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Fluorescent nano-particles for multi-photon thermal sensing
D. Jaque
a,
n
, L.M. Maestro
a
, E. Escudero
a
, E. Martı
´n Rodrı
´guez
b
, J.A. Capobianco
b
, F. Vetrone
c
,
A. Juarranz de la Fuente
d
, F. Sanz-Rodrı
´guez
d
, M.C. Iglesias-de la Cruz
e
, C. Jacinto
f
,
U. Rocha
f
, J. Garcı
´a Sole
´
a
a
Fluorescence Imaging Group, Universidad Auto
´noma de Madrid, Madrid 28049, Spain
b
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. W., Montreal, QC, Canada H4B 1R6
c
Institut National de la Recherche Scientifique—E
´nergie, Mate
´riaux et Te
´le
´communications, Universite
´du Que
´bec, Varennes, QC, Canada J3X 1S2
d
Departamento de Biologı
´a, Facultad de Ciencias, Universidad Autonoma de Madrid, Madrid 28049, Spain
e
Departamento de Fisiologı
´a, Facultad de Medicina, Universidad Auto
´noma de Madrid, C/Arzobispo Morcillo s/n, 29029 Madrid, Spain
f
Grupo de Fotˆ
onica e Fluidos Complexos, Instituto de Fı
´sica, Universidade Federal de Alagoas, 57072-970 Maceio
´, Alagoas, Brazil
article info
Keywords:
Bio-photonics
Two-photon luminescence
Nanothermometry
Quantum dots
Rare earth doped nano-crystals
Confocal microscopy
abstract
In this work we report on the ability of Er/Yb co-doped NaYF
4
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 [1–3].
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
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
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/jlumin
Journal of Luminescence
0022-2313/$ - see front matter &2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jlumin.2011.12.022
n
Corresponding author.
E-mail address: daniel.jaque@uam.es (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
4
nano-particles
(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
m
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
1
m
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
m in radius.
D. Jaque et al. / Journal of Luminescence ](]]]])]]]–]]]2
Please cite this article as: D. Jaque, et al., J. Lumin. (2012), doi:10.1016/j.jlumin.2011.12.022
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
3þ
ions, as already
described in many Er/Yb co-doped crystals and glasses [16–19].
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
545
/I
525
) 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
545
/I
525
ratio observed in our experimental conditions (
D
(I
525
/
I
545
)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 [20–22]. 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 [23–25]. 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
500 20
1.0
1.2
1.4
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
20
615
620
625
630
635
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
4
nano-particles at two different temperatures. (b) Temperature variation of the intensity
ratio between the thermally coupled emission of Er ions in NaYF
4
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
Please cite this article as: D. Jaque, et al., J. Lumin. (2012), doi:10.1016/j.jlumin.2011.12.022
Er
3þ
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
2
).
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/
cm
2
) 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
1
m
m spot. The combination of high power and small spot allowed
us to achieve excitation intensities as high as 100 MW/cm
2
inside
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
2
, since those intensity levels
would cause intracellular temperatures above 45 1C.
4. Conclusions
In summary, the ability of both Er/Yb co-doped NaYF
4
nano-
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
4
0.0
0
10
20
30
40
50
Excitation Intensity (MW/cm2)
Cell Temperature (ºC)
Intensity (Arb. Units)
-40
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
4
nano-particles. (b) Spatial
variation of the two-photon emission generated by the Er/Yb co-doped NaYF
4
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.)
20
0
10
20
30
40
50
60
70
80
90
100
110
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
4
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
MW/cm
2
), the local temperature increments could be large
enough to cause cell death.
Acknowledgments
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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