Noradrenaline-Specific, Efficient Visualization in Brain Tissue Triggered by Unique Cascade Nucleophilic Substitution

Article abstract of DOI:10.1021/acs.analchem.8b04836

Noradrenaline as one of the most important neurotransmitters in the sympathetic nervous system plays key roles in all of the forebrain activities such as perception, memory, learning, and homeostasis, and its dysfunction is closely related to both neurodegenerative diseases and central nervous system disorders. The very similar structures and properties of the three coexisting catecholamine neurotransmitters - dopamine, epinephrine, and noradrenaline - make it almost impossible to design fluorescent probes that respond specifically to noradrenaline, which restricted its physiological and pathological studies greatly. Fantastic design turned dreams into reality in this work: the cascade nucleophilic substitution reactions between noradrenaline and fluorophore coupling carbonic ester, which formed a five-membered ring catechol-containing compound with the release of the fluorophore accompanied by unique fluorescent responses, allowed us to develop a fluorescent probe to detect and visualize noradrenaline over dopamine and epinephrine in brain tissue. Instead of the noradrenaline synzyme immunofluorescence labeling path, the present fluorescent probe can visualize noradrenaline directly with comparable specificity.

Full text of DOI:10.1021/acs.analchem.8b04836

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Article
Noradrenaline Specific, Efficient Visualization in Brain Tissue
Triggered by Unique ‘Cascade’ Nucleophilic Substitution
Yongkang Yue, Fangjun Huo, and Caixia Yin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04836 • Publication Date (Web): 28 Dec 2018
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Analytical Chemistry
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Noradrenaline Specific, Efficient Visualization in Brain Tissue
Triggered by Unique ‘Cascade’ Nucleophilic Substitution
Yongkang Yue,† Fangjun Huo,‡ Caixia Yin†,*
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†Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for
Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University. Taiyuan 030006,
China. E-mail: yincx@sxu.edu.cn. Tel/Fax: +86-351-7011022.
‡Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China.
ABSTRACT: Noradrenaline as one of the most important neurotransmitters in sympathetic nervous system plays key roles in all of
the forebrain activities such as perception, memory, learning, and homeostasis and its dysfunction is closely related to both
neurodegenerative diseases and central nervous system disorders. The very similar structures and properties of the three co-existed
catecholamine neurotransmitters: dopamine, epinephrine, noradrenaline make it almost impossible to design fluorescent probes that
respond specifically to noradrenaline which astricted its physiological and pathological studies greatly. Fantastic design turned
dreams into reality in this work: the cascade nucleophilic substitution reactions between noradrenaline and fluorophore coupling
carbonic ester which formed a five-membered ring catechol-containing compound with the release of the fluorophore accompanied
by unique fluorescent responses allow us to develop fluorescent probe to detect and visualize noradrenaline over dopamine and
epinephrine in brain tissue. Instead of the noradrenaline synzyme immunofluorescence labeling path, the present fluorescent probe
can visualize noradrenaline directly with comparable specificity.
Perception, memory, learning, and homeostasis are
mediated by the release and accumulation of neurotransmitters
in our brain.1 Typically, catecholamine derivates such as
dopamine, noradrenaline (norepinephrine), and epinephrine
are principal neurotransmitters in the sympathetic nervous
system. In neurons, the three siblings are synthesized from
tyrosine via a series of enzymatic reaction and stored in the
acidic synaptic vesicles (pH 5.0-5.5) with high concentration
(~mM, Figure 1).2-4 As one of the most important
neurotransmitters, noradrenaline plays key roles in all of the
forebrain activities and its dysfunction is closely related to
both neurodegenerative diseases and central nervous system
disorders.5-7 Besides, noradrenaline has been used as first-line
vasopressor agents in the treatment of shock.8,9 Promoted by
its vital functions and increasing incidence of noradrenaline
related diseases, the physiological and pathological studies
attracted more and more attention in recent years.10-14
based on this design strategy, they can also react with generic
amines such as glutamate featuring even more pronounced
fluorescence responses compared with their reaction with
catecholamines because of the quenching nature of the
catechol group in catecholamine neurotransmitters through
photo-induced electron transfer process.22-24 Besides, the
discrimination of dopamine and noradrenaline cannot be
realized by this strategy, which may block its application in
tissue specific noradrenaline imaging. In 2014, the Kleinfeld
group modified the G-protein with α1A adrenergic receptor to
detect noradrenaline release indirectly via the activation of
receptor triggered cytosolic Ca2+ increase which was detected
by a genetically encoded FRET based Ca2+ sensor.14 For this
strategy based Ca2+ probes, the cellular sliced
neurotransmitters cannot be directly detected. Thus, new
strategy for noradrenaline specific fluorescent probes design is
imperative to elucidating the effect of noradrenaline in brain.
In 2009, Sames and coworkers designed fluorescent false
neurotransmitters to visualize the release of dopamine in the
striatum under stimulation and made excellent progresses
based on this strategy.15-18 Recently, they developed the
FFN270 as fluorescent false neurotransmitter to noradrenergic
synapses in brain tissue specifically.19 These substrates can
image the neurotransmitters release and the activity of
synapses in living tissue. However, the in situ noradrenaline
analysis which is extremely important for its biological roles is
restricted. Fluorescent probes with specific responses toward
noradrenaline are supra tools for its further study.14,19
Representatively, the Glass group utilized coumarin aldehyde
derivates (NeuroSensor 521 and NeuroSensor 715) as
fluorescent probes for monoamines neurotransmitters
detection via the formation of Schiff base.20,21 For the probes
Figure 1. Catecholamine neurotransmitters and their biosynthesis.
Tyrosine hydroxylase (TH), aromatic amino acid decarboxylase
(AADC), dopamine-β-hydroxylase (DBH), phenylethanolamine
N-methyltransferase (PNMT).
For the three siblings, the inherent 2-aminoethanol moiety
of noradrenaline may react with fluorophore coupling carbonic
ester via cascade nucleophilic substitution reactions to form a
five-membered ring compound with the release of the
fluorophore (Figure 2). The reaction process can promote the
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specificity of fluorescent probes toward noradrenaline over
UV-vis spectrophotometer was employed to measure UV-vis
spectra. Shanhai Huamei Experiment Instrument Plants, China
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other generic amines, dopamine, and epinephrine. Besides, the
cleavage detection mechanism can effectively avoid the
fluorescent quench caused by the aforementioned
intramolecular PET process. Holding this in mind, we firstly
synthesized a coumarin based carbonic ester derivate CNE.
CNE featured strong fluorescent emission at 394 nm which
was induced by the weak electron-withdrawing effect of
carbonic ester moiety and non-fluorescent response displayed
upon the addition of noradrenaline (Figure S1). To effectively
modulate the fluorescence of the probe during the detection
process, we forecasted that fluorescein derivates with spiro
ring-closing and -opening process might be suitable candidates
for probes construction. Indeed, the introduction of ethyl
carbonate to fluorescein quenched its fluorescent emission
(FHE, Figure S2). But noradrenaline addition did not cause
significant fluorescent change of FHE containing detection
system within one hour. To activate the reaction site, we
further combined 4-methylthiophenol with fluorescein through
carbonyl to synthesize probes FHS and FCS for noradrenaline
detection. The thiophenol moiety as the primary leaving group
1
provided a PO-120 quartz cuvette (10 mm). H NMR and 13C
NMR experiments were performed with
a BRUKER
AVANCE III HD 600 MHz and 151 MHz NMR spectrometer,
respectively (Bruker, Billerica, MA). Coupling constants (J
values) are reported in hertz. HR-MS determinations were
carried out on an Thermo Scientific Q Exactive Instruments.
The brain tissue imaging experiments were measured by
Olympus Fluo View FV1000 laser-scanning microscope with
an objective lens (×20).
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Synthesis. Compounds were synthesized according to the
procedure in Scheme S1-S3 in the Supporting Information.
Synthesis of CNE. m-Aminophenol (5 mmol) was dissolved
in ethyl acetate and the solution was refluxed for 30 min. Then,
ethyl chloroformate (0.5 equiv.) was added dropwise. The
solution was cooled to room temperature after white solid
precipitated. After filtration and washing with ethyl acetate,
compound 1 was obtained as white solid and used for further
synthesis directly.
In the compound 1 (1 mmol) and ethyl acetoacetate (2
equiv.) system, concentrated sulfuric acid (2 drops) was added.
The mixture was stirred for 10 h under room temperature.
After that, H2O was added and the mixture was filtrated to
obtain probe CNE as white solid. 1H NMR (600 MHz,
DMSO-d6) δ 10.14 (s, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.55 (s,
1H), 7.41 (d, J = 8.6 Hz, 1H), 6.23 (s, 1H), 4.17 (q, J = 7.0 Hz,
2H), 2.39 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H). 13C NMR (151
MHz, DMSO-d6) δ 160.5, 154.3, 153.8, 153.7, 143.3, 126.5,
114.7, 114.7, 112.3, 104.8, 61.2, 18.5, 14.9.
could facilely be replaced by amino which initiated the
25-27
detection process.
Exhilaratingly, both FHS and FCS
could react with noradrenaline exhibited turn-on fluorescent
responses. Featuring better water solubility, FCS was used for
further spectroscopic evaluation and brain tissue noradrenaline
imaging.
Synthesis of FHE. In a fluorescein (1 mmol) contained THF
(10 mL) system, Et3N (2 equiv.) was added. Then, ethyl
chloroformate (3 equiv.) was added dropwise under ice bath.
The mixture was stirred for 10 h under room temperature
before filtration. The obtained filtrate was concentrated under
reduced pressure and purified by column chromatography
using ethyl acetate / petroleum ether (1 / 6) as eluent to give
1
FHE as a white solid. H NMR (600 MHz, DMSO-d6) δ 8.07
(d, J = 7.6 Hz, 1H), 7.83 (t, J = 7.3 Hz, 1H), 7.77 (t, J = 7.4 Hz,
1H), 7.42 (d, J = 8.2 Hz, 3H), 7.06 (d, J = 8.7 Hz, 2H), 6.92 (d,
J = 8.7 Hz, 2H), 4.28 (q, J = 7.0 Hz, 4H), 1.30 (t, J = 7.1 Hz,
6H). 13C NMR (151 MHz, DMSO-d6) δ 168.8, 152.8, 152.6,
152.6, 151.2, 136.5, 131.1, 129.8, 125.8, 125.5, 124.6, 118.5,
116.9, 110.5, 81.3, 65.5, 14.4.
O
O
OH
R
R
HO
S
O
S
O
O
HO
NH2
OH
R
N
O
NH2
H
OH
HO
Synthesis of FRS. To a solution of p-toluenethiol (10 mmol)
and triphosgene (0.5 equiv.) in CH2Cl2 (20 mL) at 0 °C,
pyridine (1 equiv. in 5 mL CH2Cl2) was added dropwise. The
mixture was stirred for 1 h at 0 °C and then poured into 100
mL H2O. The organic layer was separated, washed with H2O,
dried with sodiumfulfate, and concentrated under reduced
pressure. The obtained crude product of compound 2 was used
for further synthesis directly.
HO
OH
OH
HO
HO
HO
HO
R = fluorophore
NH
NH
O
HO
R
+
O
H
O
O
O
R
Figure 2. Fluorescent probes design and the detection mechanism
of FRS toward noradrenaline.
Fluorescein or 2',7'-dichlorofluorescein (1 mmol) and Et3N
(2 equiv.) was dissolved in 20 mL CH2Cl2. Compound 2 (2
equiv. in 5 mL CH2Cl2) was then added at 0 °C. The mixture
was gradually warmed to room temperature and continued to
react for 10 h. After that, the solvent was removed under
reduced pressure and the residue was separated by column
chromatography using ethyl acetate / petroleum ether (1 / 6) as
eluent to give FRS as a white solid.
EXPERIMENTAL SECTION
Materials and Methods. All chemicals were purchased
from commercial suppliers and used without further
purification. All solvents were purified prior to use. Distilled
water was used after passing through a water ultra-purification
system. TLC analysis was performed using precoated silica
plates. Hitachi F−7000 fluorescence spectrophotometer was
employed to measure fluorescence spectra. Hitachi U-3900
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Analytical Chemistry
FHS: 1H NMR (600 MHz, DMSO-d6) δ 8.06 (d, J = 7.6 Hz,
MAB308; Millipore) for 2 h followed by a secondary antibody
cocktail of Goat Anti-Rabbit IgG / Cy3 (1:250, bs-0295G-Cy3;
Bioss Antibodies) and Goat Anti-Mouse IgG / ALexa Fluor
350 (1:100, bs-0296G-AF350; Bioss Antibodies) for 30 min,
which were then stained with FCS (5 μM) for 30 min. All
these experiments were processed at 37 °C.
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1H), 7.80 (t, J = 7.1 Hz, 1H), 7.75 (t, J = 7.4 Hz, 1H), 7.53 (d,
J = 8.1 Hz, 4H), 7.44 (d, J = 2.3 Hz, 2H), 7.37 (d, J = 7.6 Hz,
1H), 7.30 (d, J = 8.0 Hz, 4H), 7.07 (dd, J = 8.7, 2.3 Hz, 2H),
6.92 (d, J = 8.7 Hz, 2H), 2.33 (s, 6H). 13C NMR (151 MHz,
DMSO-d6) δ 168.8, 168.4, 152.5, 152.5, 151.2, 140.8, 136.5,
135.2, 131.1, 130.7, 129.9, 125.8, 125.6, 124.6, 123.1, 118.6,
117.3, 110.7, 81.1, 21.3.
RESULTS AND DISCUSSION
FCS: 1H NMR (600 MHz, DMSO-d6) δ 8.05 (d, J = 7.6 Hz,
1H), 7.86 – 7.74 (m, 4H), 7.54 (d, J = 8.1 Hz, 4H), 7.46 (d, J =
7.6 Hz, 1H), 7.32 (d, J = 8.1 Hz, 4H), 7.12 (s, 2H), 2.35 (s,
6H). 13C NMR (151 MHz, DMSO-d6) δ 168.4, 167.9, 151.7,
149.7, 148.6, 141.0, 136.6, 135.6, 135.2, 131.4, 130.8, 129.4,
126.1, 125.8, 124.5, 122.6, 122.1, 118.9, 113.8, 79.9, 21.3. HR
MS [M+H]+: m/z Calcd 701.0257, Found 701.0259.
Detection Properties of FCS Toward Noradrenaline
in Solution. The aforementioned fluorescent detection
mechanism of FCS toward noradrenaline was firstly validated
upon HR-MS characterization of the reaction mixture. As
shown in Figure S18 (detailed in Scheme S4), the signals of
both the intermediate products (compound 3 and 4) and the
ultimate products (compound 5 and 2',7'-dichlorofluorescein)
were occurred distinctly. Besides, the UV-vis absorption
spectra comparison of the FCS-noradrenaline system and 2',7'-
dichlorofluorescein demonstrated the production of 2',7'-
dichlorofluorescein in the detection process (Figure S13). The
results sustained our design assumption.
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Preparation of Stock Solutions of Probes and
Analytes. Stock solutions of probe CNE, FHE, FHS, and
FCS (2 mM) were prepared in DMSO. Stock solutions of 100
mM dopamine, epinephrine, noradrenaline, and glutamate
were prepared by dissolution in PB (pH 5.0, 50 mM Na2S2O3,
120 mM NaCl) separately. Stock solutions of 100 mM GSH
and Gly were prepared by dissolution in PB (pH 5.0). All
chemicals used were of analytical grade.
The detection property of FCS toward noradrenaline was
then valued systematically. FCS itself displayed non-
fluorescent emission in the detection system. Upon
noradrenaline addition, distinct fluorescent emission at 530 nm
enhanced gradually (Figure 3a). Correspondingly, in the UV-
vis spectra, a new absorption peak at 511 nm enhanced (Figure
3b). As for noradrenaline probes, the most challenging
parameter was the specificity over dopamine and epinephrine.
Thus, we added equal amount of dopamine or epinephrine to
FCS containing system under the same condition with
noradrenaline detection. As we designed, both dopamine and
epinephrine induced one-eighth fluorescent intensities
enhancement compared with noradrenaline (Figure 3c and
Figure S3, S4). Besides, the responses of FCS toward 5 mM
other generic amines such as glutamate, glycine, the major
reducing substance GSH, and 500 μM lysine, 2-aminoethanol
containing amino acid such as threonine and serine were
General Fluorescence Spectra Measurements. All
the detection experiments were measured in PB−DMSO (pH
5.0, 1 / 1, v / v). The procedure was as follows: into a
PB−DMSO (pH 5.0, 1 / 1, v / v) solution, containing 5 μM
FCS, an analyte sample was added. The process was
monitored by fluorescence spectrometer (λex = 510 nm, slit: 5
nm / 2.5 nm).
Brain Tissue Preparation. All animal protocols were
approved by the Department of Science & Technology of
Shandong Province. The SD mice (6- to 7-week-old, female)
were obtained from Yantai Raphael Biotechnology SPF
barrier environment animal house and acclimatized to the
animal facility for at least 5 days. After quarantine, qualified
animals were maintained under 12 h light / dark cycles at
room temperature 23 ± 3 °C with 40−70 % humidity. Mice
were anesthetized with thiobutabarbital (100 mg/kg
intraperitoneal injection) and intracardially perfused with PBS,
4 % paraformaldehyde sequentially. The brains were collected
and fixed for 1-2 hours and then cryopreserved in 30 %
sucrose at 4 °C. Then, the coronal 40 μm sections were
collected and stored in a cryoprotectant at -20 °C for
immunohistochemical treatment.
valued and none of them induced distinct fluorescent change
20,21,28
of FCS in the detection system (Figure S5-S10).
Cys
with similar mercaptoethylamine moiety was also evaluated
and 50 μM cysteine did not induce significant fluorescent
response (the cellular concentration of unbounded cysteine is
nM~μM, Figure S11).29,30 Because of the readily oxidized
nature of noradrenaline, we set 320 min as the reaction period
in the quantitative studies to minimize the oxidation induced
noradrenaline consumption. The fluorescent intensities of the
reaction system at 530 nm were linear with the concentrations
of noradrenaline (0-1 mM, Figure 3d and Figure S12). These
results demonstrated that FCS could be used for noradrenaline
specific detection in vitro.
Immunohistofluorescence.
The
sections
were
processed either for TH and DBH dual labeling, or DBH and
PNMT dual labeling which were then stained with FCS before
imaging, respectively. For TH and DBH dual labeling
experiments, the sections were processed with a primary
antibody cocktail of Rabbit Anti-Tyrosine Hydroxylase
Polyclonal Antibody (1:500, bs-0016R; Bioss Antibodies) and
Anti-Dopamine β Hydroxylase (1:1000, MAB308; Millipore)
for 2 h followed by a secondary antibody cocktail of Goat
Anti-Rabbit IgG
/ Cy3 (1:250, bs-0295G-Cy3; Bioss
Antibodies) and Goat Anti-Mouse IgG / ALexa Fluor 350
(1:100, bs-0296G-AF350; Bioss Antibodies) for 30 min,
which were then stained with FCS (5 μM) for 30 min. For
PNMT and DBH dual labeling experiments, the sections were
processed with a primary antibody cocktail of Rabbit Anti-
PNMT Polyclonal Antibody (1:500, bs-3912R; Bioss
Antibodies) and Anti-Dopamine β Hydroxylase (1:1000,
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Figure 4. FCS labels noradrenergic neurons in brain tissue. a)
Immunofluorescence Cy3 labeled TH positive region (green); b)
Immunofluorescence AF350 labeled DBH positive region (blue);
c) FCS labeled brain tissue (red); d) Merged image of a and c; e)
Figure 3. Spectroscopic responses of FCS toward noradrenaline,
dopamine, and epinephrine. Time-dependent fluorescent (a) and
UV-vis (b) responses of 5 μM FCS toward 5 mM noradrenaline in
PB−DMSO (pH 5.0, 1:1, v/v) system. (c) Time-dependent
fluorescent intensities changes of 5 μM FCS toward 5 mM
noradrenaline, 5 mM dopamine, and 5 mM epinephrine at 530 nm
in PB−DMSO (pH 5.0, 1:1, v/v) system, respectively. (d)
Quantitative fluorescent intensities at 530 nm upon addition of 0,
50 μM, 100 μM, 200 μM, 500 μM, and 1 mM noradrenaline to 5
μM FCS containing detection system, respectively. The
fluorescent intensity data were obtained 340 min after
noradrenaline addition. Error bars represent standard deviations, n
= 3; λex = 510 nm, slit: 5 nm / 2.5 nm.
Merged image of b and c; f) Bright field of the tissue. Cy3: λex
=
548 nm, λem = 560-610 nm; AF350: λex = 405 nm, λem = 430-480
nm; FCS: λex = 500 nm, λem = 510-560 nm; bar = 100 μm.
Noradrenaline Visualization in Brain Tissue.
Supported by the spectroscopic data of FCS for noradrenaline
detection, we wondered whether the probe could be used for
noradrenergic neurons labeling in brain tissue. Noradrenergic
neurons distribute in brainstem nucleus locus coeruleus. They
can be distinguished over dopaminergic neurons via dual
immunolabeling for DBH and TH which are essential enzymes
for noradrenaline and its precursor dopamine synthesis,
respectively. At the same time, adrenergic neurons can be
visualized specifically by immunolabeling for PNMT which
convert noradrenaline to epinephrine.2,15,31 So we preformed
dual immunofluorescence labeling experiments to visualize
TH and DBH, or DBH and PNMT positive regions in coronal
respectively which was then stained with FCS before imaging.
As shown in Figure 4, the overall pattern of FCS (red, c) and
DBH-AF350 (blue, b) fluorescent signals showed extensive
overlap (pink, e) which demonstrated the excellent
noradrenergic neurons specific labeling property of FCS. As a
continuous work, Figure 5 displayed the discriminatory
property of FCS toward noradrenaline over epinephrine. As
shown in Figure 5d, FCS labeled noradrenergic neurons which
were DBH positive could exactly be discriminated over
adrenergic neurons (additional tissue images are shown in
Figure S14-S17). These results mean that FCS represents an
easier tool for noradrenergic neurons specific labeling over
dopaminergic neurons and adrenergic neurons in brain tissue
compared with the time consuming and complicated
immunofluorescence labeling.
Figure 5. FCS labels noradrenergic neurons in brain tissue. a)
Immunofluorescence Cy3 labeled PNMT positive region (green);
b) Immunofluorescence AF350 labeled DBH positive region
(blue); c) FCS labeled brain tissue (red); d) Merged image of a
and c; e) Merged image of b and c; f) Bright field of the tissue.
Cy3: λex = 548 nm, λem = 560-610 nm; AF350: λex = 405 nm, λem
=
430-480 nm; FCS: λex = 500 nm, λem = 510-560 nm; bar = 100 μm.
CONCLUSIONS
In conclusion, we utilized the cascade nucleophilic
substitution reactions between noradrenaline and fluorophore
coupling carbonic ester which formed a five-membered ring
catechol-containing compound with the release of the
fluorophore to develop a new generation of noradrenaline
specific fluorescent probes design strategy. Promoted by the
structural particularity of noradrenaline, the probes based on
this strategy can effectively avoid the fluorescent quench
caused by the catechol group through intramolecular PET
process in NeuroSensor 521 with improved specificity. The
accordingly synthesized probe FCS can detect and label
noradrenaline both in aqueous solution and in brain tissue over
dopamine and epinephrine. The present work provides an
efficient tool for noradrenergic neurons visualization. To
develop probes with fast and ratiometric fluorescent responses
toward noradrenaline are on-going in our lab. We believe this
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Analytical Chemistry
(9) De Backer , D.; Biston , P.; Devriendt , J.; Madl , C.; Chochrad
probe will promote the physiological and pathological studies
of noradrenaline in brain.
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(10) Pham, L.; Baker, M. R.; Shahanoor, Z.; Romeo, R. D.
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(11) Nakatsuka, N.; Andrews, A. M. Differentiating siblings: The
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AUTHOR INFORMATION
Corresponding Author
*E-mail: yincx@sxu.edu.cn. Tel./Fax: +86-351-7011022.
ORCID
Caixia Yin: 0000-0001-5548-6333
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There are no conflicts to declare.
ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China (No.
21672131, 21775096, 21705102), Talents Support Program of
Shanxi Province (2014401), Shanxi Province Foundation for
Returness (2017-026) and Scientific Instrument Center of Shanxi
University.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Synthesis of probes and the structure characterizations,
additional fluorescence and UV-vis spectra, and additional
fluorescence images (PDF)
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