Accepted Manuscript

CaTiO3:Er3+:Yb3+ Upconversion from 980 nm and 1550 nm Excitation and its

Potential as Cells Luminescent Probes

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/[Link].2018.11.018

Reference: MAC 21102

To appear in: Materials Chemistry and Physics

Received Date: 14 August 2018

Accepted Date: 10 November 2018

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/[Link].2018.11.018

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 ACCEPTED MANUSCRIPT

CaTiO3:Er3+:Yb3+ Upconversion from 980 nm and 1550 nm Excitation

and its Potential as Cells Luminescent Probes

R.V. Perrellaa,I. C. Ribeiroa, P.H.A. Campos-Juniorb, M.A. Schiavona,E. Pecoraroc,

S.J.L. Ribeiroc, J.L. Ferrari a,d*

a – Grupo de Pesquisa em Química de Materiais – (GPQM), Departamento de Ciências

Naturais, Universidade Federal de São João del Rei (UFSJ), Campus Dom Bosco, Praça
Dom Helvécio, 74, 36301-160, São João del Rei, MG, Brazil

b –Departamento de Ciências Naturais, Universidade Federal de São João del Rei

(UFSJ), Campus Dom Bosco, Praça Dom Helvécio, 74, 36301-160, São João del Rei,
MG, Brazil

c – UNESP, Institute of Chemistry, P.O. Box 355, 14800-970, Araraquara, SP, Brazil

d –Desenvolvimento de Materiais Inorgânicos com Terras Raras (DeMITeR), Instituto

de Química – (IQ), Universidade Federal de Uberlândia – (UFU), Av. João Naves de
Ávila, 2121 – Bairro Santa Mônica, CEP: 38400-902, Uberlândia, MG, Brazil

*Prof. Dr. Jefferson Luis Ferrari (Group’s Head)

Desenvolvimento de Materiais Inorgânicos com Terras Raras (DeMITeR)
Laboratório de Materiais Fotoluminescentes (LAMAF)
Universidade Federal de Uberlândia
Instituto de Química – Bloco 1D
Av. João Naves de Ávila, 2121 – Santa Mônica
Uberlândia – MG, Brazil
Tel.: +55 34 3391-8349
E-mail address: jeffersonferrari@[Link], jeffersonferrari@[Link]

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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

a function of rare earths concentration through XRD and SEM techniques.

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

CaTiO3:Er3+:Yb3+ were successfully applied as animals cells cytoplasm staining

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

window (1350 - 2300 nm).

Keywords: calcium titanate; erbium; ytterbium; upconversion; fluorescent cell dyeing

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1. Introduction

Luminescent upconversion nanoparticles (UCNP) have been investigated for

applications in solid state lasers, temperature sensors, photodynamic therapy, and

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

nm and 1550 nm) leading to color tuning.

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.

Improvements in radiation penetrability and resolution is observed compared with

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

NaYF4:Er3+@NaYF4 core-shell nanostructures, and demonstrated their applicability

toward disease-targeted diagnostic [9].

Yin et al. investigated the UC mechanisms in Y2Ti2O7:Yb3+:Er3+ under 1550 nm

and 980 nm excitation [11]. Xianliang, et. al. studied SrY2O4:Er3+:Yb3+ [12]. Derén et

al. showed intense green UC emission in CaTiO3:Er3+, prepared by sol-gel process

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

excited states [18–21]. Moreover UC emission not involving UV excitation, which

causes autofluorescence of endogenous compounds from biological tissues.

Aiming its potential as cell staining and imaging, this study presents the

synthesis of CaTiO3:Er3+:Yb3+ by sol-gel process and the UC emission mechanisms

under excitation at 980 nm and 1550 nm, as well as some in vitro tests.

2. Experimental procedure

2.1. Synthesis of CaTiO3:x%Er3+:y%Yb3+

CaTiO3:x%Er3+:y%Yb3+ (x = 0.3 mol%; y = 1.0, 1.5 and 2.0 mol%) were

synthesized by sol-gel process. The starting materials used for the synthesis were

CaCl2.2H2O (Vetec - 99%), tetrabutyl orthotitanate (Ti(OC4H9)4 - Fluka - ≥ 97,,0%), 2-

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

stoichiometric amount of CaCl2.6H2O was dissolved in 10 mL of 2-etoxyethanol.

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

also dissolved in 10 mL of 2-ethoxyethanol. To this final mixture, 200µL of HCl (0.27

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

of 5 °C min-1 and air atmosphere operating an EDG 1300 furnace.

2.2. Characterization

Samples in powder form were characterized by X-ray diffraction (XRD), using a

Shimadzu XRD 6000 diffractometer, with Cu Kα radiation λ = 1.5418 Å, graphite

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

Williansom-Hall´s equations [23], respectively. Scanning Electron Microscopy (SEM,

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

InGaAs detector sensitive from 800 to 1700 nm.

2.3. HEK-293 cells viability in the presence of CaTiO3:0.3%Er3+:1.5%Yb3+

HEK-293 (2 x 106 cells per mL) cells were incubated with

CaTiO3:0.3%Er3+:1.5%Yb3+ at different concentrations (0 - control, 0.05, 0.1 and 0.2

mg mL-1) in Hepes Medium (Ingamed, Paraná, Brazil), supplemented with 10%wt of

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)

as previously described [24].

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2.4. Cell staining analysis

To evaluate the feasibility of CaTiO3:0.3%Er3+:1.5%Yb3+ as cell probe, the

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

well petri dishes with 0.10 [Link]-1 of CaTiO3:0.3%Er3+:1.5%Yb3+ for 12 h, and

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

Olympus FV300 confocal system in an up-right configuration (BX1WI microscope),

with a 60X objective. Excitation was performed by Ar+ laser 488 nm line. Besides

confocal microscopy, quantitative analyses were performed using cytometer (BD

FACSaria) to set the percentage of dyeing cells, and the data are presented according to

each detection filter.

3. Results and discussions

3.1. Structural and Morphological Characterization

Figure 1 shows XRD patterns obtained for CaTiO3:0.3%Er3+:y%Yb3+ samples

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

hydrolysis of titanium alkoxide [25,26]. Nevertheless, it is important to note that the

photoluminescent emissions were not affected by the presence of this secondary phase.

No crystalline phases corresponding to Er2O3 or Yb2O3 were detected, which indicates

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].

The broadening of the most intense diffraction peak, centered at 2θ = 33.22°,

corresponding to the Miller plane (121), was used to calculate the average crystallite

size of the co-doped samples. According to Scherrer's equation (Equation 1) [22]:

Kλ
Dhkl = (1)
βcosθ
where, Dhkl is the crystallite average size; K is the shape factor (0.89 for the present

study); : λ correspond to the wavelength of X-ray photons (1.5418 Å); θ is the

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].

The crystalline microstructure was further investigated using Williamson-Hall

method. The microstrain for different concentrations of RE3+ was calculated using

Equation (3), known as the Williamson-Hall´s equation [23]:

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

corresponds to FWHM (corrected by equation 2) of the diffraction peaks and 

represents microstrain. Table 1 shows the results of microstrain calculations for

CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0 mol%) samples, heat treated at 1200 °C

for 4h.

There is no significant variation on microstrain as Yb3+ concentration increases

from 1.0 up to 2.0 mol%, which means such parameter is not sensitive for this range of

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concentration. The intrinsic results could be related to charge compensation process,

when a RE3+ replaces Ca2+.

Figure 2 shows SEM images for CaTiO3:0.3%Er3+:y%Yb3+ (y = 1.0, 1.5 and 2.0

mol%) heat treated at 1200 °C for 4 h. The micrographs show aggregates of

microparticles as a result of the sintering process [33]. In addition, there are no

significant differences on morphology and particle size distribution regarding to Yb3+

concentration.

3.2. Upconversion emission under excitation at 980 nm and 1550 nm

Figure 3 shows the UC emission spectra for CaTiO3:0.3%Er3+:y% of Yb3+ (y =

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.

Thus, it is suggests that no relevant clustering of RE3+ is induced by concentration or

heat treatment, which could leads to deactivation of Er3+ excited states by non-radiative

energy transfer processes.

The UC log–log plots of intensity dependence are shown in Figure 4, which

provide important information about the number of photons involved on UC emission

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

mechanisms proposed for CaTiO3:0.3%Er3+:y%Yb3+ UC emissions are: Excited State

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.

The UC emissions were also detected by excitation at 1550 nm and the

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

UC phenomenon under excitation at 1550 nm is a three-photon absorption process

[7,8,11,35], as depicted on Figure 5b. Initially, Er3+ is excited to 4I13/2 energy level by

absorbing a1550 nm photon. Then, a second 1550 nm photon is absorbed which

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

suppression of UC emission intensity. This condition may indicate the presence of

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+

clustering than that at 980 nm .

Figure 7 shows the CIE chromaticity diagram for CaTiO3:0.3%Er3+:y%Yb3+ (y =

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

is observed under excitation at 1550 nm.

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3.3. Emission at 1550 nm

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

1519 nm, and is assigned to 4I13/2 → 4I15/2 transition of Er3+.

The mechanism involved in such emission can be summarized by electronic

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

remaining samples. As for UC mechanisms, Yb3+ may be acts as a sensitizer and

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

be the highest concentration indicates to explore the emission at 1519 nm in

CaTiO3:0.3%Er3+:y%Yb3+ heat treaded xerogels.

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3.4. Cell Viability Analysis

To explore Er3+:Yb3+ co-doped CaTiO3 as a potential inorganic cell staining, first

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.

3.5. Cell staining

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

neoplasm involving the ovaries of cattle) by means of confocal fluorescence

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

way as two 980 nm or three 1550 nm photons.

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

CaTiO3:0.3%Er3+:1.5% Yb3+ for in vitro cytoplasm staining. At this point, it is not

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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

selective cell membrane dye (PKH) was used.

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+:1.5% Yb3+ dyed cells, show only red emission from 4F

9/2 → 4I15/2 transition of

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

intracellular environment, probably act as fluorescence-quenching centers, due to their

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

explore further the possibilities of CaTiO3:Er3+:Yb3+ for cell labeling.

4. Conclusions

CaTiO3:0.3%Er3+:y%Yb3+. (y = 1.0, 1.5 and 2.0 mol%) were successfully

obtained by the sol-gel process. As shown by XRD analyses, orthorhombic lattice

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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

heat-treated xerogels obtained displayed UC emission under excitation at 980 nm and

1550 nm. The UC emission mechanism under excitation at 980 nm was interpreted as a

result of two-photon absorption process, while at 1550 nm it was proposed as a three-

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

emission at 1550 nm on these xerogels, aiming for applications on optical imaging of

biological systems.

Acknowledgments

This work was supported by the FAPEMIG; FINEP, CAPES; CNPq. This work

is a collaboration research project between members of the RedeMineira de Química

(RQ-MG) supported by FAPEMIG (Project: CEX - RED-00010-14). The authors also

acknowledge Dra. Roseli Marins Balestra for SEM images and Dra Ana Maria de Paula

(UFMG) for confocal imaging.

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Table contents

Table 1. Crystallite size and microstrain data for CaTiO3:0.3%Er3+:y%Yb3+, calculated

by Scherrer and Williamson-Hall equations, respectively.

1.0 mol% Yb3+ 1.5 mol% Yb3+ 2.0 mol% Yb3+

Crystallite size (nm) 57.9 ± 2.9 69.2 ± 3.5 65.9 ± 3.3

Microstrain (x10-3) 2.7 ± 0.5 3.0 ± 0.4 3.0 ± 0.2

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Table 2. Values of x and y coordinates, calculated based on the CIE chromaticity

diagram for CaTiO3:0.3%Er3+:y% Yb3+ (y = 1.0, 1.5 and 2.0 mol%)

Excitation (nm) Yb3+ concentration (mol%) Coordinates

x y

1.0 0.244 0.736

980 1.5 0.251 0.727

2.0 0.266 0.714

1.0 0.598 0.394

1550 1.5 0.587 0.404

2.0 0.645 0.344

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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

treated at 1200 °C for 4 h.

Figure 2. SEM images of CaTiO3:0.3%Er3+ co-doped with: (a) 1.0, (b) 1.5 and (c) 2.0

mol% of Yb3+ heat treated at 1200 °C for 4 h.

Figure 3. UC emission spectra of CaTiO3:0.3%Er3+:y%Yb3+ samples heat treated at

1200 °C for 4 h, with different concentrations of Yb3+: (a) 1.0, (b) 1.5 and (c) 2.0 mol%,

under excitation at 980 nm.

Figure 4. 980 nm excitation intensity dependence on UC emission for

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,

ESA and UC emissions in CaTiO3:0.3%Er3+:y%Yb3+, under excitation at (a) 980 nm

and (b) 1550 nm.

Figure 6. UC emission spectra excited at 1550 nm for CaTiO3:0.3%Er3+:y%Yb3+ (y =

1.0, 1.5 and 2.0 mol%), heat treated at 1200 °C for 4 h xerogels.

Figure 7. CIE chromaticity diagrams for CaTiO3:0.3%Er3+:y%Yb3+ xerogels from UC

spectra under excitation at (a) 980 nm and (b) 1550 nm.

Figure 8. Room temperature near infrared emission spectra of

CaTiO3:0.3%Er3+:y%Yb3+ heat treaded xerogels, under excitation at 980 nm (300 mW).

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Figure 9. Cell viability (%) test over incubation time. The HEK-293 cells were

incubated along with 0-0.2 mg mL-1 of CaTiO3:0.3%Er3+:1.5%Yb3+ for 6, 12, 24 and 48

h.

Figure 10. Confocal images of (a) CaTiO3:0.3%Er3+:1.5%Yb3+; (b) negative control;

(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

µm; (c), (d), (e) and (f) = 5 µm.

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

correspond to CaTiO3:0.3%Er3+:1.5%Yb3+ and the purple color is related to the negative

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

 Materials displayed UC emission under excitation at 980 nm and 1550 nm.

 The potential of CaTiO3:0.3% Er3+:1.5% Yb3+ as a cell inorganic dye was assessed;

 0% of the cells were stained;

 The materials presents potential as an inorganic dye for in vitro cell dyeing.