applied
sciences
Article
Improved Imaging of Magnetically Labeled Cells
Using Rotational Magnetomotive Optical
Coherence Tomography
Peter Cimalla 1,2 , Julia Walther 1,3 , Claudia Mueller 4 , Seba Almedawar 2 , Bernd Rellinghaus 5 ,
Dierk Wittig 4 , Marius Ader 2 , Mike O. Karl 2,6 , Richard H. W. Funk 4 , Michael Brand 2 and
Edmund Koch 1, *
1
2
3
4
5
6
*
Anesthesiology and Intensive Care Medicine, Clinical Sensoring and Monitoring,
Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany;
[email protected] (P.C.); [email protected] (J.W.)
DFG-Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden,
01307 Dresden, Germany; [email protected] (S.A.); [email protected] (M.A.);
[email protected] (M.O.K.); [email protected] (M.B.)
Department of Medical Physics and Biomedical Engineering, Faculty of Medicine Carl Gustav Carus,
Technische Universität Dresden, 01307 Dresden, Germany
Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden,
01307 Dresden, Germany; [email protected] (C.M.); [email protected] (D.W.);
[email protected] (R.H.W.F.)
Institute for Metallic Materials, Leibniz Institute for Solid State and Materials Research,
01069 Dresden, Germany; [email protected]
German Center for Neurodegenerative Diseases (DZNE), 01307 Dresden, Germany
Correspondence: [email protected]; Tel.: +49-351-458-6131
Academic Editor: Michael Pircher
Received: 20 January 2017; Accepted: 14 April 2017; Published: 27 April 2017
Abstract: In this paper, we present a reliable and robust method for magnetomotive optical coherence
tomography (MM-OCT) imaging of single cells labeled with iron oxide particles. This method
employs modulated longitudinal and transverse magnetic fields to evoke alignment and rotation of
anisotropic magnetic structures in the sample volume. Experimental evidence suggests that magnetic
particles assemble themselves in elongated chains when exposed to a permanent magnetic field.
Magnetomotion in the intracellular space was detected and visualized by means of 3D OCT as well
as laser speckle reflectometry as a 2D reference imaging method. Our experiments on mesenchymal
stem cells embedded in agar scaffolds show that the magnetomotive signal in rotational MM-OCT
is significantly increased by a factor of ~3 compared to previous pulsed MM-OCT, although the
solenoid’s power consumption was 16 times lower. Finally, we use our novel method to image
ARPE-19 cells, a human retinal pigment epithelium cell line. Our results permit magnetomotive
imaging with higher sensitivity and the use of low power magnetic fields or larger working distances
for future three-dimensional cell tracking in target tissues and organs.
Keywords: optical coherence tomography; cell imaging; dynamic contrast agents; magnetic particles;
laser speckle; fluorescence microscopy
1. Introduction
Magnetomotive optical coherence tomography (MM-OCT) is a promising imaging method
for noninvasive three-dimensional (3D) tracking of magnetically labeled cells in target tissues or
organs [1–3]. In this situation, selected cells are labeled with magnetic micro- or nanoparticles as
Appl. Sci. 2017, 7, 444; doi:10.3390/app7050444
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Appl. Sci. 2017, 7, 444
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dynamic contrast agents, which are excited into motion by a modulated external magnetic field.
By analysis of OCT signal alterations, this motion can be detected and visualized for enhanced contrast
of labeled cells.
In conventional MM-OCT, the external magnetic field is generated by a solenoid close to the
sample. Ideally, magnetomotion is characterized by particle displacement towards the solenoid when
the current is switched on, and return of the particle to its initial position when the current is switched
off. Hence, besides the magnetic force, also the restoring force of the elastic tissue environment is
essential for the retrieval of appropriate magnetomotive signals. To expand MM-OCT to applications
and environments where no elastic restoring force is present, such as in liquids, dual-coil configurations
were introduced, in which the particles are attracted alternately by a primary and an opposite secondary
coil on a common axis [4]. Such a setup would also be convenient for cell imaging, since cells are
characterized by viscoelastic properties rather than a true elastic behavior [5]. However, because of the
limited space between the two solenoids, this method is restricted to small samples only. Therefore,
we introduce an alternative method called rotational magnetomotive (rMM)-OCT which uses two
magnets on the same side of the sample but with two different axes. This off-axis configuration allows
the generation of modulated longitudinal and transverse magnetic fields, which cause magnetomotion
by the alignment and rotation of anisotropic magnetic particles in the sample volume.
For our experiments, we used a multimodal imaging system presented recently by our group [6].
This system allows MM-OCT imaging in combination with light microscopy and laser speckle
reflectometry for structural and motion analysis of single cells. For the implementation of rMM-OCT,
we added a small permanent magnet to the existing setup to generate the transverse magnetic field.
After analysis of magnetic field components and particle behavior, we provide proof-of-principle
evidence for rMM-OCT imaging of single mesenchymal stem cells (MSCs) embedded in agar scaffolds
and demonstrate the magnetomotive signal enhancement compared to previous pulsed MM-OCT.
Finally, we use our method to image ARPE-19 cells [7], a human retinal pigment epithelium (RPE) cell
line, in order to demonstrate the potential of rMM-OCT to track therapeutically relevant cell types,
such as RPE cells transplanted into the retina [8,9].
2. Materials and Methods
2.1. Experimental Setup for MM-OCT
All experiments were carried out with a multimodal imaging system for MM-OCT, laser speckle
reflectometry and light microscopy, which was presented recently by our group [6]. In brief, the
setup shown in Figure 1 consists of a conventional epi-fluorescence microscope (Leica DMRB,
Leica Microsystems GmbH, Wetzlar, Germany) for bright-field transmission and multi-color
fluorescence microscopy, an integrated MM-OCT probe including an electromagnet for magnetic
field generation, and a dark-field laser illumination for laser speckle imaging. The setup allows parallel
visualization of the same cells by means of two-dimensional (2D) light microscopy and 3D OCT, as well
as investigation of sample motion by means of laser speckle variance analysis. Laser illumination of the
sample is generated by a fiber-coupled laser diode at 638 nm (Lasiris PTL-500-635-1-2.0, Coherent Inc.,
Santa Clara, CA, USA) with an output power of 1 mW. Light microscopy and laser speckle images are
detected by a video camera (1.3 megapixel monochrome CMOS, Sumix SMX-M71M, Sumix, Oceanside,
CA, USA) mounted on the microscope. The microscope-integrated OCT probe is fiber-coupled to a
self-developed spectral domain OCT system operating at 880 nm [10]. This system is equipped with
a superluminescent diode (Exalos EXS8810-2411, Exalos AG, Schlieren, Switzerland) with a center
wavelength of 876 nm and a spectral bandwidth of 64 nm (FWHM), and a linear-in-wavenumber
spectrometer. In summary, the system allows OCT imaging at an axial depth scan (A-scan) rate of
11.9 kHz with an axial resolution of 6.7 µm in air, an optical imaging depth of 3 mm and a sensitivity
of −102 dB.
Appl. Sci. 2017, 7, 444
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The
electromagnet
Appl. Sci.
2017,
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for magnetic field generation (Intertec Components GmbH, ITS-MS 5030
3 of 12
10
VDC, Hallbergmoos, Germany) is attached to the distal end of the OCT probe with parts of the OCT
imaging optics
optics incorporated
incorporated into
into aa center
center bore
bore of the solenoid’s
solenoid’s ferromagnetic
ferromagnetic core. With
With a working
imaging
distance of
and
an imaging
numerical
aperture
(NA) of
0.1 yielding
a theoretical
lateral resolution
distance
of66mm
mm
and
an imaging
numerical
aperture
(NA)
of 0.1 yielding
a theoretical
lateral
of
3.3
µm,
this
MM-OCT
probe
allows
observation
of
single
cells
in
a
three-dimensional
tissue-like
resolution of 3.3 µm, this MM-OCT probe allows observation of single cells in a three-dimensional
environment.
The field ofThe
view
of of
theview
OCTofprobe
in the
focalinplane
is 1.5plane
mm in
tissue-like
environment.
field
the OCT
probe
the focal
is diameter.
1.5 mm in diameter.
Figure 1.
1. Multimodal
Multimodal imaging
imaging setup
setup for
for magnetomotive
magnetomotive OCT
OCT (MM-OCT),
(MM-OCT), laser
laser speckle
speckle reflectometry
reflectometry
Figure
and light
light microscopy.
microscopy. (a)
(a) Light
Light microscope
microscope with
with integrated
integrated MM-OCT
MM-OCT probe
probe and
and dark-field
dark-field laser
laser
and
illumination
for
laser
speckle
imaging.
The
microscope
is
equipped
with
a
halogen
lamp
for
bright-field
illumination for laser speckle imaging. The microscope is equipped with a halogen lamp for bright-field
transmission imaging
imaging and
and aa mercury-vapor
mercury-vapor lamp
lamp for
for epi-fluorescence
epi-fluorescence imaging;
imaging; (b)
(b) the
the inset
inset shows
shows
transmission
a
detailed
view
of
the
sample
region
and
the
OCT
scanning
unit
with
attached
electromagnet.
a detailed view of the sample region and the OCT scanning unit with attached electromagnet.
The coordinate system (x, y, z) indicates the orientation of the MM-OCT probe. The x-direction is the
The
coordinate system (x, y, z) indicates the orientation of the MM-OCT probe. The x-direction is the
fast scanning direction of the OCT probe. Hence, cross-sectional OCT images (tomographic B-scans)
fast scanning direction of the OCT probe. Hence, cross-sectional OCT images (tomographic B-scans)
are created in the x–z plane Abbreviations: BS—beam splitter, C—collimator, CL—condensing
are created in the x–z plane Abbreviations: BS—beam splitter, C—collimator, CL—condensing lens,
lens, DC—dispersion compensation, DM—Vis/NIR dichroic mirror, GS—galvanometer scanners,
DC—dispersion compensation, DM—Vis/NIR dichroic mirror, GS—galvanometer scanners,
RL—reference lens, RM—reference mirror, SL—scanning lens, SMF—single-mode fiber (color online).
RL—reference lens, RM—reference mirror, SL—scanning lens, SMF—single-mode fiber (color online).
The solenoid
solenoid coil
coil (18.5
(18.5 Ω)
Ω) is usually driven
driven in pulsed operation mode by a self-developed
The
capacitive-discharge pulse generator
generator synchronized
synchronized to
to the
the OCT
OCT system.
system. Typically,
Typically, the magnetic pulse
pulse
capacitive-discharge
length
is
set
to
80
ms,
which
corresponds
to
the
acquisition
time
of
two
tomographic
images
(B-scans),
length is set to 80 ms, which corresponds to the acquisition time of two tomographic images
and the pulse
is set torate
1 Hz.
In the
the maximum
charging
voltage
of the
(B-scans),
andrepetition
the pulse rate
repetition
is set
to 1current
Hz. Insetup,
the current
setup, the
maximum
charging
capacitorofisthe
120capacitor
V yielding
peak
density
of 0.2flux
T and
an axial
fieldTgradient
of 18 field
T/m
voltage
is a120
V magnetic
yielding aflux
peak
magnetic
density
of 0.2
and an axial
in
the
sample
region.
gradient of 18 T/m in the sample region.
2.2. Additional
Additional Experimental
Experimental Setup
Setup for
for Rotational
Rotational MM-OCT
MM-OCT
2.2.
For the
the implementation
ring
magnet
(Conrad
Electronic
SE,
For
implementation of
ofrMM-OCT,
rMM-OCT,we
weadded
addeda apermanent
permanent
ring
magnet
(Conrad
Electronic
506014-62,
Hirschau,
Germany)
with
an internal
remanent
field
of 1.35
T and
a thickness
of 2ofmm
on
SE,
506014-62,
Hirschau,
Germany)
with
an internal
remanent
field
of 1.35
T and
a thickness
2 mm
top
of
the
electromagnet.
This
ring
magnet
is
a
highly
textured
NdFeB
sinter
magnet
whose
easy
axis
on top of the electromagnet. This ring magnet is a highly textured NdFeB sinter magnet whose easy
of magnetization
is parallel
to its rotational
symmetry
axis. As indicated
Figure 2,inboth
magnets
are
axis
of magnetization
is parallel
to its rotational
symmetry
axis. As in
indicated
Figure
2, both
not concentric
have a lateral
axisaoffset
ofaxis
approximately
15 mm. Hence,
as long
as theassolenoid
magnets
are notbut
concentric
but have
lateral
offset of approximately
15 mm.
Hence,
long as
is
switched
off
(Figure
1b),
the
laterally
displaced
permanent
magnet
creates
a
magnetic
field
with a
the solenoid is switched off (Figure 1b), the laterally displaced permanent magnet creates a magnetic
significant
horizontal
component
in
the
sample
region.
This
causes
the
anisotropic
magnetic
particles
to
field with a significant horizontal component in the sample region. This causes the anisotropic
align horizontally
theirhorizontally
long axis in parallel
to the
field
lines
as atocompass
However,
magnetic
particleswith
to align
with their
long
axis
in (such
parallel
the fieldneedle).
lines (such
as a
compass needle). However, when the solenoid is switched on (Figure 2c), the field of the permanent
magnet is superimposed by the solenoid’s magnetic field, which has a significant vertical component
in the sample region. Thus, the particles will rotate and align vertically. As a consequence, periodic
Appl. Sci. 2017, 7, 444
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when the solenoid is switched on (Figure 2c), the field of the permanent magnet is superimposed by
the solenoid’s magnetic field, which has a significant vertical component in the sample region. Thus,
Appl. Sci. 2017, 7, 444
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the particles will rotate and align vertically. As a consequence, periodic switching of the solenoid, such
as in pulsed operation mode, will create a nod-like motion of the particles, which should be detectable
switching of the solenoid, such as in pulsed operation mode, will create a nod-like motion of the
by OCT and laser speckle reflectometry.
particles, which should be detectable by OCT and laser speckle reflectometry.
Figure2.2.Principle
Principleof
ofrotational
rotationalmagnetomotive
magnetomotiveOCT
OCT(rMM-OCT)
(rMM-OCT)using
usingan
anelectromagnet
electromagnet(solenoid)
(solenoid)
Figure
and
a
laterally
displaced
permanent
magnet.
(a)
Top
view
and
(b,c)
side
view
of
the
dual-magnet
and a laterally displaced permanent magnet. (a) Top view and (b,c) side view of the dual-magnet
configuration.When
Whenthe
thesolenoid
solenoidisisturned
turnedoff
off(b),
(b),anisotropic
anisotropicmagnetic
magneticparticles
particlesin
inaasoft
softsample
sampleare
are
configuration.
aligned
horizontally
(y-direction)
towards
the
field
of
the
permanent
magnet;
when
the
solenoid
aligned horizontally (y-direction) towards the field of the permanent magnet; when the solenoid isis
turnedon
on(c),
(c),particles
particlesare
arealigned
alignedvertically
vertically(z-direction)
(z-direction)towards
towardsthe
thefield
fieldofofthe
thesolenoid.
solenoid.Drawn
Drawn
turned
magnetic
field
lines
are
a
guide
to
the
eye
only.
The
indicated
coordinate
system
(x,
y,
z)
is
identical
to
magnetic field lines are a guide to the eye only. The indicated coordinate system (x, y, z) is identical to
Figure
1b.
The
coordinate
origin
is
at
the
optical
axis
of
the
OCT
probe
(x
=
y
=
0)
and
the
surface
of
Figure 1b. The coordinate origin is at the optical axis of the OCT probe (x = y = 0) and the surface of the
the
solenoid
(z
=
0)
(color
online).
solenoid (z = 0) (color online).
Results
3.3.Results
3.1.Validation
Validationofofthe
theRotational
RotationalMagnetomotive
MagnetomotiveConcept
Concept
3.1.
Tovalidate
validatethe
thehypothesis
hypothesismentioned
mentionedabove,
above,we
wefirst
firstanalyzed
analyzedthe
thetransverse
transverseand
andlongitudinal
longitudinal
To
magnetic
field
components
of
our
rMM-OCT
setup
during
the
offand
on-states
of
the
solenoid.
For
magnetic field components of our rMM-OCT setup during the off- and on-states of the solenoid. For
thisexperiment,
experiment,we
weoperated
operatedthe
the
solenoid
(direct
current)
mode
successively
measured
this
solenoid
in in
DCDC
(direct
current)
mode
andand
successively
measured
the
the
field
components
(Figure
3a,b)
using
a
conventional
magnetometer
(KOSHAVA
5,
Wuntronic
field components (Figure 3a,b) using a conventional magnetometer (KOSHAVA 5, Wuntronic GmbH,
GmbH, München,
Germany).
In prevent
order tothe
prevent
the permanent
magnet
from
beingoffpushed
off the
München,
Germany).
In order to
permanent
magnet from
being
pushed
the solenoid
solenoid
thewe
on-state,
the supply
30be
V.seen
As itincan
be seen
in Figureoff
3c,
during
theduring
on-state,
limited we
the limited
supply voltage
to 30voltage
V. As ittocan
Figure
3c, switching
switching
off
the
solenoid
results
in
a
varying
magnetic
field
vector
in
the
sample
region,
i.e.,
the
the solenoid results in a varying magnetic field vector in the sample region, i.e., the focus region of the
◦ to
focusbeam.
regionWhen
of thethe
OCT
beam.isWhen
the solenoid
is angle
switched
on,field
the tilt
angle
of the from
field 32
vector
OCT
solenoid
switched
on, the tilt
of the
vector
changes
◦ , thus, from
changes
83°, thus,
there is
a dominant
vertical
field
during the
comparedfield
to a
83
there32°
is ato
dominant
vertical
field
during the
on-state
compared
to a on-state
rather horizontal
rather
horizontal
field
during
the
off-state.
The
magnitude
of
the
field
vector
amounts
to
17
mT
in
during the off-state. The magnitude of the field vector amounts to 17 mT in the off-state and 101 mT
the
and
101significant
mT in thedifference
on-state. between
This significant
between
two
states
is most
in
theoff-state
on-state.
This
the twodifference
states is most
likelythe
due
to the
empirical
likely
due
to
the
empirical
and
non-optimized
character
of
our
dual-magnet
configuration.
Ideally,
and non-optimized character of our dual-magnet configuration. Ideally, one would assume that the
one results
would will
assume
that thewith
best equal
resultsfield
willstrengths
be obtained
withstates.
equalHowever,
field strengths
in both
states.
best
be obtained
in both
the setup
presented
However,
setup presented
this work is considered
a proof-of-principle
thanthe
an
in
this workthe
is considered
more in
a proof-of-principle
rather more
than an
optimized system.rather
Related,
optimized
system.
Related,
the
corresponding
field
gradient
(in
the
direction
of
the
respective
tilt
corresponding field gradient (in the direction of the respective tilt angle) is approximately 4 T/m and
is the
approximately
4 T/m and
9 T/m in However,
the off- and
respectively.
However,
weat
believe
9angle)
T/m in
off- and on-states,
respectively.
weon-states,
believe that
the field gradient
plays
least
therole
fieldingradient
playsasat
least
a minor
in thistocontext,
as thethan
particles
to
athat
minor
this context,
the
particles
are role
supposed
rotate rather
moveare
viasupposed
a magnetic
rotate
rather
than
move
via
a
magnetic
gradient
force-induced
displacement.
gradient force-induced displacement.
Appl. Sci. 2017, 7, 444
Appl. Sci. 2017, 7, 444
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Figure 3.
3. Magnetic
Magnetic field
field in
in the
the rMM-OCT
rMM-OCT setup.
setup. (a)
(a) Transverse
Transverse magnetic
magnetic field;
field; and
and (b)
(b) longitudinal
longitudinal
Figure
magnetic field
fieldmeasured
measured
with
solenoid
turned
offonand
on The
(30 measurements
V). The measurements
were
magnetic
with
the the
solenoid
turned
off and
(30 V).
were performed
performed
in thewith
y–z plane
x = 0, as indicated
Figure
The coordinate
at the
in
the y–z plane
x = 0,with
as indicated
in Figure in
2b,c.
The2b,c.
coordinate
origin isorigin
again is
atagain
the optical
optical
of theprobe
OCT probe
= 0 mm)
surface
of the
solenoid
mm).The
The transverse
transverse
axis
of axis
the OCT
(x = y (x
= =0 ymm)
and and
the the
surface
of the
solenoid
(z (z
= =0 0mm).
field in (a)
was
measured
at
the
working
distance
of
the
OCT
probe
(z
=
6
mm);
The
longitudinal
field
(a) was measured at the working distance of the OCT probe (z = 6 mm); The longitudinal
in (b)inwas
alongalong
the optical
axis of
theofOCT
probeprobe
(x = y(x
= 0= mm).
The focus
of the
field
(b) measured
was measured
the optical
axis
the OCT
y = 0 mm).
The position
focus position
OCT
along along
the optical
axis axis
(x = (x
y = y0 =mm;
z =z 6= mm)
of
the beam
OCT beam
the optical
0 mm;
6 mm)isisindicated
indicatedby
bythe
the dashed
dashed circles;
(c) Resulting
Resulting magnetic
magnetic field
field vectors
vectorsin
inthe
thecentral
centralOCT
OCTfocus
focusposition
position(x(x==yy== 00 mm;
mm; zz == 6 mm) during
(c)
off- and
andon-states
on-statesofofthe
thesolenoid.
solenoid.The
The
field
vectors
were
calculated
from
the respective
transverse
offfield
vectors
were
calculated
from
the respective
transverse
and
and longitudinal
magnetic
components
indicated
bydashed
the dashed
circles
in (a,b).
longitudinal
magnetic
field field
components
indicated
by the
circles
in (a,b).
In aa second
second step,
step, we
we proved
proved magnetic
magnetic particle
particle alignment
In
alignment and
and rotation
rotation in
in the
the switchable
switchable field.
field.
We
dispersed
magnetic
particles
in
glycerin
(0.5
mg/mL)
and
investigated
them
using
the bright-field
bright-field
We dispersed magnetic particles in glycerin (0.5 mg/mL) and investigated them using the
microscopy channel
channel of
of our
our multimodal
multimodal imaging
imaging setup
setup in
in combination
combination with
with aa high-magnification
high-magnification
microscopy
objective
(Leica
C
Plan
L
40×/0.50).
The
particles
were
identical
to
the
ones
used
for
cellused
experiments
objective (Leica C Plan L 40×/0.50). The particles were identical to the ones
for cell
in
our
previous
study
[6];
they
are
sub-micron
particles
with
a
nominal
size
of
200
nm
(nano-screen
experiments in our previous study [6]; they are sub-micron particles with a nominal size of 200 nm
MAG/R-DXS, MAG/R-DXS,
Chemicell GmbH,
Berlin,GmbH,
Germany)
consisting
of aconsisting
magnetiteofcore,
a fluorescent
(nano-screen
Chemicell
Berlin,
Germany)
a magnetite
core,
shell
emitting
in
the
red
visible
spectral
range,
and
a
coating
made
of
dextran-sulfate
for
a fluorescent shell emitting in the red visible spectral range, and a coating made of dextran-sulfate for
biomolecule
coupling.
biomolecule coupling.
As shown
areare
rather
homogeneously
distributed
in the
As
shown in
in Figure
Figure 4a,
4a,the
themagnetic
magneticparticles
particles
rather
homogeneously
distributed
in
freshly
prepared
sample.
However,
after
30
min
exposure
to
the
permanent
magnetic
field,
the
the freshly prepared sample. However, after 30 min exposure to the permanent magnetic field,
particles
have
formed
long
chains
that
magnet
the
particles
have
formed
long
chains
thatare
aretransversally
transversallyaligned
alignedtowards
towards the
the permanent
permanent magnet
(y-direction,
Figure
4b).
Finally,
when
the
electromagnet
is
switched
on,
these
chains
rotate
and
align
(y-direction, Figure 4b). Finally, when the electromagnet is switched on, these chains rotate and align
longitudinally
towards
the
solenoid
(z-direction,
Figure
4c).
A
corresponding
time-lapse
video
of
the
longitudinally towards the solenoid (z-direction, Figure 4c). A corresponding time-lapse video of the
particle movement
movement is
is shown
shown in
in Video
Video S1.
S1. This
This type
type of
of chaining,
chaining, alignment
alignment and
and rotation
rotation behavior
behavior is
is
particle
also
reported
in
the
literature
for
magnetic
particles
inside
cells.
Wilhelm,
C.
et
al.
[11]
describe
that
also reported in the literature for magnetic particles inside cells. Wilhelm, C. et al. [11] describe that
magnetic particles
particles internalized
internalized by
by cells
cells are
are accommodated
accommodated in
in “magnetic
“magnetic endosomes,”
endosomes,” i.e.,
i.e., vesicles
vesicles
magnetic
containing several
several magnetic
magnetic particles.
particles. These
These magnetic
magnetic endosomes
endosomes cluster
cluster to
to elongated
elongated chains
chains within
within
containing
single
cells
when
exposed
to
a
permanent
magnetic
field,
and
these
chains
also
rotate
when
the
single cells when exposed to a permanent magnetic field, and these chains also rotate when the
direction of
of the
the magnetic
magnetic field
field is
is changed.
changed.
direction
Interestingly,
as
Figure
4b
appears
brighter than
than Figure
Figure 4c,
4c, the
the rotation
rotation of
of the
the magnetic
magnetic particle
particle
Interestingly, as Figure 4b appears brighter
chains
also
seems
to
influence
the
transparency
of
the
sample.
chains also seems to influence the transparency of the sample.
Scheme 1. Time-lapse video of the magnetic particle movement when the solenoid is turned on and
off. (Left) Bright-field transmission image as shown in Figure 4; (Right) Three-fold magnified view of
a single particle chain. The position of the magnified view is indicated by the dark rectangle. Due to
the fluidic properties of glycerin, there is a certain drift of the particles caused by the gradient of the6 of 10
Appl. Sci. 2017, 7, 444
magnetic field.
Figure 4.
4. Magnetic
Magnetic particle
particle behavior
behavior in
in the
the switchable
switchable magnetic
magnetic field.
field. (a)
(a) Bright-field
Bright-field transmission
transmission
Figure
microscopy
of
magnetic
nanoparticles
dispersed
in
glycerin
(0.5
mg/mL)
in
a
freshly
microscopy of magnetic nanoparticles dispersed in glycerin (0.5 mg/mL) in a freshly preparedprepared
sample;
sample;
(b)min
after
30 mintoexposure
to the magnetic
permanent
magnetic
withswitched
the solenoid
switched
off;
(b)
after 30
exposure
the permanent
field
with thefield
solenoid
off; and
(c) shortly
and
(c)
shortly
after
with
the
solenoid
switched
on
(30
V).
The
dominant
direction
of
the
magnetic
after with the solenoid switched on (30 V). The dominant direction of the magnetic field is indicated
field
indicated
byupper
the symbols
upper left
corner of the
images (↑—transverse,

by
theissymbols
in the
left cornerinofthe
the images
(↑—transverse,
—longitudinal).
The indicated
—longitudinal).
coordinate
system
y) is identical
Figure 2a.views
As illustrated
by the
coordinate
systemThe
(x, indicated
y) is identical
to Figure
2a. As(x,
illustrated
by thetomagnified
(red rectangles),
magnified
thechains
particles
have parallel
formed to
long
that are
aligned
the
particlesviews
in (b)(red
haverectangles),
formed long
that in
are(b)
aligned
the chains
image plane;
when
the
parallel to
imageon
plane;
when
solenoid
switched
on in (c), thesetochains
rotate
and align
solenoid
is the
switched
in (c),
thesethe
chains
rotateisand
align perpendicular
the image
plane.
This
perpendicular
to reversible
the image across
plane. many
This rotation
effect is
across
on/off
cycles
of the
rotation
effect is
on/off cycles
ofreversible
the solenoid
(seemany
Scheme
1 and
Video
S1)
solenoid
(see Scheme 1 and Video S1) (color online).
(color
online).
Appl. Sci. 2017, 7, 444
6 of 10
3.2. Cell Imaging
Based on these findings, we applied rMM-OCT to cell imaging. For this, we magnetically
labeled mesenchymal stem cells (MSCs) with the fluorescent magnetic particles, as mentioned in
Section 3.1, and embedded them in agar scaffolds resembling the elastic properties of soft tissue, as
described in [6]. Concerning cellular particle load and its effect on cell function, recent literature
indicates that MSCs incubated with 200-nm magnetic particles coated with dextran-sulfate take up
~10 pg of iron oxide per cell, and show a cell viability of ~70% after 24 h [12]. After preparation, we
placed the samples on the object stage of our multimodal imaging setup containing the permanent
magnet and kept them under resting conditions for 30 min in order to provoke the formation and
alignment of magnetic chains inside the cells. After that, we analyzed single cells using rMM-OCT in
combination with light microscopy and laser speckle reflectometry. In this context, we applied
Scheme 1. Time-lapse video of the magnetic particle movement when the solenoid is turned on and
Scheme transmission
1. Time-lapse video
the magneticmicroscopy
particle movement
when
solenoid
is turned
and the
bright-field
and of
fluorescence
in order
to the
identify
single
cellson
inside
off. (Left) Bright-field transmission image as shown in Figure 4; (Right) Three-fold magnified view of
off. volume
(Left) Bright-field
transmission
image as
shown
Figure 4; (Right)
Three-fold
magnified
view
of
sample
and evaluated
cell labeling
using
theinfluorescent
properties
of the
magnetic
particles.
a single particle chain. The position of the magnified view is indicated by the dark rectangle. Due to
a
single
particle
chain.
The
position
of
the
magnified
view
is
indicated
by
the
dark
rectangle.
Due
to
Additionally, we applied laser speckle reflectometry as a 2D reference imaging method to evaluate
the fluidic properties of glycerin, there is a certain drift of the particles caused by the gradient of the
the fluidic
properties
glycerin,
is a certain
drift of theinparticles
the gradient
of the
the sample
motion.
Thisof time,
wethere
operated
the solenoid
pulsedcaused
modeby
(pulse
duration
80 ms;
magnetic field.
magnetic
field.
repetition rate 1 Hz) in order to induce repetitive magnetomotion. According to our previous
3.2. Cell Imaging
Based on these findings, we applied rMM-OCT to cell imaging. For this, we magnetically labeled
mesenchymal stem cells (MSCs) with the fluorescent magnetic particles, as mentioned in Section 3.1,
and embedded them in agar scaffolds resembling the elastic properties of soft tissue, as described in [6].
Concerning cellular particle load and its effect on cell function, recent literature indicates that MSCs
incubated with 200-nm magnetic particles coated with dextran-sulfate take up ~10 pg of iron oxide per
cell, and show a cell viability of ~70% after 24 h [12]. After preparation, we placed the samples on the
object stage of our multimodal imaging setup containing the permanent magnet and kept them under
Figure
4. Magnetic
in the switchable
magnetic
(a) Bright-field
transmission
resting
conditions
for 30particle
min in behavior
order to provoke
the formation
andfield.
alignment
of magnetic
chains inside
microscopy
of magnetic
nanoparticles
dispersed
in glycerin in
(0.5combination
mg/mL) in awith
freshly
the cells.
After that,
we analyzed
single cells
using rMM-OCT
lightprepared
microscopy
sample;
(b)
after
30
min
exposure
to
the
permanent
magnetic
field
with
the
solenoid
switched
off;
and laser speckle reflectometry. In this context, we applied bright-field transmission and fluorescence
and (c) shortly
after
the solenoid
switched
on the
(30 V).
The dominant
direction
of the magnetic
microscopy
in order
to with
identify
single cells
inside
sample
volume and
evaluated
cell labeling
field
is
indicated
by
the
symbols
in
the
upper
left
corner
of
the
images
(↑—transverse,

using the fluorescent properties of the magnetic particles. Additionally, we applied laser speckle
—longitudinal). The indicated coordinate system (x, y) is identical to Figure 2a. As illustrated by the
magnified views (red rectangles), the particles in (b) have formed long chains that are aligned
parallel to the image plane; when the solenoid is switched on in (c), these chains rotate and align
perpendicular to the image plane. This rotation effect is reversible across many on/off cycles of the
solenoid (see Scheme 1 and Video S1) (color online).
Appl. Sci. 2017, 7, 444
7 of 10
Appl. Sci. 2017, 7,as
444a 2D reference imaging method to evaluate the sample motion. This time, we operated
7 of 10
reflectometry
the solenoid in pulsed mode (pulse duration 80 ms; repetition rate 1 Hz) in order to induce repetitive
experiments, we set
the capacitor
to 30 V,we
expecting
the magnitude
of the
pulsed
magnetomotion.
According
to ourcharging
previousvoltage
experiments,
set the capacitor
charging
voltage
to
magnetic
field
to
be
similar
to
the
values
measured
in
DC
mode
(Figure
3a–c).
Finally,
we
retrieved
30 V, expecting the magnitude of the pulsed magnetic field to be similar to the values measured in
motion
contrast
both
OCT we
andretrieved
laser speckle
(LS)
imagesfrom
(fOCT both
= fLS OCT
= 25 Hz)
calculating
the
DC
mode
(Figurefrom
3a–c).
Finally,
motion
contrast
andby
laser
speckle (LS)
temporal
variance
of
the
OCT
signal
amplitude
and
the
laser
speckle
intensity
over
an
interval
of
images (f OCT = f LS = 25 Hz) by calculating the temporal variance of the OCT signal amplitude and the
five
B-scans
and
five
video
frames,
respectively.
laser speckle intensity over an interval of five B-scans and five video frames, respectively.
Exemplary imaging
imaging results
Exemplary
results for
for five
five labeled
labeled and
and two
two unlabeled
unlabeled control
control cells
cells are
are shown
shown in
in Figure
Figure 5.
5.
It
can
be
seen
that
labeled
cells
are
characterized
by
an
intense
red
fluorescence
signal
in the
It can be seen that labeled cells are characterized by an intense red fluorescence signal in the microscopy
microscopy
channelfrom
originating
from the
fluorescent
magnetic
particles,
as color-coded
well as significant
channel
originating
the fluorescent
magnetic
particles,
as well as
significant
motion
color-coded
motion
signals
in
the
OCT
and
laser
speckle
channels
resulting
from
magnetomotion.
signals in the OCT and laser speckle channels resulting from magnetomotion. In comparison, unlabeled
In comparison,
unlabeled cells
no fluorescence
cells
show no fluorescence
and show
no motion
contrast. and no motion contrast.
Figure 5.
laser speckle
speckle and
and rMM-OCT
rMM-OCT imaging
imaging of
of labeled
labeled and
and unlabeled
unlabeled
Figure
5. Multimodal
Multimodal light
light microscopy,
microscopy, laser
stem cells
cells(MSCs)
(MSCs)embedded
embeddedininagar
agarscaffolds.
scaffolds.The
The
labeled
cells
characterized
mesenchymal stem
labeled
cells
areare
characterized
by by
an
an intense
fluorescence
signal
in microscopy
the microscopy
images
a significantly
increased
variance
in
intense
redred
fluorescence
signal
in the
images
and and
a significantly
increased
variance
in both
bothlaser
the speckle
laser speckle
images
and
thecross-sections,
OCT cross-sections,
which
is caused
by magnetically
the
images
and the
OCT
which is
caused
by magnetically
inducedinduced
motion.
motion.
In contrast,
the unlabeled
cells no
show
no fluorescence
and
no motion
signal
(color
online).
In
contrast,
the unlabeled
controlcontrol
cells show
fluorescence
and no
motion
signal
(color
online).
Hypothetically, the pulse length of 80 ms is not long enough to allow the sub-micron particles
Hypothetically,
themagnetic
pulse length
80 ms is not
enough
to allow
the sub-micron
particles
to fully respond to the
field.ofHowever,
we long
choose
this pulse
duration
to make the
results
to
fully
respond
to
the
magnetic
field.
However,
we
choose
this
pulse
duration
to
make
the
results
comparable to previous work [6].
comparable
previous
[6]. the magnetomotive signal of five different cells imaged with
In this to
context,
wework
measured
In
this
context,
we
measured
the
signal
of five different
imaged with
rMM-OCT and compared the results
to magnetomotive
our previous pulsed
MM-OCT
method. cells
For measurement,
rMM-OCT
and
compared
the
results
to
our
previous
pulsed
MM-OCT
method.
For
measurement,
we selected a region-of-interest (ROI) representing the cell, and calculated the normalized
we
selected a region-of-interest
(ROI)
representing
cell, and
and the
calculated
the normalized
magnetomotive
signal as the quotient
of the
ROI’s meanthe
variance
average amplitude
of the
magnetomotive
signal
as
the
quotient
of
the
ROI’s
mean
variance
and
the
average
amplitude
of
OCT signal across the magnetic pulse. Similarly, we measured the magnetomotive signal in laser
the
OCTreflectometry
signal acrossby
thecalculating
magnetic the
pulse.
Similarly,
we measured
magnetomotive
signal
in
speckle
mean
laser speckle
variance the
divided
by the average
laser
laser
speckle
reflectometry
by
calculating
the
mean
laser
speckle
variance
divided
by
the
average
speckle intensity. The results in Figure 6 show that the magnetomotive signal in rMM-OCT is
laser
speckleincreased
intensity. in
The
results
Figure
show that
the magnetomotive
in rMM-OCT
is
significantly
both
OCTin
and
laser6speckle
analysis
by a factor of 2.8signal
and 2.5,
respectively.
significantly
both OCT and
laser
speckle analysis
by a factorto
ofthe
2.8 and
2.5, respectively.
We
We note thatincreased
the onlyin
differences
of the
rMM-OCT
setup compared
MM-OCT
setup are the
addition of the permanent magnetic ring and the lower charging voltage of the pulse generator
capacitor (30 V compared to 120 V, respectively). However, rMM-OCT provides a significantly
Appl. Sci. 2017, 7, 444
8 of 10
note that the only differences of the rMM-OCT setup compared to the MM-OCT setup are the addition
of the permanent magnetic ring and the lower charging voltage of the pulse generator capacitor
(30 V Appl.
compared
Sci. 2017, 7,to
444120 V, respectively). However, rMM-OCT provides a significantly improved
8 of 10
magnetomotive contrast compared to MM-OCT, although the charging voltage was four times smaller
improved
contrast
compared
to MM-OCT,
although the charging voltage was8 of
four
Appl.
Sci. 2017,magnetomotive
7, 444
10
and, thus,
the solenoid’s
power
consumption
was
16 times lower.
times smaller and, thus, the solenoid’s power consumption was 16 times lower.
improved magnetomotive contrast compared to MM-OCT, although the charging voltage was four
times smaller and, thus, the solenoid’s power consumption was 16 times lower.
6. Quantification of the cellular magnetomotive signal using OCT and laser speckle analysis.
FigureFigure
6. Quantification
of the cellular magnetomotive signal using OCT and laser speckle analysis.
In each OCT (a) and laser speckle image (b) a region of interest (ROI) representing the cell is chosen
In each OCT (a) and laser speckle image (b) a region of interest (ROI) representing the cell is chosen and
Figure
Quantification
of the cellular
using OCTsignals
and laser
specklefrom
analysis.
and the6.mean
magnetomotive
signal ismagnetomotive
determined; (c)signal
Magnetomotive
retrieved
OCT
the mean magnetomotive signal is determined; (c) Magnetomotive signals retrieved from OCT data
In
each
OCT
(a) andand
laserlaser
speckle
image
(b) (right
a region
of interest
(ROI)different
representing
cell iswith
chosen
data
(left
column)
speckle
data
column)
for five
cells the
imaged
our
(left column)
and
laser
specklesetup
data
(right
column)
for
five
different
cells
imaged
with
our
previous
and
the mean
magnetomotive
signal
is determined;
(c) novel
Magnetomotive
signals
retrieved
from
OCT
previous
pulsed
MM-OCT
(dark
bars)
and the
rMM-OCT
setup
(bright
bars)
using
a
pulsed
MM-OCT
setup
(dark
bars)
the
novel
rMM-OCT
setup
(bright
bars)
using
a capacitor
data
(left column)
and
laser
speckle
data
column)
for
different
cells
imaged
with
our
capacitor
charging
voltage
of
120 Vand
and
30 (right
V,
respectively.
Thefive
magnetomotive
signals
represent
the
charging
voltage
of 120
V and 30
respectively.
The
magnetomotive
the using
normalized
previous
pulsed
MM-OCT
setup
(dark
and
the
novel
setuprepresent
(bright
a
normalized
mean
variance
inV,the
ROI, bars)
i.e., the
mean
pixel rMM-OCT
variancesignals
divided
by the bars)
average
pixel
meancapacitor
variancecharging
in thethe
ROI,
i.e.,ofthe
mean
pixel
divided
by
the average
pixel(color
intensity
across
voltage
120
V and
30represents
V, variance
respectively.
The magnetomotive
signals
represent
the
intensity
across
magnetic
pulse.
Data
mean
values
± standard
deviation
online).
normalized
mean
variance
in the mean
ROI, i.e.,
the mean
pixel variance
divided
the average pixel
the magnetic
pulse.
Data
represents
values
± standard
deviation
(colorby
online).
In a final
step,the
wemagnetic
tested our
rMM-OCT
imaging
method
alternative
cells next
MSCs that
intensity
across
pulse.
Data represents
mean
values on
± standard
deviation
(colorto
online).
are used for retinal transplantation studies concerning the RPE, the ARPE-19 cell line [13–15]. These
In a In
final
step,
wewetested
rMM-OCTimaging
imaging
method
on alternative
cells
next to
MSCs
a final
step,
tested our
ourinrMM-OCT
method
on alternative
cells
next
to MSCs
that
cells were
labeled
and embedded
agar scaffolds identical
to MSCs,
as described
before.
Representative
that are
used
for
retinal
transplantation
studies
concerning
the
RPE,
the
ARPE-19
cell
line
[13–15].
are
used
for retinal
transplantation
concerning
RPE,intheFigure
ARPE-19
These
OCT
imaging
results
for labeled studies
ARPE-19
cells are the
shown
7. Itcell
canline
be[13–15].
seen that
the
Thesecells
cells
were
labeled
and
embedded
agar
scaffolds
as Representative
described before.
were
labeled
and embedded
in
agar scaffolds
identical
to MSCs,
as described
before.
magnetomotive
contrast
is very
similar
tointhe
MSCs
shown
inidentical
Figure
5. to MSCs,
Representative
OCT
imaging
resultsARPE-19
for labeled
ARPE-19
cells
shown
Figure
7. Itthat
can the
be seen
OCT imaging
results
for labeled
cells
are shown
in are
Figure
7. Itincan
be seen
magnetomotive
contrast
is veryissimilar
to the MSCs
shown
Figure 5.
that the
magnetomotive
contrast
very similar
to the
MSCsinshown
in Figure 5.
Figure 7. Volumetric rMM-OCT imaging of labeled ARPE-19 cells embedded in an agar scaffold.
(a) Top view and (b) side view of a recorded 3D OCT image stack showing two cells next to each
Figure
7. Volumetric
imaging
of labeled
labeled
ARPE-19
cells
embedded
in an
scaffold.
other.
(c)
3D viewrMM-OCT
ofrMM-OCT
the two cells.
All views
represent
maximum
intensity
projections
of
thescaffold.
3D
Figure
7. Volumetric
imaging
of
ARPE-19
cells
embedded
in agar
an agar
(a)
Topdata
view
andside
(b)
side
a recorded
3D OCT
image
stack
showing
twocells
cellsnext
nexttotoeach
eachother.
image
online).
(a) Top
view
and(color
(b)
viewview
of a of
recorded
3D OCT
image
stack
showing
two
of theAll
twoviews
cells. represent
All views represent
projections
of the
3D data
(c) 3Dother.
view(c)
of 3D
theview
two cells.
maximummaximum
intensityintensity
projections
of the 3D
image
4. Discussion
image data (color online).
(color online).
In conclusion, we have shown that rMM-OCT provides a higher magnetomotive signal at a
4. Discussion
lower
power consumption compared to previous pulsed MM-OCT. This improvement was mostly
4. Discussion
In conclusion,
have permanent
shown thatmagnet
rMM-OCT
a higher magnetomotive
signalsetup.
at a
achieved
by addingwe
a small
in anprovides
off-axis configuration
to the MM-OCT
In
conclusion,
we
shown
that rMM-OCT
provides
athe
higher
magnetomotive
signal
atfrom
a lower
lower
power consumption
compared
to isprevious
pulsed
MM-OCT.
This
improvement
was mostly
The
thickness
of
thehave
permanent
magnet
only 2 mm;
hence,
reduction
of working distance
achieved
by adding
small permanent
magnet
inMM-OCT.
an
off-axis
configuration
to thewas
MM-OCT
setup.
power
toofprevious
pulsed
This
improvement
mostly
achieved
6 consumption
mm (relative
tocompared
thea surface
the solenoid)
in the
original
setup
to
4 mm (relative
to the
surface
of
The thickness of the permanent magnet is only 2 mm; hence, the reduction of working distance from
6 mm (relative to the surface of the solenoid) in the original setup to 4 mm (relative to the surface of
Appl. Sci. 2017, 7, 444
9 of 10
by adding a small permanent magnet in an off-axis configuration to the MM-OCT setup. The thickness
of the permanent magnet is only 2 mm; hence, the reduction of working distance from 6 mm (relative to
the surface of the solenoid) in the original setup to 4 mm (relative to the surface of the permanent
magnet) in the new setup is still acceptable for retinal imaging in small animal models used in cell
transplantation studies, such as mice (eye length 3 mm). Further, the dual-magnet configuration of
rMM-OCT is a single-sided setup that circumvents the restrictions of previous dual-coil configurations
to small samples.
In this context, we have also shown that rMM-OCT allows robust and reliable
contrast-enhancement in different cell types, such as MSCs and transplantable ARPE-19 cells. We note
that magnetomotion in cells can also be induced by other MM-OCT methods, such as pulsed MM-OCT
using our previous setup. However, our results shown in Figure 6 imply that the magnetomotive
signal of rMM-OCT is significantly higher compared to the previous pulsed MM-OCT. Besides particle
chaining and rotation, this signal increase might be additionally supported by particle clumping
induced by the permanent magnet leading to enhanced magnetic susceptibility of the contrast agent
and, thus, improved displacement in response to the applied magnetic field. As a consequence,
rMM-OCT permits higher sensitivity, the use of low power magnetic fields or corresponding larger
working distances, and single-sided access to biological specimens. This makes it a promising method
for future 3D cell tracking in target tissues and organs, such as the retina in small animal models, under
in vivo conditions.
However, further investigation is required to adress several aspects of the method presented
herein. Most important, as the induced magnetic particle changes to elongated chains (as shown
in Figure 4) end up being very long in glycerin (~10 µm) and approach the diameters of cells,
cell viability experiments are necessary to investigate whether particle chain formation and rotation
during rMM-OCT imaging may be destructive to cells. Additionally, it is not yet clear what the size
these chains in cells may reach, how controllable this is in conjunction with the magnetization time
(currently 30 min), and what the minimum detectable concentration of magnetic particles is or the
minimum detectable size of particle aggregates. In this context, the current magnetization time of
30 min is relatively long, especially with regards to prospective in vivo applications. Therefore, we plan
further experiments in which we investigate the time-dependency of magnetic chain formation and
the correlation with magnetomotive signal enhancement in order to find the minimum magnetization
time appropriate for in vivo imaging.
As an alternative to further enhance the sensitivity of rotational magnetomotive measurements,
particles with potentially increased anisotropy such as magnetic nanostars [16,17] could be applied.
Moreover, for future in vivo application, faster imaging is desired. In this study, the magnetomotive
imaging speed was limited by the fixed pulse repetition rate of 1 Hz in order to make the results
comparable to previous work. However, the lower power consumption of the solenoid in this
work facilitates the use of higher duty cycles, and thus faster pulse repetition rates. In this context,
an adequate pulse generator for faster pulse series is the subject of current, ongoing investigation.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3417/7/5/444/s1,
Video S1: rMM-OCT magnetic particle movement.avi.
Acknowledgments: This research was supported by the TU Dresden CRTD (Center for Regenerative Therapies
Dresden) Seed Grant Program and grants from the MeDDrive program of the Faculty of Medicine Carl Gustav
Carus of the TU Dresden.
Author Contributions: All authors contributed to this work. Peter Cimalla conceived the method of rotational
MM-OCT, designed the experimental setup and performed the imaging experiments; Claudia Mueller and
Seba Almedawar performed the cell labeling experiments of the MSC and ARPE-19 cells; Peter Cimalla,
Julia Walther and Edmund Koch processed and analyzed the data; Bernd Rellinghaus characterized the magnetic
particles and analyzed the magnetic field; Dierk Wittig, Marius Ader, Mike O. Karl, Richard H. W. Funk and
Michael Brand analyzed the cell labeling and the imaging data; Peter Cimalla and Julia Walther wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Appl. Sci. 2017, 7, 444
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