers designed a class of TTA-based upconver-
Boltzmann factor (Fig. 13). The properties of a such temperature
sion nanoparticles modified with photocleavable groups that are
reporter have been fully investigated,42 but few works involved its
bound to tumor targeting peptides beforehand. Upon low power
purposeful applications, especially in the biomedical field.
irradiation (200 mW cm2) at 530 nm, upconversion emission
Recently, we used Er3+-doped upconversion nanoparticles coated
was excited and the photon energy was transferred to the
with a photothermal material to monitor the microscopic tem-
photocleavable groups to release the targeting groups, thus
perature in photothermal therapy. By deciphering the tempera-
activating the tumor targeting effect.45 Moreover, with this
ture information in upconversion luminescence, we developed
phototargeting concept, the uptake of nanocarriers in tumor
new-generation photothermal therapy strategy and realized high
will be increased which is of great significance in targeted drug
delivery.
Beyond the above mentioned function, high energy upconverted
photons can activate photosensitizers to perform photodynamic
therapy (PDT).46 For example, intentionally designed a-NaYF4:
Yb,Er(2%)@CaF2 lanthanide upconversion nanoparticles with
a high dopant concentration of Yb3+ (80%) in the host lattice
have strong red emission centred at 660 nm. This red emission
has a good overlap with the absorption of 5-aminolevulinic acid
(5-ALA), a commercial prodrug for PDT. With the combination
of upconversion nanoparticles and 5-ALA, a remarkable photo-
dynamic effect was observed under 980 nm excitation. When
compared with the excitation light in the visible region, NIR

Fig. 13 Temperature sensing mechanism based on the anti-Stokes shift

luminescence in cells. Fig. 14 Upconversion emission for photo-triggered cleavage.

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irradiation at 980 nm has a better killing effect on deep tumor material are in different stages, they all have great potential
in vivo due to its considerable tissue penetration depth (41.2 cm).47 in bio-applications if the corresponding drawbacks of each type
Such upconversion luminescence-driven devices also have of material can be overcome or neglected in specific cases. The
found a place in the bioengineering technology. Han et al. future research will focus on exploring specific applications
embedded dye-sensitized core/active shell upconversion nano- which can maximize the superiorities of anti-Stokes shift
particles in a polymer film to form an implantable device for luminescence, making the materials more valuable for practical
photogenetics.21 Upconversion luminescence generated by
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applications.
800 nm excitation can effectively activate the channel of
rhodopsin modified neuron cells to show a NIR light intensity
dependent depolarization. Their work offered a very ingenious Acknowledgements
strategy to break the predicament of poor tissue penetration in
The authors thank MOST of China (2015CB931801, 2013CB733703,
present optogenetic tools.
and 2012CB932403), National Natural Science Foundation of China
(21231004, 21375024, and 21527801), and Shanghai Sci. Tech.
Comm. (15QA1400700) for financial support.
5. Conclusions and prospect
In summary, the anti-Stokes luminescent materials indeed Notes and references
have potential in various bio-applications, but the develop-
ments are not balanced at the current stage. 1 J. R. Lakowicz, Principles of fluorescence spectroscopy,
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