Calcium Sensor for Photoacoustic Imaging

Article abstract of DOI:10.1021/jacs.7b03064

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca²⁺-dependen

Full text of DOI:10.1021/jacs.7b03064

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Communication
Calcium Sensor for Photoacoustic Imaging
Sheryl Roberts, Markus Seeger, Yuanyuan Jiang, Anurag Mishra, Felix Sigmund, Anja
Stelzl, Antonella Lauri, Panagiotis Symvoulidis, Hannes Rolbieski, Matthias Preller,
Xosé Luís Deán Ben, Daniel Razansky, Tanja Orschmann, Sabrina Desbordes,
Paul Vetschera, Thorsten Bach, Vasilis Ntziachristos, and Gil Gregor Westmeyer
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b03064 • Publication Date (Web): 25 Sep 2017
Downloaded from http://pubs.acs.org on September 25, 2017
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Razansky, Daniel; Helmholtz Zentrum München (GmbH), Institute of
Biological and Medical Imaging
Orschmann, Tanja; Helmholtz Zentrum Munchen Deutsches
Forschungszentrum fur Umwelt und Gesundheit, Institute of Developmental
Genetics
Desbordes, Sabrina; Helmholtz Zentrum Munchen Deutsches
Forschungszentrum fur Umwelt und Gesundheit, Institute of Developmental
Genetics
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Vetschera, Paul; Helmholtz Zentrum Munchen Deutsches
Forschungszentrum fur Umwelt und Gesundheit
Bach, Thorsten; Technische Universitaet Muenchen, Lehrstuhl fuer
Organische Chemie I
Ntziachristos, Vasilis ; Technische Universität München & Helmholtz
Zentrum München, Chair of Biological Imaging
Westmeyer, Gil; Technical University of Munich (TUM), Nuclear Medicine;
Helmholtz Zentrum Munchen Deutsches Forschungszentrum fur Umwelt
und Gesundheit, Institute for Biological and Medical Imaging ; Helmholtz
Zentrum Munchen Deutsches Forschungszentrum fur Umwelt und
Gesundheit, Institute of Developmental Genetics
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Calcium Sensor for Photoacoustic Imaging
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†‡¥
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‡
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‡¥
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Sheryl Roberts , Markus Seeger , Yuanyuan Jiang , Anurag Mishra , Felix Sigmund , Anja Stelzl , Antonella Lauri
,
†‡
◊
‡
‡
◊
Panagiotis Symvoulidis , Hannes Rolbieski, Matthias Preller , X. Luís DeánꢀBen , Daniel Razansky , Tanja Orschmann , Sabrina
◊
§
Ψ
§‡
†‡¥
Desbordes , Paul Vetschera , Thorsten Bach , Vasilis Ntziachristos , and Gil G. Westmeyer
†
Department of Nuclear Medicine, Technical University of Munich (TUM), Ismaninger Str. 22, 81675 Munich, Germany.
‡
¥
Institute for Biological and Medical Imaging & Institute of Developmental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Oberschleißheim, Germany.
◊
Institute for Biophysical Chemistry, CarlꢀNeubergꢀStraße 1, Hannover Medical School, 30625 Hannover.
Chair for Biological Imaging, Technical University of Munich (TUM), Trogerstr. 9, 81675 Munich, Germany.
§
Ψ
Chair of Organic Chemistry I, Technical University of Munich (TUM), Lichtenbergstraße 4, 85747 Garching, Germany
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Supporting Information
2
+
ABSTRACT: We introduce a selective and cellꢀpermeable
magnesium (Mg ) which is present at much higher intracellular
2+
4
2+
Calcium (Ca ) Sensor for Photoacoustics (CaSPA) which is a
versatile imaging technique that allows for fast volumetric mapꢀ
ping of photoabsorbing molecules with deep tissue penetration.
concentrations. Selectivity against zinc (Zn ) is also desirable,
as Zn is known to be colocalized with glutamate in neurons
and coꢀpackaged with insulin in vesicles that are released from
beta cells in a Ca ꢀdependent manner. Furthermore, transꢀ
membrane delivery of the Ca sensor should be efficient in
order to load the majority of cells in a PAI voxel and thereby
minimize partial volume effects. This set of specifications is
however not fulfilled by most current calcium sensors used for
fluorescence microscopy as those fluorophores, such as Oregon
2+
5
2+
6
2
+
To optimize for Ca ꢀdependent photoacoustic signal changes,
we synthesized a selective metallochromic sensor with high
extinction coefficient, a low quantum yield, and high photoꢀ
2+
2
+
bleaching resistance. Micromolar concentrations of Ca lead to
a robust blueshift of the absorbance of CaSPA which translated
into an accompanying decrease of the peak photoacoustic signal.
The acetoxymethyl esterified sensor variant was readily taken
up by cells without toxic effects and thus allowed us for the first
7
Green BAPTAꢀ1 operate mainly by a change in QY. Some
wellꢀknown chromophores that change their absorbance in
response to metal binding (metallochromic substances), such as
Arsenazo III and Chlorophosphonazo III, exhibit specific abꢀ
sorbance changes in response to Ca . However, these comꢀ
pounds have a lower selectivity for Ca as compared with
BAPTA while forming complexes with two dye molecules per
Ca . Furthermore, the negatively charged Ca ꢀbinding moieties
of these dyes cannot be easily modified to achieve efficient
delivery into cells. Photoacoustic signals were measured from
Arsenazo III packaged into liposomes and recently also from
cells into which the highly concentrated compound was loaded,
2
+
time to perform live imaging of Ca fluxes in genetically unꢀ
modified cells, tissue culture as well as in zebrafish larval brain
via combined fluorescence and photoacoustic imaging.
2+ 8
2+
2+
2+
■
INTRODUCTION
2+
Calcium (Ca ) is a key second messenger controlling e.g.
9
contractility in heart and skeletal muscle and triggering secretoꢀ
1
ry processes in immune, neuroendocrine, and neuronal cells. It
2+
is thus of great interest to map spatiotemporal patterns of Ca ꢀ
10,11
signaling across whole organs in intact animals. Photoacoustic
imaging (PAI) is a technique with deep tissue penetration that
provides volumetric information on the distribution of photoabꢀ
however resulting in measurable toxicity
arsenazo dyes. Recently, a Cu ꢀresponsive probe for photoaꢀ
as expected from
12
2+
2
+
coustic detection was reported based on the Cu ꢀtriggered
cleavage of a 2ꢀpicolinic ester bond that led to a change of the
photoacoustic spectrum. However, this detection mechanism is
2
sorbers with ultrasound resolution. This performance is made
possible by exploiting the photoacoustic effect which describes
the radiationless conversion of electromagnetic energy into
mechanical energy occurring when chromophores thermoelasꢀ
tically expand upon photoabsorption. The resulting mechanical
waves propagate through the surrounding tissue and can be
detected via ultrasound transducers such that imaging volumes
can be reconstructed. The key advantages of PAI are therefore
that its resolution is insensitive to photon scattering and that it
can provide volumetric information with high frame rates, beꢀ
13
not reversible.
2
+
To provide the first cellꢀpermeable and selective Ca ꢀsensor
for photoacoustics (CaSPA), we thus focused on metalꢀ
lochromic compounds that undergo a photoinduced charge
transfer (PCT) as described for furaꢀ2 , in which a Ca sensiꢀ
tive donor (aniline in BAPTA) is conjugated to an acceptor
molecule in the fluorophore (amino group) leading to a spectral
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2+
1
5,16
blueshift upon calcium binding.
We therefore reasoned that
3
cause scanning procedures are not necessary.
replacing the chromophore in furaꢀ2 with a semiꢀcyanine chroꢀ
mophore could maintain the PCT mechanism but shifted to
longer wavelengths more suitable for photoacoustics.
PAI readily detects hemodynamics and can indirectly localize
brain activity because neuronal activity is correlated with vasoꢀ
3
dilation in mammalian brains (neurovascular coupling). Howꢀ
ever, molecular signals that are directly linked to neuronal activꢀ
ity, such as intracellular calcium transients, could so far not be
effectively read out by PAI due to a lack of appropriate molecuꢀ
lar sensors.
■
RESULTS AND DISCUSSION
We here demonstrate the result of this design strategy, a new
2+
metallochromic Ca ꢀSensor for Photoacoustics (CaSPA), which
is highly selective for Ca ꢀ virtue of its BAPTA chelation
group ꢀ and is readily cell permeable in its acetomethoxy ester
2+
In distinction to fluorescence imaging, the ideal photophysical
properties of a chromophore optimized for photoacoustic detecꢀ
tion are a low quantum yield (QY) and a high molar extinction
coefficient (ε), as well as high photobleaching resistance. To
design a reversible sensor for photoacoustics based on these
prerequisites, one may search for functionalizations that alter ε
and/or QY as a function of the analyte concentration or enviꢀ
(AM) form.
Figure 1 schematizes the chemical structure of the CaSPA_550
2+
chromophore in its unbound (left) and Ca ꢀbound form (right,
Figure 1a). The semiꢀcyanine chromophore of CaSPA_550 (5,
Figure S1a in the Supporting Information) was synthesized via a
condensation reaction between 3ꢀethylꢀ1,1,2ꢀtrimethylꢀ1Hꢀ
benzo[e]indolꢀ3ꢀium iodide, containing an activated methyl
group and the appropriate aldehyde of 1,2ꢀbis(2ꢀ
2+
ronmental parameter of interest. A photoacoustic sensor for Ca
should furthermore be highly selective for the target analyte
over other biologically important divalent metals, in particular
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aminophenoxy)ethane N,N,N',N'ꢀtetraacetate skeleton resulting
in deep purple crystals with 85% yield. Subsequent
demethylation using NaOH (aq) was carried out at room
temperature and the resulting product was dried under high
vacuum to obtain the corresponding free acid in low yields
identical samples on a customꢀbuilt photoacoustic spectrometer
see Experimental Section in the Supporting Information) and
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(
observed that the peak photoacoustic signal at 550 nm also
2+
decreased by a factor of about two in response to 39 µM of Ca
(Figure 1d). The changes of the peak intensities of the absorbꢀ
ance spectra were significantly correlated with the changes of
(
≤ 5%). To improve the yield, saponification of the methyl ester
groups was performed with tetrabutylammonium hydroxide
yield of up to 20%), which increased the solubility of the acid
2
the peak photoacoustic signal (R = 0.99); the peak fluorescence
(
2
signal followed the changes in the absorbance (R = 0.99).
in polar organic solvents for further esterification reaction. The
resulting product dried in vacuo is colorless in organic solvents
and its deep purple color is only observed under polar
conditions. Further esterification of (5) was carried out by
dissolving the free acid with five equivalents of DIPEA and six
equivalents of acetoxymethyl (AM) bromide in acetonitrile.
Purification yielded purple crystals of CaSPA_550ꢀAM (Figure
S1 in the Supporting Information).
To gain insights into the mechanism of photophysical changes
2
+
upon Ca
binding, we performed quantum chemical
calculations of ground and excited electronic states of
CaSPA_550 using density functional theory (DFT) and timeꢀ
dependent DFT (TDꢀDFT) with the M06ꢀ2X hybrid exchangeꢀ
correlation functional and the M6ꢀ31(TM)** basis set. The
computed vertical absorption energies for the uncomplexed and
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Photophysical characterization of CaSPA_550 in the absence
2+
Ca ꢀbound CaSPA_550 are 2.48 eV and 2.92 eV, respectively,
2+
of Ca , revealed an absorption spectrum with a prominent peak
corresponding to vertical transition wavelengths of 500 nm (ꢀ
ꢀ
1
ꢀ1
at 550 nm with # = 77,745 M cm . As a function of
increasing Ca
5
50
2+
2+
2
+
Ca ) and 425 nm (+Ca ) (Figure S5 and Table S1). Therefore,
concentrations (0ꢀ39 µM), CasPA_550
2
+
our TDꢀDFT calculations predict a marked blue shift upon Ca
exhibited a reduction of its peak absorbance at 550 nm and the
appearance of a minor blueshifted absorbance peak at 455 nm.
The resulting maximum signal decrease at 550 nm was ~50%;
the amplitude change at 455 nm was ~65% (Figure 1b, Figure
S2).
complexation, which is in line with the observed hypsochromic
shift of the absorption band.
We determined the K values for calcium to be ~4 µM (Figure
D
2+
2+
2+
2a and Figure S3); The selectivity for Ca over Mg , Zn , and
2+
In addition, an isosbestic point at 470 nm was apparent that
could be used for ratiometric measurements correcting for
differences in concentrations of the sensor (Figure S3).
Absorbance of CaSPA_550 was not strongly affected by
lowering pH to values that may be present in endosomes and
synaptic vesicles but showed a blueshift and drop in absorbance
when the pH was lowered to very acidic (pH 3.1) values (Figure
S4). The peak fluorescence emission measured at 634 nm also
decreased to half of the intensity as a function of increasing
Cu was high as expected from BAPTA as none of these metals
induced any reduction in the absorbance (Figure 2b, Figure S6).
Furthermore, the compound proved to be very photobleaching
resistant (Figure S7).
Encouraged by the favorable photophysical properties of
CaSPA_550, we next sought to test the calcium sensor in cells.
To render the molecule cellꢀpermeable, we generated the
1
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acetoxymethyl ester variant CaSPA_550ꢀAM which we found
to be spectrally different from CaSPA_550 with a peak
2+
concentrations of Ca (Figure 1c). However, we observed no
ꢀ
1
ꢀ1
2+
absorbance at ~590 nm (#590 = 116,500 M cm ) and a peak
fluorescence amplitude at ~606 nm. Esterification also ensured
that the absorbance and fluorescence spectra were unaltered in
change in QY upon binding of Ca , which was low as desired
for photoacoustics (an absolute measurement gave: QYnoCa2+
0.01; QY39µMCa2+= 0.009; a relative measurement against
rhodamine 101 yielded: QYnoCa2+= 0.007 ± 0.0003; QY39µMCa2+
.008± 0.0008).
=
2
+
2+
=
the presense of high concentrations (1 mM) of Ca or Mg
(Figure S8).
0
We subsequently measured the photoacoustic spectra of the
We found that CaSPA_550ꢀAM was taken up by Chinese
Hamster Ovary Cells (CHO) as well as Human Embryonic
Kidney Cells (HEK 293) after incubation with low micromolar
concentrations without affecting cell viability (Figures S9, S10,
S11).
Figure 3a shows fluorescence signal trajectories obtained from
CaSPA_550 loaded CHO cells in which calcium influx was
2
+
Figure 1. Photophysical characterization of CaSPA_550 in
Figure 2. Sensitivity and selectivity of CaSPA_550 for Ca . (a) The
2
+
2+
response to Ca ions. (a) Chemical structure of CaSPA_550 in its
dissociation constant (K ) for Ca binding was determined by photoaꢀ
D
2+
unbound (left) and Ca ꢀbound (right) form (bꢀd) Spectroscopic
coustics (3.9 µM; 95% Confidence Interval (CI): 2.9ꢀ 5 µM) absorbꢀ
ance (4.0 µM; 95% CI: 3.2ꢀ 4.9 µM). (b) Selectivity of the sensor was
tested by plotting the peak of the absorbance (filled bars) and fluoresꢀ
cence spectra (striped bars) for several divalent metals added at 50 µM
to 30 mM MOPS (3ꢀ(Nꢀmorpholino)propanesulfonic acid) containing
also 100 mM KCl. Each data point is the mean of 3 experiments with
error bars representing the standard error of the mean (SEM).
analysis of CaSPa_550 (25 µM) dissolved in MOPS (2 mM) buffer
2
+
containing increasing concentrations of free Ca (0ꢀ39 µM). Spectra
were obtained for (b) absorbance, (c) fluorescence, and (d)
photoacoustic readout. Addition of 10 mM of the tight chelator
2
+
EDTA competes out Ca from CasPA_550 and reverts the signal
amplitudes (dashed line in b,c,d).
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associated with small contractions detected at the outer rim of
the heart tissue (gray shaded bars in Figure S12b).
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Figure 3c displays the maximum amplitude projections of the
optoacoustic volumes from CaSPAꢀloaded heart organoids
(magenta) overlaid on the fluorescence signals from twoꢀphoton
excitation (gray) revealing the structure of the organoid. When
we sampled at the maximum photoacoustic imaging frame rate
of 1 Hz, we observed photoacoustic signal reductions in the ROI
of up to 10% in a freqency range that we also observed vith the
standard Fluoꢀ4ꢀAM in fluorescence microscopy at
comparable sampling rate (Figure 3d, S12b).
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Next, we sought to work with the CaSPA_550ꢀAM sensor in
vivo and thus delivered it to zebrafish larval brains. We
observed that the compound spread out well from the
intraventricular injection point, and was taken up by cells
(white arrow in Figure S13a,a’). We then embedded a
CaSPAꢀinjected zebrafish larva next to a nonꢀinjected control
fish in agar and positioned it in a multispectral photoacoustic
imaging device. This imaging system is capable of fast
acquistion of photoacoustic spectra in imaging volumes over
time via the use of a tunable laser. In addition to the
photoacoustic signal time courses, we also recorded
Figure 3. Imaging of calcium fluxes in cells and heart organoids
by CaSPA_550 AM. (a) Fluorescence signal trajectories of CHO
cells incubated with CaSPA_550 (2 µM, 30 minutes) in response to
2+
2+
Ca influx triggered by addition of a Ca ionophore (BrꢀA23187, 10
2+
2+
µM in HBSS with Ca and Mg ). Insets show fluorescence images
obtained at the time points indicated. Vehicle control was 0.5%
DMSO. The average fluorescence intensities were extracted by
manual segmentation and plotted normalized to the fluorescence
intensity of each cellular ROI before addition of the ionophore or
vehicle control (error bars represent the standard error of the mean
(SEM)). (b) Analogous experiment conducted with optical resolution
photoacoustic microscopy (ORꢀPAM) in HEK cells. Photoacoustic
signal time courses were recorded after addition of vehicle control
2
+
(
0.5% DMSO) followed by Ca ionophore (BrꢀA23187, 10 µM). The
insets (a,b) show exemplary FOVs (at 0, 10 and 20 sec in (a), and 0,
00 and 440 sec in (b) with scale bars representing 20 µm (a) and 10
2
µm (b). (c) Heart organoids loaded with CaSPA_550ꢀAM and visualꢀ
ized as coꢀregistered overlay of maximum amplitude projections of
the ORꢀPAM image time series (magenta) and twoꢀphoton excitation
fluorescence volume (TPEF) (gray). The median signal differences of
the time series are shown on the map to the right. (d) Corresponding
PA signal time course averaged over the region of interest (ROI)
indicated with dashed lines in (c).
triggered by a calciumꢀspecific ionophore (BrꢀA23187) which
lead to a ~50% signal decrease as compared to vehicle control.
Corresponding data from HEK cells are shown in Figure S11.
We then conducted corresponding experiments on a customꢀ
built hybrid 2ꢀphoton and optical resolution photoacoustic
microscope (ORꢀPAM) that achieves ~1 µm axial and ~5 µm
1
8,19
Figure 4. Combined multispectral photoacoustic tomography and
fluorescence imaging of CaSPAꢀinjected larval zebrafish brain. (a)
Single imaging frame of the epifluorescence time series showing
fluorescence signal of the brain ROI of the CaSPA_550 AMꢀinjected
zebrafish (550 nm excitation, absorbing eyes highlighted with dashed
lines) as well as the nonꢀinjected control. (b) Corresponding
fluorescence signal time course of an ROI placed over the peak
fluorescence (circle with magenta broken lines) during 7 minute
baseline recording followed by two additional 7 minute measurements
after stimulations with the nuerostimulatns ethanol (EtOH) and
pentylenetetrazole (PTZ). The fluorescence signal time course
lateral resolution.
As can be seen in Figure 3b,
CaSPA_550ꢀloaded cells generated robust photoacoustic
2
+
contrast that decreased to ~50% after addition of the Ca
ionophore.
These substantial signal changes in cell experiments prompted
us to subsequently test the photoacoustic sensor in threeꢀ
dimensional tissue culture. For this purpose, we cultured heart
2
+
organoids, which spontaneously generate Ca transients, and
loaded them with CaSPA_500ꢀAM. Via epifluorescence
microscopy, we could observe fluorescent signal reductions
throughout a large stationary region of interest (ROI) that were
timeꢀlocked to small contractions detectable on the outer ring of
the organoid (gray shaded time intervals in Figure S12a) as
(errobars represent SEM) of the corresponding brain region in the nonꢀ
injected control fish is shown in green. (c) Simultanously acquired
photacoustic imaging data with 50 photoacoustics spectra (430ꢀ630
nm) obtained in each observational block. The magenta overlay
represents the voxel time courses during the baseline recording that
matched the CaSPAꢀ550 spectrum. The melanin containing eyes which
give a broad PA spectrum are overlaid in orange. An anatomical
reference is shown in gray (d) Corresponding time courses of the
normalized peak photoacoustic signal during baseline and after
stimulation with EtOH and PTZ. The signal trajectory of the control
fish is shown on the bottom. Errorbars represent SEM and may be
smaller than the symbols.
2
+
expected for a Ca response of the weakly fluorescent
metallochromic sensor. Conversely, when we loaded organoids
with the commonly used green fluorescent calcium indicator
Fluoꢀ4ꢀAM, we observed increases of the fluorescent signal
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fluorescence signal changes with a camera mounted on the
We are grateful for support from the Helmholtz Alliance
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ICEMED (AM, GGW), the European Research Council under
grant agreements ERCꢀStG: 311552 (GGW), the Priority proꢀ
gram SP1665 of the German Research Foundation (DFG), as
well as the Laura Bassi Award of TUM (SR), grant agreements
CRC 1123 (Z1), and Reinhard Koselleck project (NT 3/9ꢀ1).
opposite side from the photacoustic sensor array. As displayed
in Figure 4a, the brain of the CaSPAꢀinjected fish showed a
strong fluorescent signal (excitation at 550 nm) over 7 minutes
of baseline observation, whereas no fluorescent signal was
detected from the brain of the control fish. When we applied a
2
0
low concentration of ethanol (1% in fish water
), a
neurostimulant with fast cellular diffusion, to both agarꢀ
embedded fish, we observed a signal decay and increased signal
fluctuations (Figure 4b). Subsequently we superfused the potent
■
(1)
(2)
(3)
(4)
REFERENCES
Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Nat Rev
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Taruttis, A.; Ntziachristos, V. Nature Photon 2015, 9 (4),
2
1
neurostimulant pentylenetetrazole (PTZ, 5 mM in fish water
)
0
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2
+
which is commonly used to induce strong Ca signaling in the
brain and observed an overall fluorescent signal decrease in the
brain region of the CaSPA injected fish. Throughout the
experiment, the autofluorescence signals from the swim
bladders of both fish observed at shorter excitation wavelenghts
remained constant (Figure S13b,b’b’’). When we then analyzed
the simultaneoulsy acquired photoacoustic data, we located the
CaSPA_550 spectrum (Fig. S13c) at the location with strong
fluorescence signal in the CaSPAꢀinjected fish (Fig. 4c, magenta
2
19.
Gottschalk, S.; Fehm, T. F.; DeánꢀBen, X. L.; Razansky, D.
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(8)
overlay). Over time, we observed
a decrease of the
photoacoustic signal peak around 550 nm during the stimulation
with EtOH which slightly increased before further signal
reduction after addition of PTZ (Figure 4d). The brain of the
control fish generated only low signal around 550 nm and signal
amplitudes obtained from the melanin containing eyes of both
fish remained constant over time (Fig. 13d,d’).
(
9)
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Cooley, E. J.; Kruizinga, P.; Branch, D. W.; Emelianov, S.
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1843 (10), 2284.
(
■
CONCLUSIONS
(13)
(14)
These experiments jointly demonstrate that the metallochromic
2+
Ca ꢀsensor for photoacoustics (CaSPA_550) which we
introduce here is the first probe that possesses the necessary
photophysical and biochemical properties to enable imaging of
(
15)
16)
2+
intracellular Ca
transients with photoacoustics in cells,
(
organotypic tissue culture and in vivo in larval zebrafish brain.
The semiꢀcyanine scaffold we chose may serve as a versatile
platform to generate metallochromic sensors selective for other
biologically relevant metals or small analytes to enable
molecular and dynamic photoacoustic imaging with photoꢀ
scatteringꢀindependent resolution
(
17)
Tsien, R. Y. Nature 1981, 290 (5806), 527.
Seeger, M.; Karlas, A.; Soliman, D.; Pelisek, J.; Ntziachrisꢀ
tos, V. Photoacoustics 2016.
Soliman, D.; Tserevelakis, G. J.; Omar, M.; Ntziachristos,
V. Sci Rep 2015, 5, 12902.
Ikeda, H.; Delargy, A. H.; Yokogawa, T.; Urban, J. M.;
Burgess, H. A.; Ono, F. Plos One 2013, 8 (5), e63318.
Randlett, O.; Wee, C. L.; Naumann, E. A.; Nnaemeka, O.;
Schoppik, D.; Fitzgerald, J. E.; Portugues, R.; Lacoste, A.
M. B.; Riegler, C.; Engert, F.; Schier, A. F. Nat. Methods
2015, 12 (11), 1039.
(18)
19)
(20)
21)
(
■
ASSOCIATED CONTENT
Supporting Information.
(
Supporting Tables and Figures, as well as Experimental Methꢀ
ods. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
AUTHOR INFORMATION
Keywords: Imaging agents • Photoacoustic Imaging • Optoaꢀ
coustic Imaging • metallochromic agent • calcium imaging
Corresponding Author
* gil.westmeyer@tum.de
ORCID
Gil Gregor Westmeyer: 0000ꢀ0001ꢀ7224ꢀ8919
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGEMENTS
We thank Dr. Robert Pal (Durham University) for relative QY
measurements, Dr. Andreas Bauer (Technical University of
Munich) for absolute QY measurements, and Christian Hundꢀ
shammer for help with HPLC. We thank Prof. Dr. Oliver Pletꢀ
tenburg for helpful comments on the manuscript.
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