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The importance of high-resolution intracellular thermal sensing and imaging in the field of modern biomedicine has boosted the development of novel nanosized fluorescent systems (fluorescent nanothermometers) as the next generation of probes for intracellular thermal sensing and imaging. This thermal mapping requires fluorescent nanothermometers with good biocompatibility and high thermal sensitivity in order to obtain submicrometric and subdegree spatial and thermal resolutions, respectively. This review describes the different nanosized systems used up to now for intracellular thermal sensing and imaging. We also include the later advances in molecular systems based on fluorescent proteins for thermal mapping. A critical overview of the state of the art and the future perspective is also included.
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Fluorescent nano-particles for multi-photon thermal sensing
D. Jaque
, L.M. Maestro
, E. Escudero
, E. Martı
´n Rodrı
, J.A. Capobianco
, F. Vetrone
A. Juarranz de la Fuente
, F. Sanz-Rodrı
, M.C. Iglesias-de la Cruz
, C. Jacinto
U. Rocha
, J. Garcı
´a Sole
Fluorescence Imaging Group, Universidad Auto
´noma de Madrid, Madrid 28049, Spain
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. W., Montreal, QC, Canada H4B 1R6
Institut National de la Recherche ScientifiqueE
´nergie, Mate
´riaux et Te
´communications, Universite
´du Que
´bec, Varennes, QC, Canada J3X 1S2
Departamento de Biologı
´a, Facultad de Ciencias, Universidad Autonoma de Madrid, Madrid 28049, Spain
Departamento de Fisiologı
´a, Facultad de Medicina, Universidad Auto
´noma de Madrid, C/Arzobispo Morcillo s/n, 29029 Madrid, Spain
Grupo de Fotˆ
onica e Fluidos Complexos, Instituto de Fı
´sica, Universidade Federal de Alagoas, 57072-970 Maceio
´, Alagoas, Brazil
article info
Two-photon luminescence
Quantum dots
Rare earth doped nano-crystals
Confocal microscopy
In this work we report on the ability of Er/Yb co-doped NaYF
nano-crystals and CdTe Quantum Dots as
two-photon excited fluorescent nano-thermometers. The basic physical phenomena causing the
thermal sensitivity of the two-photon excited emission bands have been discussed and the maximum
thermal resolution achievable in each case has been estimated. The practical application of both
systems for thermal sensing at the micro-scale in biological systems is demonstrated. In particular, they
have been used to evaluate the thermal loading induced by tightly focused laser beams in both living
cells and fluids.
&2011 Elsevier B.V. All rights reserved.
1. Introduction
There is currently a great interest in the development of novel
systems capable of thermal sensing at the nano-scale [13].
Among the different scenarios in which they could be used,
thermal sensing in optically excited biological systems seems to
be one of the most challenging ones. The exact knowledge of the
local temperature of biological systems (such as living cells) is
required in order to understand the dynamical behavior and state
of the bio-system [4]. For instance, it is already known that cancer
cells, due to their faster metabolism, usually present larger
intracellular temperatures than healthy ones [5,6]. Intracellular
temperature is also a good indicator of the presence of active
intracellular chemical reactions. Fundamental processes, such as
ATP (adenosine triphosphate) hydrolysis, take place in the pre-
sence of significant thermogenesis and thus, can be monitored
through changes in the intracellular temperature [7]. Further-
more, temperature is also known to play a crucial role in
determining cell division rates and tissue response against exter-
nal interactions [8]. In addition, the exact knowledge of cellular
temperature is also required during external treatments such as
optical trapping, laser surgery, cell squeezing and photoporation
of cancer cells and photo-therapy [9].
Despite the interest in this area from both a fundamental or an
applied point of view, the study of intracellular thermal sensing
systems remains an under-explored area. In part, this is because it
is a difficult task to realize. It requires the incorporation of
thermal sensors into the cells without causing modification in
the cell’s activity, such that they can be considered as ‘‘non-
invasive thermal sensors’’. Typical cell sizes ( E10
m) restrict
the size of the thermal sensors to be used in the intracellular
incorporation down to the few nanometers since this would
ensure an efficient uptake through the cell membrane. Further-
more, thermal reading should be carried out ‘‘remotely’’ so that
direct (mechanical) interaction is avoided. This important point
typically restricts the use of micro/nano-thermocouplers.
Optical nano-thermometers (NTs) are constituted by lumines-
cent nano-particles whose luminescence features are strongly
influenced by small changes induced in their local temperature.
When incorporated in living cells the analysis of their lumines-
cence properties provides a direct measurement of the intracel-
lular temperature without any interaction with the cell under
study. During the last years numerous number of examples of
intracellular thermal sensing based on optical NTs can be found.
The most studied types are molecular nano-thermometers (MNTs)
[10]. They produce a characteristic visible luminescence when
excited in the UV or visible that is strongly temperature depen-
dent because of the existence of thermal driven structural phase
transitions. Although very good results have been obtained using
MNTs, its real application in bio-imaging is restricted because they
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Journal of Luminescence
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Corresponding author.
E-mail address: (D. Jaque).
Please cite this article as: D. Jaque, et al., J. Lumin. (2012), doi:10.1016/j.jlumin.2011.12.022
Journal of Luminescence ](]]]])]]]]]]
can only be optically excited in the UV–visible spectral range. At
these wavelengths tissue absorption is high, leading to reduced
penetration depths [11]. In addition, optical excitation in the
visible or UV makes bio-imaging difficult due to the undesired
auto-fluorescence and also because it leads to a remarkable local
heating [12]. All these disadvantages are avoided with the use of
multi-photon nano-thermometers (MPNTs) since the optical exci-
tation of the thermal sensitive luminescence can be done in the
near-infrared (700–1000 nm). In this excitation range, tissue
absorption is minimal allowing for large penetration depths. In
addition, multi-photon excitation leads to a superior spatial
resolution since the fluorescent volume is restricted to the focus
spot of the infrared excitation beam [13,14]. Furthermore near-
infrared (NIR) excitation minimizes drastically the presence of the
undesired auto-fluorescence [13,14].
In this work we summarize recent results concerning the use
of both lanthanide-doped upconverting nano-particles (UCNPs)
and semiconductor quantum dots (QDs) as MPNTs. The different
mechanisms at the basis of the thermal sensitivity of fluorescence
bands are discussed and the thermal resolution achievable in each
case is discussed. Finally, we report on recent experiments
showing the ability of both UCNPs and QDs to measure local
temperature increments caused by tightly focused laser beams in
living cells.
2. Experimental
The CdTe-QDs investigated in this work were provided by
Plasmachem Inc., and were fabricated through an aqueous synth-
esis without phase transfer. The mean size of the CdTe-QDs used
was 3.6 nm, leading to a broad emission band centered at 660 nm.
The UCNPs used throughout this work were NaYF
(18 nm diameter in average) co-doped with erbium (2 mol%) and
ytterbium ions (18 mol%) synthesized via the solvothermal pro-
cess, as described elsewhere [15]. Both UCNPs and QDs were
dispersed in distilled water with no evidence of precipitation over
a period of months.
The thermal sensitivity of the two-photon (TP) excited emission
generated from both UCNPs and QDs was measured diluting them
in water and then placing the solutions on a heating microscope
plate with a thermal resolution of 1 1C in the range of 20–100 1C.
The solutions were excited by a Ti:Sapphire mode-locked laser
(Tsunami Spectra Physics) that provides 100 fs pulses with tunable
wavelength between 670 and 980 nm. The optimum excitation
wavelengths in our experimental conditions were 920 and 880 nm
for UCNPs and CdTe-QDs, respectively. The TP spectra were
registered using a fiber coupled high-resolution spectrometer.
For the intracellular thermal measurements, non-functionalized
UCNPs were incorporated in living HeLa cancer cells by endocytosis.
For this purpose the HeLa cancer cells were incubated for 3 h in a
PBS solution containing UCNPs at a concentration of 0.3% in mass.
After incubation the cells were placed in a confocal microscope. A
50 (NA 0.55) microscope objective was used to focus the 920 nm
excitation beam into the cell. The TP emission generated from the
UCNPs was collected using the same microscope objective and,
after passing through several filters and apertures, was spectrally
analyzed by a fiber coupled high-resolution spectrometer. The cells
were placed on a motorized stage so that it was possible to scan the
920 nm excitation spot within the cell.
For the experiments devoted to evaluating the pump induced
thermal loading caused by tightly focused beams, the experi-
mental setup designed is illustrated in Fig. 1. The solution
containing the MPNT was placed into a closed micro-chamber
(with an approximate volume of 2
l). The beam delivered by a
diode pumped fiber laser operating at 1090 nm was tightly
focused into the liquid using a 100 microscope objective (NA
1.2). The 1090 nm spot radius has been estimated to be around
m (see the optical transmission picture of the locally heated
liquid included in Fig. 1). The MPNT were optically excited by a
second beam that was focused from the opposite face of the
micro-chamber using a long work distance 100 microscope
objective with a NA of 0.85. The visible luminescence generated
by the MPNT dispersed in solution (carrying information about
local temperature) was collected by the same long work distance
objective. After passing through a confocal aperture, the collected
luminescence was analyzed using a high-resolution spectrometer.
The excitation spot was placed exactly at the center of the
1090 nm excitation spot adjusting the position of the 100
focusing objective.
Fig. 1. Schematic diagram of the experimental setup used for the evaluation of the thermal loading produced by tightly focused laser beams in water. The optical
microscope on the right shows the 1090 nm spot focused into the micro-chamber by the 100 high NA microscope objective. From this image we have estimated a spot
size close to 1
m in radius.
D. Jaque et al. / Journal of Luminescence ](]]]])]]]]]]2
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3. Results and discussion
3.1. Thermal sensitivity of two-photon emissions
Fig. 2 shows the TP emission of both UCNPs (a) and QDs
(c) solutions obtained at two different temperatures (25 and
75 1C). As can be observed both emissions are strongly modified
when the temperature is increased. In the case of UCNPs
(Fig. 2(a)), the temperature increment causes a remarkable
change in the ratio between the intensities of the fluorescence
bands at around 525 and 545 nm. These two emission bands are
originated from thermally coupled states of Er
ions, as already
described in many Er/Yb co-doped crystals and glasses [1619].
As postulated previously, when temperature is modified the
population of these thermally coupled states suffers a strong
redistribution. As a consequence of this re-distribution, the
relative intensities of the fluorescence bands generated from
these states are changed. Indeed, the ratio between the emitted
intensities (I
) increases monotonously with temperature
(see Fig. 2(b)), so that from the knowledge of this ratio the
temperature of UCNPs can be determined. Data included in
Fig. 2(b) can be also used to determine the thermal sensitivity
of the UCNPs based MPNT using the experimental uncertainty of
the I
ratio observed in our experimental conditions (
)E5%). We have estimated that the thermal sensitivity of
UCNPs is approximately 711C. On the other hand, the case of
QDs is completely different. In this case temperature changes are
monitored by a thermally induced spectral shift of the broad
emission band [2022]. It is clear from Fig. 2(c) and (d) that a
temperature increment causes a red-shift that is proportional to
the temperature change. This red-shift is very likely caused by a
combination of different phenomena such as temperature
induced changes in the band gap, in the quantum confinement
energy as well as in the electron–phonon coupling [2325]. The
magnitude of this temperature induced spectral shift depends on
both the particular semiconductor material from which QDs are
fabricated as well as on the QD size. For the QDs under study
(CdTe-QDs, 3.6 nm in diameter) the peak wavelength shifts at a
rate of 0.3 nm/1C. Based on this rate, and taking into account the
accuracy in the determination of the peak wavelength, we have
estimated a temperature resolution of 70.5 1C for CdTe-Qds. This
is remarkably lower than that achieved using UCNPs.
3.2. Application of QDs and UCNPs in the determination of pump
induced thermal loading in bio-systems
The data included in Fig. 2 clearly reveals the potential use of
both UCNPs and QDs for thermal sensing at the micro/nano-scale.
In this section we will use this potential to determine how
relevant is the pump induced thermal loading in bio-imaging
experiments. In such experiments, the biological system under
study (such as living cells or tissues) is optically excited by a
tightly focused laser beam required for the TP excitation. The
presence of tightly focused IR beams could cause relevant local
heating because of the existence of residual absorptions (mostly
due to water) and also because the presence of multi-phonon
relaxation in the optically excited nano-particles. In imaging
applications this is an undesirable effect, since it constitutes a
perturbation of the system under study (the dynamic of any
biological system is strongly modified by temperature). In other
applications (such as laser photo-therapy) this pump induced
local heating is desired. In the following we focus our attention to
the in-situ determination of the temperatures achieved in biolo-
gical systems at the focus of a laser beam.
Fig. 3(a) shows an optical transmission micrograph of an
individual HeLa cancer cell, which has been incubated for 2 h in
a PBS solution containing Er/Yb co-doped UCNPs. The intracellular
incorporation of the UCNPs is evidenced when a 920 nm laser
beam is scanned through the cell (the scanning direction is
indicated in Fig. 3(a) by the dashed arrow) and the TP excited
500 20
Intensity (Arb. Units)Intensity (Arb. Units)
Wavelength (nm)
Wavelength (nm)
Temperature (ºC)
Temperature (ºC)
Peak wavelength (nm) I540/I520
25 ºC
60 ºC
25 ºC
60 ºC
520 540 560 580 30 40 50 60
600 700 800 30 40 50 60 70 80
Fig. 2. (a) Two-photon emission spectra generated from Er/Yb co-doped NaYF
nano-particles at two different temperatures. (b) Temperature variation of the intensity
ratio between the thermally coupled emission of Er ions in NaYF
nano-particles. (c) Two-photon emission spectra generated from CdTe-QDs obtained at two different
temperatures. (d) Peak position of the two-photon emission from CdTe-QDs as a function of temperature.
D. Jaque et al. / Journal of Luminescence ](]]]])]]]]]] 3
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emission is registered. Fig. 3(b) shows the UCNPs emitted
intensity profile indicating that the UCNPs are located in a small
area within the HeLa cancer cell. In order to investigate intracel-
lular pump induced thermal loading we focused our 920 nm
excitation spot in this small area (red circle in Fig. 3(a)). While
keeping the excitation spot fixed, the 920 nm excitation intensity
was varied and both the TP emission intensity and the band ratio
(giving us the on-focus temperature) were determined for each
excitation intensity. Results are shown Fig. 3(c) and (d). From
Fig. 3(c) it is clear that the emitted intensity of the UCNPs
increases quadratically with the 920 nm excitation intensity; this
being in accordance with the TP nature of the excitation process
[26,27]. The evolution of the focus temperature (calculated from
the ratio between the emitted intensities at 525 and 545 nm) as a
function of the 920 nm excitation intensity (included in Fig. 3(d))
denotes only a slight pump induced thermal increment (less than
41C when the excitation intensity is increased up to 2 MW/cm
This minimum thermal loading was indeed expected since at this
excitation wavelength (920 nm) the residual water absorption is
at minimum. In fact, 920 nm lies within the so-called biological
window which is characterized by a minimum residual absorp-
tion. Data included in Fig. 3 clearly denote that high contrast
images of biological systems can be achieved while causing
minimum intracellular heating.
Data included in Fig. 3 clearly denote that for the excitation
intensities usually employed in TP bio-imaging experiments,
thermal loading at focus is negligible. Nevertheless, for other
applications such as photo-therapy, optical trapping and tissue
ablation much larger optical intensities are used (tens of MW/
) so that in that case it is expected that local heating cannot be
neglected. In order to verify this assumption we have measured
the temperature increment induced at the focus of a 1090 mm
laser beam tightly focused inside a water solution of CdTe-QDs.
For this purpose the experimental setup schematically illustrated
in Fig. 1 has been used. In this case we used a multi-Watt
1090 nm heating laser beam that was tightly focused down to a
m spot. The combination of high power and small spot allowed
us to achieve excitation intensities as high as 100 MW/cm
the solution. By placing the low-power excitation beam where the
high power 1090 nm laser beam was focused, we were able to
collect the luminescence of the CdTe-QDs such that the analysis of
their visible luminescence allowed us to determine the tempera-
ture achieved at focus. Results are shown in Fig. 4. It is clear that
for such large excitation intensities, pump induced thermal
loading cannot be neglected, since it leads to temperature incre-
ments of the order of several tens of degrees. This large heating
can be detrimental in bio-systems. Indeed, based on Fig. 4, laser
induced cell death is expected to occur for 1090 nm excitation
intensities larger than 40 MW/cm
, since those intensity levels
would cause intracellular temperatures above 45 1C.
4. Conclusions
In summary, the ability of both Er/Yb co-doped NaYF
particles and CdTe-QDs as thermal sensors has been evaluated.
Both systems showed a large thermal sensitivity in their two-
photon excited emission. In the case of Er/Yb co-doped NaYF
Excitation Intensity (MW/cm2)
Cell Temperature (ºC)
Intensity (Arb. Units)
Emitted Intensity (Arb. Units)
Position (µm)
0.1 0.2 0.3 0.4 0.5-20 0 20 40
Fig. 3. (a) Optical transmission image of a HeLa cancer cell, which has been incubated for 3 h in a PBS solution containing Er/Yb co-doped NaYF
nano-particles. (b) Spatial
variation of the two-photon emission generated by the Er/Yb co-doped NaYF
nano-particles obtained along the scan direction indicated by an arrow in (a). (c) Variation of
the two-photon emitted intensity as a function of the 920 nm excitation intensity. (d) Variation of the intracellular temperature as a function of the 920 nm excitation
intensity. Data included in (c) and (d) were obtained at the intracellular location leading to the maximum two-photon luminescence (indicated by a red spot in (a)).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Focus Temperature (°C)
Pump Intensity (MW/cm2)
40 60 80 100
Fig. 4. On-focus local temperature increment induced in water by a 1090 nm
tightly focused laser beam (see Fig. 1).
D. Jaque et al. / Journal of Luminescence ](]]]])]]]]]]4
Please cite this article as: D. Jaque, et al., J. Lumin. (2012), doi:10.1016/j.jlumin.2011.12.022
nano-particles this thermal sensitivity is given by relative inten-
sity changes between thermally coupled emissions. On the other
hand, the CdTe-QDs temperature induced changes in dot size,
quantum confinement and electron–phonon coupling lead to a
remarkable temperature induced spectral shift of its emission.
The maximum temperature sensitivity of both systems has been
evaluated concluding that temperature sensitivities as high as
1 and 0.5 1C can be achieved using NaYF
nano-particles and
CdTe-QDs as nano-thermometers, respectively. Finally, the appli-
cation of both types of nano-thermometers for the measurement
of the temperature increments caused in biological systems
by tightly focused laser beams has been demonstrated. We have
found that for the typical excitation intensities used in bio-
imaging experiments, pump induced thermal loadings can
be neglected whereas for larger excitation intensities (tens of
), the local temperature increments could be large
enough to cause cell death.
This work was supported by the Universidad Auto
´noma de
Madrid and Comunidad Autonoma de Madrid (Projects CCG087-
UAM/MAT-4434 and S2009/MAT-1756), by the Spanish Minis-
terio de Educacion y Ciencia (MAT 2010-16161) by a Banco
Santander CEAL-UAM project and by the Brazilian agencies CAPES
(grant Nr. 02727/09-9 - PNPD-CAPES) CNPq and FAPEAL (through
the PRONEX-09-006).
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... 1,4,5,15,16 Moreover, the spectroscopic parameter considered as a thermometric parameter should reveal high thermal sensitivity and a high signal-to-noise ratio. 4,10,[17][18][19] Aiming at the development of highly sensitive luminescent thermometers (LTs) meeting the requirements described above, the optical properties of inorganic host materials doped with transition metal (TM) ions attract particular attention, among which Cr 3+ , Mn 2+/3+/4+ , Ti 3+/4+ , V 3+,4+,5+ , Fe 3+ , and Ni 2+ ions were thoroughly analyzed. [20][21][22][23][24][25][26][27][28][29][30][31][32] The unique spectroscopic properties of TM ions including the high thermal susceptibility of their emission intensity and the possibility of tuning their spectroscopic properties by the modification of the crystal field strength are their main advantages. ...
The growing interest in luminescent thermometry and the desire to improve the thermometric properties of luminescent thermometers prompts the search for new solutions including new materials and types of luminescent...
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Lanthanide luminescence nanothermometers (LNTs) provide microscopic, highly sensitive, and visualizable optical signals for reporting temperature information, which is particularly useful in biomedicine to achieve precise diagnosis and therapy. However, LNTs with efficient emissions at the long-wavelength region of the second and the third near-infrared (NIR-II/III) biological window, which is more favourable for in vivo thermometry, are still limited. Herein, we present a lanthanide-doped nanocomposite with Tm3+ and Nd3+ ions as emitters working beyond 1200 nm to construct a dual ratiometric LNT. The cross-relaxation processes among lanthanide ions are employed to establish a strategy to enhance the NIR emissions of Tm3+ for bioimaging-based temperature detection in vivo. The dual ratiometric probes included in the nanocomposite have potential in monitoring the temperature difference and heat transfer at the nanoscale, which would be useful in modulating the heating operation more precisely during thermal therapy and other biomedical applications. This work not only provides a powerful tool for temperature sensing in vivo but also proposes a method to build high-efficiency NIR-II/III lanthanide luminescent nanomaterials for broader bio-applications.
This work begins with a study of the synthesis of multi-color luminescence phosphors, and it is followed by a discussion of the temperature sensitivity of AgBi(MoO4)2:Yb3+/Er3+. Uniform and approximately spherical...
In this work, the synthesis and characterization of the β-BaB2O4 (β-BBO) phase single-doped with Nd³⁺ and co-doped with Nd³⁺/Yb³⁺ were performed. This work focuses on luminescence nanothermometry application in the near-infrared (NIR) window and the temperature range between 25 and 55 °C to evaluate potential technological applications in nanomedicine. The polymeric precursor method was used for the synthesis of Nd³⁺ doped and Nd³⁺/Yb³⁺ co-doped β-BBO nanoparticles (0.2 mol% of each doping ion) crystallized after a heat-treatment. The structural and thermal properties of these nanoparticles were characterized using thermal analysis (Thermogravimetry and Differential Scanning Calorimetry) to specify the temperature of heat treatment (20 h at 750 °C) for the formation of pure crystalline phases characterized by X-ray diffraction and Raman spectroscopy. Transmission Electron Microscopy was used to assess the shape (parallelepiped needles) of the particles, their length (∼214 nm) and average width (∼18 nm), as well as the orientation of the atomic planes of the crystalline planes by electron diffraction. Diffuse reflectance spectroscopy allowed to specify the transparency range of the pure crystalline matrix and to investigate the absorption bands of the doping ions, as well as to determine the bandgap using the Kubelka-Munk equation. In addition, the temperature dependence of the luminescence intensity ratios of Stark components from both Nd³⁺ and Yb³⁺ ions were analyzed. This led to a relative thermal sensitivity value of 0.06%·oC⁻¹ for single-doped Nd³⁺ sample and 0.23%·oC⁻¹ for Nd³⁺/Yb³⁺ co-doped sample from emissions in the range 1050.5 nm–1067.5 nm. Finally, a value of 0.28%·oC⁻¹ was found for Nd³⁺/Yb³⁺ co-doped sample from the intensity ratio between emissions at 979.0 nm and 1058.5 nm. Thus, the relative thermal sensitivity of the β-BBO phase co-doped with the ions Nd³⁺ and Yb³⁺ at the concentration of 0.2 mol.% was superior to that of some matrices found in the literature. This allows us to conclude that Nd³⁺/Yb³⁺ co-doped β–BBO nanoparticles can be used as an efficient luminescent nanothermometer in the near-infrared.
Upconversion (UC) is a nonlinear and unique phenomenon, which is usually observed in trivalent lanthanide‐doped materials. The trivalent lanthanides possess a large number of closely spaced energy levels, which are responsible for both radiative and nonradiative transitions. The radiative transitions emit photons and give rise to intense and sharp emissions. The external temperature affects the radiative transitions that provide opportunity to develop optical thermometers. On the other hand, nonradiative transitions are a key factor for internal heating of the system, known as “optical heating.” If this heating is produced by using a laser, this effect is known as laser‐induced optical heating. Thus, the effect of radiative and nonradiative transitions on upconversion nanoparticles (UCNPs) and their applications in optical thermometers and optical heaters are discussed in this chapter.
Temperature is the most intuitive and widespread in various physical quantities. Violent changes in temperature usually implies the appearing of fluctuations in physical properties of an object. Therefore, temperature is often an important indicator. With the development of science and technology, the scales in many fields are being more and more miniaturized. However, there are no mature temperature measurement systems in the case where the spatial scale is less than 10 μm. In addition to the requirement for spatial resolution, the sensor ought to exert no dramatic influence on the object to be measured. The nitrogen vacancy (NV) center in diamond is a stable luminescence defect. The measurements of its spectrum and spin state can be used to obtain the information about physical quantities near the color center, such as temperature and electro-magnetic field. Owing to its stable chemical properties and high thermal conductivity, the NV center can be applied to the noninvasive detection for nano-scale researches. It can also be used in the life field because it is non-toxic to cells. Moreover, combined with different techniques, such as optical fiber, scanning thermal microscopy, NV center can be used to measure the local temperatures in different scenarios. This review focuses on the temperature properties, the method of measuring temperature, and relevant applications of NV centers.
In this research, we have synthesized carbon dots (CDs) co-doped with nitrogen and sulfur by facile hydrothermal method, using citric acid and cysteine as carbon source. The effect of solid-state thermic treatment (STT) at 303 to 453 K on the size, surface, fluorescence and cellular cytotoxicity of the CDs were systematically investigated. Through a simple STT, it was possible to tune surface states and the average size of the CDs, causing a permanent red shift. Initially, CDs showed a decrease in cell viability with increasing concentration. However, after STT, its viability remained constant with an increase in concentration. Here, we show the possibility to label the cells cytoplasm according to the CDs fluorescence emission before (blue emission) and after STT (red emission). The CDs studied in this paper show selective luminescence properties, which are fundamental for any cell imaging application.
Mn²⁺ activated glass derived from ion-exchanged USY zeolites was prepared by spark plasma sintering (SPS) to investigate its temperature-dependent luminescence properties. The luminescence intensity ratio showed a high sensitivity in the temperature range from 4 K to 548 K. The absolute sensitivity and the relative sensitivity values can reach 2 × 10⁻² K⁻¹ and 0.66% K⁻¹ at 498 K, respectively. After 3 consecutive thermal cycles tests, the repeatability was 96.6%, and the temperature uncertainty is less than 0.1 K in the range of 270–548 K. The Mn²⁺ activated USY-zeolite derived glass with high sensitivity, wide temperature range and high thermal conductivity is promising for remote temperature sensing applications.
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Intracellular thermometry provides important information about the physiological activity of single cells and has been implemented using diverse temperature-sensitive materials as nanoprobes. However, measuring the temperature of specific organelles or subcellular structures is challenging because it requires precise positioning of the nanoprobes. Here, it is shown that dispersed fluorescent nanodiamonds (FNDs) endocytosed in living cells can be aggregated into microspheres using optical forces and used as intracellular temperature probes. The aggregation of the FNDs and electromagnetic resonance between individual nanodiamonds in the microspheres lead to a sevenfold intensity enhancement of 546-nm laser excitation. With the assistance of a scanning optical tweezing system, the FND microspheres can be precisely patterned and positioned within the cells. By measuring the fluorescence spectra of the microspheres, the temperatures at different locations within the cells are detected. The method provides an approach to the constructing and positioning of nanoprobes in an intracellular manner, which has potential applications in high-precision and flexible single-cell analysis.
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Near-infrared (NIR) fluorescent probes offer advantages of high photon penetration, reduced light scattering and minimal autofluorescence from living tissues, rendering them valuable for noninvasive mapping of molecular events, assessment of therapeutic efficacy, and monitoring of disease progression in animal models. This review provides an overview of the recent development of the design and optical property of the different classes of NIR fluorescent nanoprobes associated with in vivo imaging applications.
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Semiconductor quantum dots of few nanometers have demonstrated a great potential for bioimaging. The size determines the emitted color, but it is also expected to play an important role in the image brightness. In this work, the size dependence of the fluorescence quantum yield of the highly thermal sensitive CdTe quantum dots has been systematically investigated by thermal lens spectroscopy. It has been found that an optimum quantum yield is reached for 3.8-nm quantum dots. The presence of this optimum size has been corroborated in both one-photon excited fluorescence experiments and two-photon fluorescence microscopy of dot-incubated cancer cells. Combination of quantum yield and fluorescence decay time measurements supports that the existence of this optimum size emerges from the interplay between the frequency-dependent radiative emission rate and the size-dependent coupling strength between bulk excitons and surface trapping states.
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In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.
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The "gold standard" for nanothermometry: The application of ultrasmall, near-IR-emitting fluorescent gold nanoclusters (AuNCs) for temperature sensing has been explored. AuNC-based fluorescent nanothermometry features excellent thermal sensitivity and simultaneous temperature sensing and imaging in HeLa cells.
The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real-time. Both the thermo-sensitive fluorophore, β-diketonate chelate europium (III) thenoyltrifluoroacetonate, and the thermo-insensitive fluorophore, rhodamine 101 that was used as a self-reference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the Z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca(2+) burst induced by the Ca(2+) ionophore, ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca(2+), suggesting that the activity and/or number of heat sources are dependent on the Ca(2+) concentration.
The future perspective of fluorescence imaging for real in vivo application are based on novel efficient nanoparticles which is able to emit in the second biological window (1000-1400 nm). In this work, the potential application of Nd(3+) -doped LaF3 (Nd(3+) :LaF3 ) nanoparticles is reported for fluorescence bioimaging in both the first and second biological windows based on their three main emission channels of Nd(3+) ions: (4) F3/2 →(4) I9/2 , (4) F3/2 →(4) I11/2 and (4) F3/2 →(4) I13/2 that lead to emissions at around 910, 1050, and 1330 nm, respectively. By systematically comparing the relative emission intensities, penetration depths and subtissue optical dispersion of each transition we propose that optimum subtissue images based on Nd(3+) :LaF3 nanoparticles are obtained by using the (4) F3/2 →(4) I11/2 (1050 nm) emission band (lying in the second biological window) instead of the traditionally used (4) F3/2 →(4) I9/2 (910 nm, in the first biological window). After determining the optimum emission channel, it is used to obtain both in vitro and in vivo images by the controlled incorporation of Nd(3+) :LaF3 nanoparticles in cancer cells and mice. Nd(3+) :LaF3 nanoparticles thus emerge as very promising fluorescent nanoprobes for bioimaging in the second biological window.
Controlling and monitoring temperature at the single cell level has become pivotal in Biology and Medicine. Indeed, temperature influences many intracellular processes and is also involved as an activator in novel therapies. Aiming to assist such developments, several approaches have recently been proposed to probe cell temperature in vitro. None of them have so far been extended to a living organism. Here we present the first in vivo intracellular temperature imaging. Our technique relies on measuring the fluorescence polarization anisotropy of Green Fluorescent Protein (GFP) on a set of GFP expressing neurons in Caenorhabditis elegans (C. elegans). We demonstrate fast and non-invasive monitoring of sub-degree temperature changes on a single neuron induced by local photo-heating of gold nanoparticles. This simple and biocompatible technique is envisioned to benefit several fields including hyperthermia treatment, selective drug delivery, thermal regulation of gene expression and neuron laser ablation.
An accurate method for measuring intracellular temperature is potentially valuable because the temperature inside a cell can correlate with diverse biological reactions and functions. In a previous study, we reported the use of a fluorescent polymeric thermometer to reveal intracellular temperature distributions, but this polymer required microinjection for intracellular use, such that it was not user-friendly; furthermore, it could not be used in small cells or cells with a cell wall, such as yeast. In the present study, we developed several novel cationic fluorescent copolymers, including NN-AP2.5 and NN/NI-AP2.5, which exhibited spontaneous and rapid entry (≤ 20 min) into yeast cells and subsequent stable retention in the cytoplasm. The fluorescence lifetime of NN-AP2.5 in yeast cells was temperature-dependent (6.2 ns at 15°C and 8.6 ns at 35°C), and the evaluated temperature resolution was 0.09-0.78°C within this temperature range. In addition, NN-AP2.5 and NN/NI-AP2.5 readily entered and functioned within mammalian cells. Taken together, these data show that our novel cationic fluorescent polymeric thermometers enable accurate and practical intracellular thermometry in a wide range of cells without the need for a microinjection procedure.
Photothermal effects are studied under laser irradiation of aqueous suspensions of gold nanorods (in vitro experiments) and mice-inoculated Erlich carcinoma after intravenous injection of gold nanorods with the size 40 × 10 nm and plasmon resonance at the wavelength 810 nm (in vivo experiment). In 24 hours after the injection the polyethylene-glycol-coated nanoparticles accumulated in the tumour with the concentration three — four times greater than in healthy muscle tissue. At concentrations, attained as a result of passive accumulation of nanoparticles in the tumour (4 μg per 1 g of tumour), the efficiency of the tumour heating was higher than that in aqueous solutions having the same concentration of nanoparticles. Various mechanisms of this effect are discussed including the difference in thermal physical parameters of water and biotissue, the aggregation of nanoparticles in tissues, the influence of multiple scattering in biotissue, and the nonuniform accumulation of particles in the tumour. Using the Monte Carlo method for simulating multiple scattering of light, it is shown that there are such proportions between the biotissue scattering coefficient and the absorption coefficient of nanoparticles, at which the fraction of absorbed photons in the tissue is higher than that in a transparent medium containing the same nanoparticles. The conclusion is made that the regime of hyperthermia is less efficient for antineoplastic therapy than the thermal damage due to fast short-time heating of the tissues up to the destruction temperature.
In this study, a novel fluorescent and temperature responsive block copolymer has been designed and synthesized by a reversible addition–fragmentation chain transfer (RAFT) polymerization method in terms of the strategy that N-isopropylacrylamide (NIPAm), maleic anhydride (MAn) and 7-amino-4-methylcoumarin (AMC) act as the temperature responsive unit, the hydrophilic unit and the fluorescent unit, respectively. The successfully synthesized block copolymer was characterized by gel permeation chromatography (GPC) and nuclear magnetic resonance (1H NMR) spectroscopy. Meanwhile, the self-aggregation behaviour in aqueous solution and the thermo-responsive property of the block copolymer were demonstrated by particle size measurement, transmission electron microscopy (TEM) observations and lower critical solution temperature (LCST) determination, respectively. Then the variation of fluorescence intensity with temperature was confirmed. With increasing temperature, shrinking of PNIPAm chains caused the block copolymer to become more hydrophobic above the LCST, assembling larger aggregates with lower interfacial curvature. Thus a part of the fluorescent groups would be embedded inside the enlarged block copolymer micelles, resulting in lower fluorescence intensity. Furthermore, the superior hydrophilicity and biocompatibility of the block copolymer as a thermometer have been demonstrated by application in intracellular temperature sensing of MDCK cells ranging from 24 °C to 38 °C.