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Per Rella 2019
Per Rella 2019
R.V. Perrella, I.C. Ribeiro, P.H.A. Campos-Junior, M.A. Schiavon, E. Pecoraro, S.J.L. Ribeiro, J.L.
Ferrari
PII: S0254-0584(18)30976-3
DOI: 10.1016/j.matchemphys.2018.11.018
Please cite this article as: R.V. Perrella, I.C. Ribeiro, P.H.A. Campos-Junior, M.A. Schiavon, E.
Pecoraro, S.J.L. Ribeiro, J.L. Ferrari, CaTiO3:Er3+:Yb3+ Upconversion from 980 nm and 1550 nm
Excitation and its Potential as Cells Luminescent Probes, Materials Chemistry and Physics (2018),
doi: 10.1016/j.matchemphys.2018.11.018
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c – UNESP, Institute of Chemistry, P.O. Box 355, 14800-970, Araraquara, SP, Brazil
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Abstract
CaTiO3:xEr3+:yYb3+ (x = 0.3mol%; y = 1.0, 1.5 and 2.0 mol%) was synthesized by the
sol-gel process. Crystal phase purity, crystallite sizes and microstrain were evaluated as
Upconversion (UC) spectra were recorded at 300 K under excitation at 980 nm and
1550 nm. The samples showed higher intensity of green emission assigned to the
upconversion phenomena compared to the red one, when excited at 980 nm and the
opposite was observed when excitation was performed at 1550 nm. The nanoparticles
luminescent probes, with low cytotoxic effect being observed. Er3+ emission at 1550 nm
was also proposed for in vitro or in vivo optical imaging analysis in the so-called second
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1. Introduction
luminescent imaging [1-5]. Er3+ is widely used since it can be efficiently excited to the
2H 4S and 4F9 upper energy levels by using commercial 980 nm or 1550 nm lasers
11/2, 3/2
[6–8]. Normally, Yb3+ is used together with Er3+ for improving the 980 nm absorption
cross-section, and sensitizing the Er3+ upper energy levels via energy transfer. In fact,
upconversion (UC) mechanisms can be different for the two different excitations (980
On the other side, the Er3+ 1550 nm emission falls in the so-called second
transparency window (1350 - 2300 nm) considered for imaging in biological tissues.
systems that use visible light [9,10]. Naczynski et al., reported real-time short-
wavelength infrared (1525 nm) in vivo imaging with anatomical resolution, by using
and 980 nm excitation [11]. Xianliang, et. al. studied SrY2O4:Er3+:Yb3+ [12]. Derén et
under excitation at 980 nm [13]. However no study has been conducted to investigate
the UC mechanism through 1550 nm excitation in this last host. The CaTiO3 host
exhibits favorable properties such as chemical stability (better than fluorides and
sulfides) and low phonons energy, approximately 700 cm-1 [14], that is lower than
YVO4 (890 cm-1) and LaPO4 (about 1050 cm-1) [15–17]. UC from compounds doped
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with Er3+ have shown potential application for biological tissue imaging/probing. In
general, trivalent rare earths ions (RE3+) display some advantages when compared to
organic dyes (OD) or quantum dots (QD), such as higher photostability, higher
chemical stability, lower toxicity, narrower emission bandwidth and longer lifetime of
Aiming its potential as cell staining and imaging, this study presents the
under excitation at 980 nm and 1550 nm, as well as some in vitro tests.
2. Experimental procedure
synthesized by sol-gel process. The starting materials used for the synthesis were
ethoxyethanol (Sigma-Aldrich - 99%) and ethanolic solutions of ErCl3 and YbCl3, both
0.1 mol L-1, obtained after dissolution of the respective oxides (Er2O3 and Yb2O3 -
Aldrich - 99,,99%) in hot HCl solution (1 mol L-l). The concentration of the RE3+
solutions was confirmed by titration with EDTA (0.01 mol L-1). Initially, a
Afterwards the RE3+ solutions were added and mixture was kept under stirring at room
temperature for 15 min. This solution was added slowly over the solution of Ti(OC4H9)4
mo L-1) was added. After 60 min. under stirring, the sol turned into a transparent gel.
After drying at 100 °C for 24 h, the obtained xerogels were grounded and transferred to
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alumina crucibles. The heat treatment was carried out at 1200 °C for 4h, at heating rate
2.2. Characterization
monochromator, scan speed of 0.75 deg min-1, and 2θ ranging from 20° to 80°. The
crystallites average size and the microstrain were calculated by Scherrer´s [22] and
TM-3000, Hitachi) was used to assess the influence of heat treatment on the
morphology of the samples. UC emission spectra (500-750 nm) were recorded with a
Spectra pro 300i Acton Research Co. spectrometer. Fiber-coupled diode lasers (FDL)
operating at 980 nm and 1550 nm were used for excitation. The emission spectra
between 1350 and 1700 nm were recorded with the same spectrometer and using a
Fetal Bovine Serum (FBS, Ingamed, Paraná, Brazil). Trypan Blue (Sigma Aldrich)
exclusion test was performed in all samples at 6, 12, 24 and 48 h of incubation at 37 ºC,
in order to evaluate the number of viable cells present in a cell suspension. The numbers
of viable and unviable cells were assessed using a haemocytometer (Neubauer chamber)
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interaction with bovine granulosa cells was analyzed. The cells were withdrawn from
fresh ovaries collected on a slaughterhouse. After the third passage, the isolated cells
were fixed using Formalin Neutral Buffer 4%wt (20 min, room temperature). Cells (2 x
106 per mL) were incubated in Hepes Medium (Ingamed, Paraná, Brazil), supplemented
with 10%wt of Fetal Bovine Serum (FBS, Ingamed, Paraná, Brazil) at 37C using four
washed three times with PBS (Phosphate-buffered saline). As negative control, cells
were also incubated only with PBS. Additionally, cell membrane dye (PKH- Sigma)
was used, according to the supplier's instructions. Analyses were performed using an
with a 60X objective. Excitation was performed by Ar+ laser 488 nm line. Besides
FACSaria) to set the percentage of dyeing cells, and the data are presented according to
heat treated at 1200 °C for 4 h. The diffraction peaks and the lattice parameters of the
unit cell (a = 5.439 Å, b = 7.642 Å, c = 5.384 Å) are in good agreement with the
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orthorhombic phase of CaTiO3 (ICSD 96258) belonging to the space group Pbnm.
Reflections related to a secondary phase around 2θ = 27°, which can be attributed to the
rutile phase of TiO2 (ICSD 62677), was also detected. It is known that for temperatures
above 800°C, that the metastable anatase phase of TiO2 irreversibly converts to rutile
phase. The presence of such secondary phase can be associated to the high rate of
photoluminescent emissions were not affected by the presence of this secondary phase.
that RE3+ were dissolved into CaTiO3 host. As the ionic radii of Ca2+ (rCa2+ = 1.34 Å;
coordination number = 12) [27], Er3+ (rEr3+ = 1.19 Å; coordination number = 12) and
Yb3+ (rYb3+ = 1.17 Å; coordination number = 12) [28] are similar, it is expected to RE3+
replace Ca2+ in the CaTiO3 structure. This behavior was also suggested by Mazzo et al.
[29]. That replacement is based on charge compensation, which happens due the event
´ •
of point defects, such as Ca2+ (VCa) and/or O2- vacancies (VO) [29–31].
corresponding to the Miller plane (121), was used to calculate the average crystallite
Kλ
Dhkl = (1)
βcosθ
where, Dhkl is the crystallite average size; K is the shape factor (0.89 for the present
diffraction angle of the most intense peak, and β stands for the full width at the half
maximum (FWHM) of the most intense peak. The β values were corrected in
accordance to Equation 2:
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2 2
β = 𝛽exp ‒ 𝛽inst(2)
where, βins tis the instrumental FWHM and βexp is the experimental FWHM of the most
intense peak. The values for instrumental broadening were obtained from XRD of a
silicon standard sample, with particle size of 325 meshes (44µm) (Shimadzu).
The crystallites sizes are listed in Table 1. There is an increase in the crystallites
size of about 20% from 1.0 to 1.5 mol% of Yb3+, after what it remains virtually
constant. This result may be explained by interactions between RE3+ and the surface of
the crystals, over nucleation and growth stages. Concentrations higher than 0.3 mol% of
Er3+ and 1.5 mol% of Yb3+ and heat treatment, may induce RE3+ clustering at the
surface of the crystallites, which interferers on the mechanism of crystal growth [32].
method. The microstrain for different concentrations of RE3+ was calculated using
Kλ
𝛽cos𝜃 = + 4𝜀sin𝜃 (3)
𝐷ℎ𝑘𝑙
where, Dhkl correspond to the average size of the crystallite; K is the shape factor, λ
stands for the wavelength of the X-ray photons (1.5418 Å), θ is the diffraction angle; β
CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0 mol%) samples, heat treated at 1200 °C
for 4h.
from 1.0 up to 2.0 mol%, which means such parameter is not sensitive for this range of
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Figure 2 shows SEM images for CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0
concentration.
1.0, 1.5 and 2.0 mol%) heat treated at 1200 °C for 4 h, from 500 to 750 nm under
excitation at 980 nm, with laser power ranging from 100 to 500 mW in steps of 100
mW. The emissions can be attributed to intraconfigurational f-f transitions of Er3+. The
2H 4 and 4S3/2→4I15/2 transitions between 510 and 580 nm (correspondent to the
11/2→ I15/2
green spectral range) are more intense than the 4F9/2→4I15/2 transition, around 660 nm,
which correspond to the red spectral range. No significant changes are detected either in
the relative intensities, or in the spectral profile as Yb3+ concentration goes higher.
heat treatment, which could leads to deactivation of Er3+ excited states by non-radiative
process. The intensity of UC emission bands are related to the power excitation
𝑛
according to the expression 𝐼 ∝ 𝑃 , where I is the integrated emission area, 𝑃 is the
power of the excitation source and 𝑛 is the number of photons involved in the excitation
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process [34]. According to the results the UC emission occurs by means of two-photon
absorption.
The energy levels diagram for Er3+ and Yb3+ and the mechanisms proposed to
UC process, under excitation at 980 nm, are shown in Figure 5a. The prevailing
Absorption (ESA) and Energy Transfer Upconversion (ETU). The first mechanism
involves the sequential absorption of two photons of 980 nm by the Er3+. This process
populates the 2H11/2, 4S3/2 and 4F9/2 upper energy levels, from which the characteristic
green and red emissions happen. In contrast, ETU is an electronic excitation process in
which energy transfer succeeds between two nearby rare earth ions. In such context,
ETU process starts with the absorption of 980 nm photons by Yb3+, once it shows
higher absorption cross-section at 980 nm than the Er3+. Next, energy transfer process
from 2F5/2 state of Yb3+ to higher energy levels of Er3+ occur giving rise to the emissions
in visible region.
corresponding spectra are shown in Figure 6. The red emission is more intense than the
green one. This result is opposite to that observed under excitation at 980 nm. This
behavior shows that UC mechanisms are dependent on the excitation wavelength. The
[7,8,11,35], as depicted on Figure 5b. Initially, Er3+ is excited to 4I13/2 energy level by
populates the 4I9/2 level. Furthermore, cross-relaxation (CR) process (4I13/2 + 4I13/2 →
4I + 4I15/2) can also populate the 4I9/2 level. From this point, two electronic excitation
9/2
processes can occur. In the first, the absorption of one photon from the 4I9/2 energy level
populates the 4F7/2 level, which decay by non-radiative processes to the lower 2H11/2 and
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4S
3/2 states. The radiative decays from these states give rise to emissions in green region
The second proposed mechanism is a non-radiative decay from4I9/2 level to the 4I11/2
level, followed by electronic excitation to 4F9/2 state. From this level, Er3+ returns to the
ground state by emitting red radiation. It is noteworthy that the energy gap between the
4I and 4I11/2 states is too small, about 2300 cm-1, and most of the ions in the 4I9/2 level,
9/2
rapidly decay to the 4I11/2 state by a multi-phonon relaxation process. Then, these ions
will be excited to the 4F9/2 state [7,8]. Therefore, the intensity of red emission is higher
than the green one due to higher electron population at the 4I11/2 level as compared with
the 4I9/2 level. Meanwhile,Yb3+ does not contribute to the photon absorption process
under 1550 nm excitation, since its single excited level (2F5/2), absorbs only photons at
980 nm. In addition, as shown in Figure 6, the increase in Yb3+ concentration causes a
energy transfer (ET) processes from 4I11/2 or 4I9/2 levels of Er3+ to the 2F5/2 state of Yb3+.
Such ET can be enhanced by reducing distances among the ions within the crystal
structure of CaTiO3, which point out to this process been more sensitive to RE3+
1.0, 1.5 and 2.0 mol%) xerogels, heat treated at 1200 °C for 4 h. The CIE diagrams were
composed by UC emission data, under excitation at 980 nm and 1550 nm. The x and y
color coordinates are presented in Table 2. They show a red shift for both excitation
wavelengths as Yb3+ goes higher, which was also detected by Yin et al., in the
compound Y2Ti2O7:Er3+:Yb3+ [11]. The numbers also reflect the UC spectra behavior
regarding the relative intensity for the green and red emissions, in which the green
emission is more intense than the red one under excitation at 980 nm, while the opposite
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Apart from the UC visible emissions, these compounds also showed broad
emission band in the near infrared region, around 1550 nm. Such emission is especially
important for applications as optical probing through the second window for in vivo
imaging (1350 - 2300 nm) in biological systems [10]. Figure 8 shows the room
temperature emission spectra from 1300 to 1700 nm, for xerogels heat treated at 1200°C
for 4 h, with excitation at 980 nm (300 mW). The band (composed by multiple peaks,
due to the energy level splitting cause by crystalline environment) has his maximum at
excitation of Yb3+ to its single excited level (2F5/2), followed by energy transfer from
4I of Er3+. Next, the electron from 4I11/2 level undergoes a non-radiative decay to the
11/2
lowest Er3+excited state (4I13/2), leading to that multicomponent emission with maximum
at 1519 nm [36]. Moreover, it is also possible to assess the effect of the Yb3+
concentration upon FWHM. The xerogel containing 1.5 mol% of Yb3+ shows the largest
FWHM values, approximately 8.0 nm, and the most intensity emission, relative to two
absorbs photons at 980 nm more efficiently. For 2.0 mol% of Yb3+, there is a drop in the
relative emission intensity and also to FWHM, which can be associated to concentration
quenching due RE3+ clustering. Therefore, in the present study,Yb3+1.5 mol% proved to
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it is necessary to assess if it is cytotoxic at any level. In this sense, Trypan Blue dye
exclusion assay, with respect to human embryonic kidney 293 cells (HEK-293), was
used and the results compared with cell-only control. Figure 9 shows that upon
incubation for 6, 12, 24 and 48 h, no significant toxic effects (p > 0.05) were observed
in the presence of 0-0.2 mg mL-1 of CaTiO3:0.3% Er3+:1.5Yb3+, i.e., cell viability was
similar (about 45%) for all concentrations, including the negative control. This result
indicates that the compound is suitable for in vitro cell staining experiments.
The potential of CaTiO3:0.3% Er3+:1.5% Yb3+ being used in cell staining was
explored by assessing interactions with bovine granulosa cells (the most common
microscopy. The images were collected under laser excitation at 488 nm and brightfield
(Figure 10). The 488 nm excitation populates the 4F7/2 energy level of Er3+ in the same
The images in Figure 10 (each blue or red dot represents a cell) shows that
CaTiO3:0.3% Er3+:1.5% Yb3+ was able to incorporate into the cytoplasm of granulosa
cells, demonstrated by Er3+ red emission (Figure 10a), while the negative control under
the same condition shows no emission at all (Figure 10b). At higher magnification,
(Figure 10 c and d), it is possible to see the red fluorescent dots completely scattered
into the cytoplasm, evidencing the cell uptake of the particles and allowing the
observation of the cellular morphology. The results illustrate the potential to use
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possible to claim that CaTiO3:0.3% Er3+:1.5%Yb3+ could be envisage for cell labeling.
The results showed it is not selective for a specific biomolecule, as revealed by the
images in Figure 10 c and d, and by a positive control (Figure 10 e and f), where a
The percentage of dyed cells was assessed through flow cytometry (Figure 11).
The results indicate that approximately 70% of granulosa cells were dyed with in
CaTiO3:0.3% Er3+:1.5% Yb3+. The fluorescence was only detected in APC-A filter (650
- 670 nm) and it was not observed for the control group, which means that CaTiO3:0.3%
Er3+. The green emission was not detected by flow cytometry, which indicates the
suppression of radiative emission from 2H11/2 and 4S3/2 levels. Organic groups, such as
COO-, -NH2, -CO, as well as water molecules and -OH groups, which are present in the
vibration frequencies. In this way, when the electrons are in the 2H11/2 and 4S3/2 states
part of the energy is transferred to the organic groups, and then, they decay non-
radiatively to the 4F9/2 state, from which the emission in the red region originates. Thus,
the properties presented by CaTiO3:0.3% Er3+:1.5% Yb3+ show its potential for in vitro
cell dyeing in biological systems, once it presents low cytotoxicity and emits in specific
spectral region, allowing, for instance, multiplex imaging. However, the specific sites of
interaction with the compound and the cellular components will be investigated, to
4. Conclusions
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structure is formed after CaTiO3 heat treatment at 1200 ºC for 4 h. The small amounts of
secondary phase do not influence the photoluminescent behavior of the samples. All
1550 nm. The UC emission mechanism under excitation at 980 nm was interpreted as a
photon process. The potential of CaTiO3:0.3% Er3+:1.5% Yb3+ as a cell inorganic dye
was assessed and the results indicated that the material is not cytotoxic, regarding HEK-
293 cells, and is able to incorporate and distribute into cell cytoplasm. Approximately
70% of the cells were stained, which suggest that CaTiO3:0.3% Er3+:1.5% Yb3+ presents
potential as an inorganic dye for in vitro cell dyeing. However, further studies must be
carried out, in order to understand the dynamics of interaction of the compound with the
cells, for future in vivo assays. Er3+ emission around 1550 nm was also observed and
CaTiO3:0.3% Er3+:1.5% Yb3+ proved to be the better concentration ratio, to explore the
biological systems.
Acknowledgments
This work was supported by the FAPEMIG; FINEP, CAPES; CNPq. This work
acknowledge Dra. Roseli Marins Balestra for SEM images and Dra Ana Maria de Paula
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Table contents
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x y
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Figure Captions
Figure 1. XRD patterns of CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0 mol%) heat
Figure 2. SEM images of CaTiO3:0.3%Er3+ co-doped with: (a) 1.0, (b) 1.5 and (c) 2.0
1200 °C for 4 h, with different concentrations of Yb3+: (a) 1.0, (b) 1.5 and (c) 2.0 mol%,
CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0 mol%), heat treated at 1200 °C for 4h.
Figure 5. Energy levels diagrams for Er3+ and Yb3+, depicting the characteristics ETU,
1.0, 1.5 and 2.0 mol%), heat treated at 1200 °C for 4 h xerogels.
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Figure 9. Cell viability (%) test over incubation time. The HEK-293 cells were
h.
(c) dark field under excitation at 488 nm; (d) brightfield of bovine granulosa cells
stained; (e, f) cell membranes dyed by PKH fluorescent dye. Bars in (a) and (b) = 100
Figure 11. Flow cytometry analysis: (a) amount of dyed cells (%) and (b) APC - A
filter used to detect the emission of Er3+ in CaTiO3:0.3%Er3+:1.5%Yb3+. The green color
control.
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Highlights
CaTiO3:0.3%Er3+:y%Yb3+. (y = 1.0, 1.5 and 2.0 mol%) obtained by the sol-gel process
The potential of CaTiO3:0.3% Er3+:1.5% Yb3+ as a cell inorganic dye was assessed;
The materials presents potential as an inorganic dye for in vitro cell dyeing.