Methods and Applications in Fluorescence
PAPER • OPEN ACCESS
NIR induced modulation of the red emission from erbium ions for
selective lanthanide imaging
To cite this article: Stefan Krause et al 2018 Methods Appl. Fluoresc. 6 044001
 
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Methods Appl. Fluoresc. 6 (2018) 044001 https://doi.org/10.1088/2050-6120/aadef1
PAPER
NIR inducedmodulation of the red emission from erbium ions for
selective lanthanide imaging
StefanKrause1,3 ,MadsKoerstz1 , RiikkaArppe-Tabbara1, Tero Soukka2 andTomVosch1,3
1 Nano-Science Center/Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100Copenhagen, Denmark
2 Department of Biotechnology, University of Turku,MedisiinaD6, Kiinamyllynkatu 10, 20520Turku, Finland
3 Authors towhomany correspondence should be addressed.
E-mail: stefan.krause@chem.ku.dk and tom@chem.ku.dk
Keywords: lanthanides, photophysics, upconversion, luminescencemodulation, imaging
Supplementarymaterial for this article is available online
Abstract
Upon direct excitationwith green light (522 nm), Er3+ ion doped nanoparticles feature a number of
radiative and non-radiative decay pathways, leading to distinct and sharp emission lines in the visible
and near-infrared (NIR) range. Herewe apply, in addition to continuous 522 nm irradiation, a
modulatedNIR irradiation (1143 nm) to actively control andmodulate the red emission intensity
(around 650 nm). Themodulation of red Er3+ ion emission at a chosen frequency allows us to
reconstruct fluorescence images from the Fourier transform amplitude at this particular frequency.
Since only the emission from the Er3+ ion ismodulated, it allows to selectively recover the lanthanide
specific signal, removing any non-modulated auto-fluorescence or background emission resulting
from the continuous 522 nmexcitation. Themodulated emission of specific lanthanides can open up
newdetection opportunities for selective signal recovery.
Introduction
Lanthanide ion-based emitters have enabled a variety
of new methods for optical investigations [1–6]. In
upconversion nanoparticle materials, the sequential
absorption of multiple photons leads to an anti-Stokes
emission signal—referred to as upconversion [7].
Hence, autofluorescence background appearing on
the Stokes side of the excitation can be easily
suppressed. Due to the small absorption cross-section
of the lanthanide-ions, a large number must be
combined in a nanoparticle scaffold typically consist-
ing of NaYF4 in order to create detectable emission [4,
8–12]. Sensitizing these particles with, e.g. ytterbium
or organic dyes, further increases the absorption
probability [13–16]. The resulting upconversion nano
particles (UCNPs) have beenwidely applied in numer-
ous fields of research, such as emissive labels
[3, 17, 18], photodetectors [19], multiplexing [20],
molecular sensing [21], immunoassays [22], energy
transfer [23–26], nanoscopy [27, 28], photodynamic
therapy [29], cross-correlation spectroscopy [30],
photoacoustic imaging [31] and thermometry
[32–35]. The sharp emission and absorption lines of
lanthanide ions are often advantageous and in combi-
nation with the long excited state lifetimes, specific
excited states can be populated by excitation engineer-
ing [36, 37]. Moreover, it has been shown that
depletion of specific lanthanide excited states is
possible and can be exploited for optical nanoscopy
[27, 28, 38].
Here we demonstrate for the first time a combina-
tion of direct excitation in the visible range and addi-
tional NIR excitation to enhance andmodulate the red
emission of Er3+ UCNPs. We demonstrate the poten-
tial of this approach for emission modulation-based
imaging by recovering the selective lanthanide signal
from environments containing bright organic fluor-
ophores. This selective emission modulation at a con-
stant frequency allows for efficient discrimination of
the non-modulated background [39–42].
Results and discussion
Details on the Er3+ UCNPs synthesis and size char-
acterization can be found in the supporting informa-
tion and figure S1 which are available online at stacks.
OPEN ACCESS
RECEIVED
25 June 2018
REVISED
24August 2018
ACCEPTED FOR PUBLICATION
5 September 2018
PUBLISHED
21 September 2018
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iop.org/MAF/6/044001/mmedia. Figure 1(a) shows
the electronic energy diagram of the Er3+ ions and the
decay pathways that occur upon direct excitation with
522 nm light [43, 44]. The 522 nm laser excites Er3+
from the ground state to the 2H11/2 state from which
the Er3+ ions undergo non-radiative transitions to the
subsequent energy levels 4S3/2 and
4F9/2. Besides non-
radiative transitions, two emission lines appear on the
Stokes side in the visible range at about 550 nm
(4S3/2–
4I15/2) and 660 nm (
4F9/2–
4I15/2) [43]. Addi-
tional emission lines, such as theNIR emission around
810 nm (4I9/2–
4I15/2), 850 nm (
4S3/2–
4I13/2), 980 nm
(4I11/2–
4I15/2) and 1530 nm (
4I13/2–
4I15/2), should also
be present [43, 44]. The 4S3/2–
4I13/2 transition is of
particular importance for populating the 4I13/2 state
which we will use to modulate the red emission. In
order to increase and, hence, modulate the red
emission of the 4F9/2–
4I15/2 transition, we have chosen
a secondary NIR wavelength of 1143 nm that can
optically pump the Er3+ ions back from the 4I13/2 to
the 4F9/2 excited state (See figure 1(b)). In accordance
with the electronic diagram proposed by Anderson
et al and Würth et al this repopulation of 4F9/2 from
4I13/2 should not have an effect on the 850 nm
emission (4S3/2–
4I13/2) [43, 44]. The 810 nm emission
(proposed to originate from energy transfer ET1 of
4S3/2–
4I13/2 and
4I15/2–
4I9/2 and energy transfer ET2
of 4I13/2–
4I9/2 and
4I13/2–
4I15/2 in figure 1(a)) [43, 44]
might be affected if ET2 and the depopulation of 4I13/2
contributes significantly to the 810 nm emission. We
measured the 810 nm and 850 nm emission as can be
seen in figure S2. The intensity of the 850 nm and
810 nm emission remains unchanged upon NIR
modulation which indicates that ET2 does not sig-
nificantly contribute to the 810 nm emission or is not
affected by NIR modulation. Besides emission on the
Stokes side, the 522 nm excitation also generates
emission on the anti-Stokes side (see figure S3),
however this is not of interest for this paper. In order
to check the efficiency by which the Er3+ UCNPs can
be pumped back from 4I13/2 to
4F9/2, we recorded
spectra with and without the additional 1143 nm light
(See figure 1(c)) of a sample of Er3+UCNPs on a glass
coverslip. Figure 1(c) shows that at a secondary NIR
excitation intensity of 15 kW cm−2, the emission of
the 660 nm transition increases more than twofold,
while the spectral shape remains unchanged. The
amount of additional 660 nm emission is dependent
on the decay time of the 4I13/2 state (4.4 ms in D2O)
and the intensity of the 1143 nm laser [44]. The latter
Figure 1. (a)Energy diagramof Er3+ ions and decay pathways upon direct excitationwith 522 nm light. (b) Same as (a)with an
additional excitationwavelength of 1143 nm.Green and red arrows represent the transitionsmonitored in figure 1(c). Cyan and the
black upward straight arrow represent the 522 nmprimary and 1143 nm secondary excitationwavelength, respectively. The black
downward arrow represents the 850 nm radiative transition from 4S3/2–
4I13/2 and the 810 nm radiative transition from
4I9/2–
4I15/2.
The pairs of dashed and dotted lines represent energy transfer processes that can occur inUCNPs (ET1 and ET2) (c)Emission spectra
of 4S3/2–
4I15/2 and the
4F9/2–
4I15/2 transition of Er
3+ ions with (red) andwithout (black) 1143 nmNIR secondary excitation. (d)NIR
excitation power dependency of the red 4F9/2–
4I15/2 transition integrated over the spectral range from645 to 675 nm. The solid line
represents a linearfit to the data. The black dashed vertical line indicates the approximate beginning of the deviation from linearity.
2
Methods Appl. Fluoresc. 6 (2018) 044001 SKrause et al
was proven by changing the 1143 nm laser intensity
while measuring the 660 nm emission as shown in
figure 1(d). The dependency follows a linear trend up
to about a NIR laser intensity of 12 kW cm−2. After-
wards, the emission intensity seems to deviate from
linearity, which might indicate saturation of the
optical pumping. Furthermore, the unchanged green
emission intensity in figure 1(c) shows that we observe
direct optical pumping and not amultiphoton absorp-
tion process. Additional factors like temperature,
particle size and composition will also have an effect
on the observed emission of the UCNPs, but they were
not the focus of this investigation [34, 35].
After we demonstrated spectrally that our proof of
principle works, we continued with measuring the
integrated intensity of the red emission shown in
figure 1(c) with a long pass/short pass filter combina-
tion (creating a transmission window from 633 nm to
750 nm) on an avalanche photo diode (APD) as a func-
tion of time. During the measurements, the 1143 nm
laser light was modulated on and off by a chopper
wheel with adjustable frequency. The resulting inten-
sity time traces can be seen in figure 2(a) for fre-
quencies of 50, 100 and 200 Hz. The ratio between red
emission intensities measured with and without
1143 nm irradiation is similar as in the experiment in
figure 1(c). From the time traces, we calculated the
Fourier transform which is displayed in figure 2(b). As
expected, a clear peak in the Fourier transform spec-
trum can be seen at the frequency that coincides with
the frequency settings of the chopper wheel. Although
it is possible to modulate the red emission of the Er3+
ions at even higher frequencies, problems might arise
when approaching a frequency that is close to the
inverse of the transit time from 2H11/2–
4I13/2
[36, 37, 45].
In order to evaluate the Er3+modulation for selec-
tive lanthanide signal recovery and imaging, we pre-
pared a sample which consisted on one side of Er3+
UCNPs embedded in polystyrene (PS) and on the
other side of an organic fluorophore (ATTO 633)
embedded in polyvinyl alcohol (PVA). A scheme of the
sample is shown in figure 3(a). A piezo scanner was
used to raster scan the sample while acquiring single
photonmacro times (time elapsed since the start of the
experiment) for each detected single photon with
time-correlated single photon counting hardware. In
this example, the sample was excited with the 522 nm
primary and the 100 Hz modulated 1143 nm second-
ary excitation wavelength. The intensity image in the
spectral bandpass range (633 nm–750 nm) can be seen
in figure 3(b) and shows that the ATTO 633 dye (on
the left side) displays a homogeneous and, on average,
brighter emission than the Er3+ UCNPs (on the right
side). Since the emission of the ATTO 633 dye is not
modulated by the 1143 nm laser, removal of this
‘unwanted’ emission can be achieved by constructing
an image of the amplitude of the 100 Hz contribution
in the FT spectrum. An example of the intensity time
trace and FT spectrum at each pixel (time the scanner
moves over one pixel is 0.4 s) is given in figure 3(c). For
image reconstruction the maximum FT amplitude in
the range from 95 to 105 Hz was determined and plot-
ted. The resulting FT amplitude image can be seen in
figure 3(d) and shows that the amplitude of the 100 Hz
modulation image recovers selectively only the red
emission of the Er3+ ions, with complete removal of
the ATTO 633 emission. Scan time per pixel andmod-
ulation frequency will determine the total time and
quality of the FT amplitude image.
Figure 2. (a) Intensity time traces of Er3+ doped nanoparticle emissionmodulated at frequencies 50, 100 and 200 Hz. The binwidths
have been chosen to be one tenth of the chopping periodicity. (b)Amplitudes of the Fourier transforms of the signals shown in (a) as a
function of frequency.
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Methods Appl. Fluoresc. 6 (2018) 044001 SKrause et al
Conclusion
We have demonstrated the use of multiple excitation
wavelengths for manipulating the 4F9/2 and
4I13/2
excited states in Er3+ UCNPs. This allowed us to
selectively modulate the amplitude of the red emission
from the 4F9/2 excited state. Plotting of the corresp-
onding Fourier transform amplitude of the pixel-wise
acquired, modulated time traces allowed for selective
lanthanide signal recovery and removal of ‘unwanted’
background emission. Application of more powerful
visible and NIR laser systems will allow for expanding
the presented approach towide-fieldmicroscopy. This
will enable for fast selective lanthanide signal recovery
and imaging on the seconds time-scale. Our work
shows how active control of excited state populations
in lanthanides can open up new imaging and sensing
applications for this interesting class of elements.
Acknowledgments
We gratefully acknowledge financial support from the
‘Center for Synthetic Biology’ at Copenhagen Univer-
sity funded by the UNIK research initiative of the
Danish Ministry of Science, Technology and Innova-
tion (Grant 09-065274), bioSYNergy, University of
Copenhagen’s Excellence Programme for Interdisci-
plinary Research, the Villum Foundation (Project
number VKR023115), the Carlsberg Foundation
(CF14-0388), the Danish Council of Independent
Research (Project numberDFF-7014-00027).
ORCID iDs
StefanKrause https://orcid.org/0000-0002-
7062-8472
MadsKoerstz https://orcid.org/0000-0001-
5813-6232
TomVosch https://orcid.org/0000-0001-
5435-2181
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