Two-photon fluorescent probes for long-term imaging of calcium waves in live tissue

Article abstract of DOI:10.1002/chem.200701453

2-Acetyl-6-(dimethylamino)-naphthalene-derived two-photon fluorescent Ca²⁺ probes (ACa1-ACa3) are reported. They can be excited by a 780 nm laser beam, show 23-50-fold enhancement in one- and two-photon excited fluorescence in response to Ca²⁺, emit fourfold stronger two-photon excited fluorescence than Oregon Green 488 BAPTA-1 upon complexation with Ca²⁺, and can selectively detect intracellular free Ca²⁺ ions in live cells and living tissues with minimum interference from other metal ions and membrane-bound probes. Moreover, these probes are capable of monitoring calcium waves at a depth of 120-170 μm in live tissues for 1100-4000 s using two-photon microscopy with no artifacts of photobleaching.

Full text of DOI:10.1002/chem.200701453

FULL PAPER
DOI: 10.1002/chem.200701453
Two-Photon Fluorescent Probes for Long-Term Imaging of Calcium Waves in
Live Tissue
Hwan Myung Kim,[a] Bo Ra Kim,[a] Myoung Jin An,[a] Jin Hee Hong,[b]
Kyoung J. Lee,[b] and Bong Rae Cho*[a]
Abstract: 2-Acetyl-6-(dimethylamino)-
naphthalene-derived two-photon fluo-
rescent Ca2+ probes (ACa1–ACa3) are
reported. They can be excited by a
780 nm laser beam, show23–50-fold
enhancement in one- and two-photon
excited fluorescence in response to
Oregon Green 488 BAPTA-1 upon
complexation with Ca2+, and can selec-
tively detect intracellular free Ca2+
ions in live cells and living tissues with
minimum interference from other
metal ions and membrane-bound
probes. Moreover, these probes are ca-
pable of monitoring calcium waves at a
depth of 120–170 mm in live tissues for
1100–4000 s using two-photon micro-
scopy with no artifacts of photobleach-
ing.
Keywords: calcium
·
fluorescent
probes · imaging agents · live tissue ·
two-photon microscopy
Ca2+
, emit fourfold stronger two-
photon excited fluorescence than
Introduction
bis(2-aminophenyl)ethyleneglycol-N,N,N’,N’-tetraacetic acid
(BAPTA) as the Ca2+-selective chelator and benzofuran,
benzoindole, and fluorescein as the fluorophore.[3–6] Howev-
er, the application of these probes with one-photon micro-
scopy (OPM) is limited to use near the tissue surface (<
80 mm).
Calcium is a ubiquitous intracellular messenger; it is an es-
sential regulator of many cellular processes including fertili-
zation, cell death, sensory transduction, muscle contraction,
motility, exocytosis, and fluid secretion.[1,2] Calcium triggers
exocytosis within microseconds, drives the gene transcription
and proliferation in minutes to hours, and the concentration
varies from 100 nm at rest to 1 mm upon activation.[1,2] Defec-
tive Ca2+ signaling is a feature of diverse diseases including
hypertension and immunodeficiencies. To understand these
functions, fluorescence imaging with one-photon (OP) fluo-
rescent probes has most often been used.[3,4] The most
widely used probes are fura-2, indo-1, Fluo-3, Calcium
Green, and Oregon Green 488 BAPTA-1 (OG1) with O,Oꢀ-
To observe cellular events deep inside the tissue, it is cru-
cial to use two-photon microscopy (TPM). TPM employing
two near-infrared (NIR) photons for excitation offers a
number of advantages over OPM, including increased pene-
tration depth (>500 mm), localized excitation, and pro-
longed observation time.[7–9] The extra penetration depth
that TPM affords is particularly useful for tissue imaging be-
cause surface preparation artifacts, such as damaged cells,
extend >70 mm into the brain slice interior.[9,10] However,
most of the OP fluorescent probes presently used for TPM
have small two-photon (TP) action cross sections (Fd) that
limit their use in TPM. Another limitation associated with
tissue imaging is a mistargeting problem, which results from
membrane-bound probes.[3,11,12] As the probes can be accu-
mulated in any membrane-enclosed structure within the cell,
and as the fluorescence quantum yield should be higher in
the membrane than in the cytosol, it is practically difficult
for the signals from membrane-bound probes to be separat-
ed from those of the probe–Ca2+ complex. Therefore, there
is a need to develop efficient TP probes with 1) enhanced
Fd values for brighter TPM images and 2) larger spectral
shifts in different environments for better discrimination be-
tween the cytosolic and membrane-bound probes.
[a] H. M. Kim, B. R. Kim, M. J. An, Prof. B. R. Cho
Molecular Opto-Electronics Laboratory
Department of Chemistry
Korea University
1-Anamdong, Seoul 136-701 (Korea)
Fax : ACHTREUNG(+82)2-3290-3121
E-mail: chobr@korea.ac.kr
[b] Dr. J. H. Hong, Prof. K. J. Lee
National Creative Research Initiative Center for Neurodynamics
and Department of Physics
Korea University
1-Anamdong, Seoul 136-701 (Korea)
Supporting information for this article contains additional figures and
movies of Figures 6 and 7 and is available on the WWW under http://
www.chemeurj.org/ or from the author.
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To address these critical needs, we have developed a
series of TP probes for Ca2+ derived from 2-acetyl-6-(dime-
thylamino)naphthalene (Acedan) as the TP chromophore
and BAPTA as the Ca2+-selective binding site. Acedan is a
polarity-sensitive fluorophore that has been successfully em-
ployed in the design of TP fluorescent probes for the mem-
brane and metal ions,[13,14] and BAPTA is the receptor for
OG1, a well-known OP fluorescent probe for Ca2+ ions.[3]
These findings led us to design Acedan-derived TP fluores-
cent Ca2+ probes (ACa1–ACa3) with the expectation that
the probes would emit strong two-photon excited fluores-
cence (TPEF) on forming complexes with Ca2+. Moreover,
if the probe–Ca2+ complex emits TPEF in a widely different
wavelength range depending on the polarity of the environ-
ment, the emission resulting from membrane-bound probes
could be excluded from that of the probe–Ca2+ complex by
using different detection windows. We recently reported an
efficient TP probe that can vis-
meability, the carboxylic acid moieties were converted to
acetoxymethyl esters (ACa1-AM–ACa3-AM).[15]
The absorption and emission spectra of ACa1 and ACa2
are almost the same (Table 1), probably because the elec-
tron-donating effect of the N-methyl group is nullified by
the electron-withdrawing ability of the amide group between
the fluorophore and BAPTA. ACa3 shows a slight batho-
chromic shift compared to ACa2, indicating an enhanced in-
tramolecular charge transfer (ICT) by the N-methyl group.
All compounds showgradual bathochromic shifts with the
solvent polarity in the order 1,4-dioxane<DMF<EtOH<
H2O (see Figure S1 and Table S1 in the Supporting Informa-
tion), and the effects are greater for the emission (72–
82 nm) than those for the absorption spectra (17–19 nm).
This indicates the potential utility of these compounds as
fl
max
polarity probes. In addition, the l
values of the AM
esters in DMF are similar to those of the membrane-bound
ualize the calcium waves in live
tissues.[14b] Here, we extend our
earlier work and present
a
series of TP fluorescent probes
for Ca2+ (ACa1–ACa3) that ex-
hibit high selectivity toward
Ca2+
,
emit strong TPEF on
forming complexes with Ca2+
,
and are capable of monitoring
the calcium signals in living as-
trocyte cells and in live mouse
tissues at a depth of >100 mm
for a long period of time (1100–
4000 s) without photobleaching
and mistargeting problems.
Scheme 1. Synthesis of ACa1–ACa3. a) MeNH2·HCl, Na2S2O5, NaOH, H2O; b) i) BrCH2CO2Me, Na2HPO4,
NaI, MeCN; ii) KOH, EtOH; c) i) 5-amino-BAPTA-tetramethyl ester, EDCI, DMAP, DMF; ii) KOH, EtOH,
H2O; d) NH4OH, Na2S2O5; e) i) 5-methyl-5’-formyl-BAPTA-tetramethyl ester, (MeCO2)3BHNa, DCE;
ii) KOH, EtOH, H2O (ACa2); iii) C, MeI, proton sponge, MeCN; iv) KOH, EtOH, H2O (ACa3);
f) BrCH2OCOCH3, Et3N, CHCl3. EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; DMAP: 4-dimethyl-
Results and Discussion
The synthesis began with the aminopyridine; DCE: 1,2-dichloroethane.
preparation of TP chromo-
phores A and B (Scheme 1). A
Table 1. Photophysical data for ACa1–ACa3 and OG1.
was prepared by the reaction of
(1)[b]
fl[c]
(2)[g]
Compound[a]
lmax
lmax
F[d]
KOd P/KTdP[e]
FEF/TFEF[f]
lmax
d[h]
Fd[i]
1 with MeNH2 in the presence
ACa1-AM
ACa1
362
365
365
362
362
362
370
375
375
494
495
498
498
494
495
495
499
500
517
523
0.060
0.012
0.49
0.014
0.010
0.42
0.032
0.015
0.38
nd[j]
nd[j]
780
nd[j]
nd[j]
780
nd[j]
nd[j]
780
800
nd[j]
nd[j]
230
nd[j]
nd[j]
210
nd[j]
nd[j]
250
37
nd[j]
nd[j]
110
nd[j]
nd[j]
90
of Na2S2O5 and NaOH.[14] Cou-
pling of
A
with 5-amino-
ACa1+Ca2+
ACa2-AM
ACa2
0.27[k]/0.25
40/44
42/50
BAPTA-tetramethyl ester fol-
lowed by hydrolysis produced
ACa1 in 53% yield. ACa2 was
prepared in 74% yield by the
reductive amination of B with
5-methyl-5’-formyl-BAPTA-tet-
ramethyl ester. Alkylation of
ACa2 with methyl iodide af-
forded ACa3 in 90% yield
(Scheme 1). ACa2 and ACa3
were obtained in higher yields
and with fewer steps relative to
ACa2+Ca2+
ACa3-AM
ACa3
0.14[k]/0.16 nm
nd[j]
nd[j]
95
ACa3+Ca2+
OG1+Ca2+
0.13[k]/0.14 nm
25/23
0.66
0.17[l]/nd
14[l]/nd
24
[a] All data were measured in 30 mm MOPS, 100 mm KCl, 10 mm EGTA, pH 7.2 in the absence and presence
(39 mm) of free Ca2+. [b,c] lmax of the OP absorption and emission spectra, respectively, in nm. [d] Fluorescence
quantum yield, ꢀ10%. [e] Dissociation constants for Ca2+ in mm measured by OP (KOd P) and TP (KdTP) process-
es, ꢀ14%. [f] Fluorescence enhancement factor, (FꢁFmim)/Fmin, measured by OP (FEF) and TP (TFEF) pro-
cesses. [g] lmax of the TP excitation spectra in nm. [h] The peak TP cross section in 10ꢁ50 cm4 sphotonꢁ1 (GM),
ꢀ15%. [i] TP action cross section. [j] nd: not determined; the TPEF intensity was too weak to measure the
cross section accurately. [k] KOd P values of ACa1, ACa2, and ACa3 for Mg2+ are 6.8ꢀ0.7, 6.2ꢀ0.7, and 6.0ꢀ
ACa1. To enhance the cell per- 0.6 mm, respectively. [l] Reference [3].
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Fluorescent Probes for Imaging in Live Tissue
FULL PAPER
a slope of 1.0, indicating 1:1 complexation (Figure 2c and
Figure S4b in the Supporting Information).[17]
The dissociation constants (KOd P) were calculated from the
fluorescence titration curves (Figure 2d and Figure S4c in
the Supporting Information).[17,18] The KdOP values of ACa2
and ACa3 for Ca2+ were approximately twofold smaller
than that of ACa1 (Table 1), indicating higher affinity for
Ca2+, which can be attributed to the enhanced electron den-
sity at the metal-ion binding sites by the p-Me group. Simi-
lar values were determined by the TP process (Table 1).
This indicates the capabilities of ACa2 and ACa3 to detect
lower concentrations of Ca2+ than ACa1 by TPM. More-
over, the KOd P values of ACa1–ACa3 for Mg2+ are in the
range of 6.0–6.8 mm (Table 1 and Figure S6 in the Support-
ing Information), thus negating the possibility of Mg2+ inter-
ference.
ACa2 and ACa3 showed a modest response toward Zn2+
and Mn2+, a much weaker response toward Mg2+, Fe2+, and
Co2+, and no response toward Cu2+ (Figure 3a and Figure
S5a in the Supporting Information). A similar result was re-
ported for ACa1.[14b] As the intracellular free-ion concentra-
tion of Mn2+ is negligible,[19] these probes can detect the in-
tracellular Ca2+ concentration ([Ca2+]i) in the regions in
which the chelatable Zn2+ concentration ([Zn2+]i) is much
lower than KTdP (Ca2+). Furthermore, all probes were pH-in-
sensitive in the neutral pH range, although the FEF de-
creased slightly at pH <6.5 (Figure 3b, Figure S5b in the
Supporting Information, and reference [14b]).
The TP action spectra of the Ca2+ complexes with ACa1–
ACa3 in buffer solutions indicated the Fd values of 90–
110 GM at 780 nm, approximately fourfold larger than that
of OG1–Ca2+ (Table 1). Thus, TPM images for samples
stained with ACa1–ACa3 would be much brighter than
those stained with a commercial probe. In addition, the two-
photon fluorescence enhancement factors (TFEFs) estimat-
Figure 1. OP absorption (a) and emission (b) spectra of 1 mm ACa2
(30 mm MOPS, 100 mm KCl, 10 mm EGTA, pH 7.2) in the presence of
free Ca2+ (0–39 mm). EGTA: ethylene glycol tetraacetic acid.
probes (Figure 4b and Figure S2 and Table S1 in the Sup-
porting Information), indicating that they could be used as
models for the latter (see below).
ed from the TP titration curves were in the range of 23–50
2+
(Table 1), values that could allowdetection of Ca
TPM.
by
When Ca2+ was added to ACa2 in 3-(N-morpholino)pro-
panesulfonic acid (MOPS) buffer solution (30 mm, pH 7.2),
the fluorescence intensity increased gradually with the
metal-ion concentration without affecting the absorption
spectra (Figure 1), probably due to the blocking of the pho-
toinduced electron-transfer (PET) process by the metal-ion
complexation.[16] Similar results were observed for ACa1
and ACa3 in OP and TP processes (Figure 2b, Figures S3
and S4 in the Supporting Information, and reference [14b]).
The pseudocolored TPM images of cultured astrocytes la-
beled with 2 mm ACa2-AM showed intense spots and homo-
geneous domains (Figure 4a). We attributed the image to
the TPEF emitted from the intracellular ACa2–Ca2+ com-
plex and membrane-bound probes, because the fluorescence
quantum yields of ACa2–Ca2+ in MOPS buffer (0.42) and
ACa2-AM in DMF (0.37) are much higher than those of
ACa2 (0.010) and ACa2-AM (0.014) in MOPS buffer
(Table 1 and Table S1 in the Supporting Information). In ad-
dition, ACa2-AM in DMF has been assumed to be a good
The fluorescence-enhancement factors [FEF=(FꢁFmin)/Fmin
]
of ACa1–ACa3 measured by the titration curves were in the
range of 23–50 (Table 1). The FEF of ACa2 is nearly identi-
cal to that of ACa1, while that of ACa3 is appreciably small-
er as a result of the larger fluorescence quantum yield (F)
in the absence, and smaller F in the presence, of excess
Ca2+ than for the others. All compounds showlarger FEFs
than the 14 reported for OG1, indicating a greater sensitivi-
ty of these probes to the change in the Ca2+-ion concentra-
tion.[3] Moreover, Hill plots for Ca2+ binding, measured by
OP and TP processes, showed good linear relationships with
model for the membrane-bound probes due to the similarity
fl
max
in l
(see above). The TPEF spectra of the intense spots
and homogeneous domains showed emission maxima at 445
(Figure 4b, blue curve) and 500 nm (red curve), respectively.
Moreover, the blue emission band was unsymmetrical and
could be fitted to two Gaussian functions with maxima at
440 and 488 nm (sky blue curve), respectively, whereas the
red emission band could be fitted to one Gaussian function
with maximum at 500 nm (brown curve). Compared with
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B. R. Cho et al.
Figure 3. a) Relative fluorescence intensity of 1 mm ACa2 in the presence
of 2 mm Mg2+ and 100 mm Zn2+, Fe2+, Mn2+, Cu2+, or Co2+ (empty bars)
followed by addition of 100 mm of Ca2+ (filled bars). b) Effect of pH on
*
the OP fluorescence intensity of 1 mm ACa2 in the presence of 0.0 (
)
2+
*
and 39 mm ( ) of free Ca in 30 mm MOPS and 100 mm KCl. The data
at [Ca2+]=0 mm were determined by adding 10 mm EGTA. These data
were measured in 30 mm MOPS buffer (100 mm KCl, 10 mm EGTA,
pH 7.2). The excitation wavelength was 365 nm.
the emission spectra recorded in MOPS buffer (Figure 1b),
the shorter-wavelength band of the dissected spectrum was
significantly blue-shifted, while the longer-wavelength band
remained similar (Table 1). The spectral shift suggests that
the probes may be located in two regions of differing polari-
ty. Furthermore, the intense spot exhibited an excited-state
lifetime of 1.2 ns, which was much longer than the upper ex-
treme of the lifetime distribution curve centered at 0.6 ns
(Figure 5). From these results, we hypothesize that the
probes are located in two different environments, the more-
polar one is cytosol (red emission with a shorter lifetime)
~
&
*
Figure 2. a) TP action spectra of ACa2 ( ), ACa3 ( ), and OG1 ( ) in
the presence of 39 mm free Ca2+. b) TP emission spectra of ACa2 (30 mm
MOPS, 100 mm KCl, 10 mm EGTA, pH 7.2) in the presence of free Ca2+
(0–39 mm). c) Hill plots for the complexation of ACa2 with free Ca2+ (0–
*
*
*
*
39 mm) measured by OP ( ) and TP ( ) processes. d) OP ( ) and TP ( )
fluorescence titration curves for the complexation of ACa2 with various
concentrations (0–39 mm) of free Ca2+. The excitation wavelengths for
OP and TP processes were 365 and 780 nm, respectively.
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Fluorescent Probes for Imaging in Live Tissue
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2.2 msꢁ1 (n=8 astrocytes; Fig-
ure 6a and b, and Movie S1 in
the Supporting Information).
The spontaneous increase in
[Ca2+]i was also propagated be-
tween astrocytes, as indicated
by the delayed activity in the
neighboring astrocyte (Fig-
ure 6a and c). The speed of
propagation of spontaneously
occurring waves was 2.1ꢀ
1.5 msꢁ1 (n=10 astrocytes).
Nearly identical results were re-
ported for ACa1-AM-labeled
astrocytes.[14b] Furthermore,
a
closer examination of Fig-
ure 6a–c reveals that ACa2 can
also detect Ca2+ in the nucleus.
Thus, these probes are clearly
capable of visualizing the intra-
and intercellular calcium waves
in cultured astrocytes by using
TPM.
We further investigated the
utility of this probe in tissue
imaging. Figure 7 reveals the
TPM images of individual as-
trocytes in acute hypothalamic
slices from postnatal one-day
rat incubated with 10 mm ACa2-
AM for 30 min at 378C. The
spontaneous Ca2+ waves in the
somas (1–3) of neurons and as-
trocytes could be simultaneous-
ly visualized with an average
Figure 4. Pseudocolored TPM images of ACa2-AM-labeled (2 mm) astrocytes collected at a) 360–620, c) 360–
460, and d) 500–620 nm. b) TPEF spectra from the hydrophobic (blue) and hydrophilic (red) domains of
ACa2-AM-labeled astrocytes. The excitation wavelength was 780 nm. Cells shown are representative images
from replicate experiments. Scale bar: 30 mm.
and the less-polar one is membrane-associated (blue emis-
sion with a longer lifetime).
frequency of about 11 mHz (n=4 slices) for more than
4000 s without appreciable decay (Supporting Information,
Movie S2). Both the intensity and the frequency of the sig-
nals varied significantly depending on the cells. Similarly,
ACa1 was capable of detecting the spontaneous Ca2+ waves
in the hypothalamic slice at a depth of ꢂ170 mm for longer
than 1100 s.[14b] In contrast, OPM images of the tetrodotox-
in-treated thalamus slice stained with fura-2[20] revealed
damaged cells on the tissue surface and was not as clear as
the TPM image presented here. Also, the fluorescence in-
tensity decayed appreciably after 500 s.[20] The improved
TPM image of ACa2-labeled tissue obtained at a depth of
ꢂ120 mm for a prolonged observation time underlines the
high photostability and lowphototoxicity of this probe, in
addition to the capability of deep-tissue imaging. Finally, it
has to be mentioned that the use of laser power higher than
that of OP fluorescence microscopy (ꢂ10 mW versus
ꢂ20 mW) does not in any way cause appreciable damage to
the cell and tissue. This can be attributed to the negligible
absorption by the sample at 780 nm, which can only lead to
vibrational and not the electronic excitation.
The intracellular Ca2+ could be detected with minimum
errors from the membrane-bound probes. The spectrum of
the shorter-wavelength band in the dissected Gaussian func-
tion (Figure 4b, sky blue curve) decreased to baseline at
ꢂ500 nm, indicating that the TPEF emitted from the mem-
brane-bound probes contributes negligible interference at
l>500 nm. Similarly, if one considers ACa2-AM in DMF as
a model for the membrane-bound probe (see above), the
fluorescence at l>500 nm accounts for only 5% of the total
emission band (Figure S2 in the Supporting Information).
Consistently, the TPEF image collected at 500–620 nm is ho-
mogeneous without the intense spots (Figure 4d), whereas
that collected at 360–460 nm clearly shows them (Figure 4c).
Therefore, one can selectively detect the intracellular Ca2+
by using the detection window at 500–620 nm.
To demonstrate the utility of this probe, we monitored
[Ca2+]i waves in live cells and tissue. The TPM images of
cultured astrocytes labeled with 2 mm ACa2-AM revealed
spontaneous Ca2+ signal propagation from the astrocytic
process (1) to soma (3) to terminal (5) with a speed of 7.2ꢀ
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B. R. Cho et al.
Synthesis of ACa1–ACa3: The synthe-
sis of ACa1 has been reported.[14b] 6-
Acyl-2-hydroxynaphthalene[22] and 5-
methyl-5’-formyl-BAPTA-tetramethyl
ester[6a] were prepared by literature
methods. The synthesis of other com-
pounds is described below.
6-Acyl-2-naphthylamine (B):
A mix-
ture of 6-acyl-2-hydroxynaphthalene
(1; 5.2 g, 28 mmol), Na2S2O5 (13.3 g,
70 mmol), and NH4OH (150 mL) in a
steel-bomb reactor was stirred at
1408C for 96 h. The product was col-
lected by filtration, washed with water,
and purified by flash-column chroma-
tography by using hexane/ethyl acetate
(1:1) as the eluent. It was further puri-
fied by recrystallization from MeOH.
Yield: 3.9 g (75%); m.p. 1708C;
1H NMR (400 MHz, CDCl3): d=8.30
(d, J=1.6 Hz, 1H), 7.91 (dd, J=8.8,
2.0 Hz, 1H), 7.74 (d, J=8.6 Hz, 1H),
7.58 (d, J=9.2 Hz, 1H), 6.97 (dd, J=
8.6, 2.2 Hz, 1H), 6.95 (d, J=2.2 Hz,
1H), 4.05 (brs, 2H), 2.66 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=
198.1, 147.0, 137.8, 131.6, 131.4, 130.7,
126.7, 126.2, 124.9, 118.9, 108.1,
26.7 ppm; IR (KBr): n˜ = 3436, 3345,
Figure 5. a) OP fluorescence intensity image and b) pseudocolored lifetime image of ACa2-AM-labeled (2 mm)
astrocytes. The excitation wavelength was 405 nm. c) Lifetime distribution; the average lifetime was ꢂ0.6 ns.
d) Single-point analysis of the region indicated by the white arrow gave a lifetime of 1.2 ns. Scale bar: 30 mm.
1662 cmꢁ1
; elemental analysis calcd
(%) for C12H11NO: C 77.81, H 5.99, N
7.56; found: C 77.78, H 5.86, N 7.59.
Compound C: Sodium triacetoxyboro-
Conclusions
hydride (1.24 g, 5.85 mmol) was added to
a mixture of B (0.64 g,
3.46 mmol) and 5-methyl-5’-formyl-BAPTA-tetramethyl ester (1.68 g,
2.92 mmol) in dichloroethane (30 mL), and the solution was stirred for
12 h under N2. The solvent was removed in vacuo and the product was
purified by column chromatography by using chloroform/ethyl acetate/
hexane (3:1:1) as the eluent. Yield: 1.6 g (74%); m.p. 868C; 1H NMR
(400 MHz, CDCl3): d=8.29 (d, J=1.8 Hz, 1H), 7.91 (dd, J=8.8, 2.0 Hz,
1H), 7.71 (d, J=8.6 Hz, 1H), 7.58 (d, J=8.6 Hz, 1H), 6.92 (m, 3H), 6.73
(m, 5H), 4.51 (brs, 1H), 4.36 (s, 2H), 4.25 (m, 4H), 4.15 (s, 4H), 4.10 (s,
4H), 3.58 (s, 6H), 3.52 (s, 6H), 2.66 (s, 3H), 2.25 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=198.0, 172.2, 172.1, 150.8, 150.5, 138.8, 138.1,
136.9, 132.6, 132.4, 131.2, 131.1, 130.6, 126.3, 125.0, 122.0, 120.7, 119.4,
119.1, 118.7, 114.3, 114.2, 112.7, 112.6, 104.8, 67.4, 67.1, 53.6, 53.5, 51.9,
51.7, 47.9, 26.7, 21.2 ppm; IR (KBr): n˜ = 3343, 1756, 1663 cmꢁ1; elemen-
tal analysis calcd (%) for C40H45N3O11: C 64.59, H 6.10, N 5.65; found: C
64.49, H 6.15, N 5.54.
We have developed a series of TP probes (ACa1–ACa3)
that show23–50-fold TPEF enhancement in response to
Ca2+, dissociation constants (KTdP) in the range of (0.14ꢀ
0.02)–(0.25ꢀ0.03) mm, and emit fourfold stronger TPEF
than OG1 upon complexation with Ca2+. The TP probes
can selectively detect dynamic levels of intracellular free
Ca2+ in live cells and living tissues without interference
from other metal ions and membrane-bound probes. More-
over, these probes are capable of monitoring the calcium
waves at a depth of 120–170 mm in live tissues for longer
than 1100–4000 s by using TPM with no artifacts of photo-
bleaching.
ACa2: A mixture of C (0.50 g, 0.67 mmol) and KOH (0.30 g, 5.36 mmol)
in EtOH/H2O (50/10 mL) was stirred for 6 h. The ethanol was evaporated
in vacuo, the residue was diluted with ice-water (30 mL), and concentrat-
ed HCl aq. was added slowly at <58C until pH 3 had been reached. The
resulting precipitate was collected and washed with distilled water. Yield:
Experimental Section
1
0.27 g (59%); m.p. 1518C; H NMR (400 MHz, CD3OD): d=8.33 (d, J=
1.6 Hz, 1H), 7.80 (dd, J=8.8, 1.6 Hz, 1H), 7.71 (d, J=9.2 Hz, 1H), 7.50
(d, J=8.6 Hz, 1H), 7.09 (d, J=2.0 Hz, 1H), 7.06 (dd, J=9.0, 2.0 Hz, 1H),
6.94 (dd, J=8.8, 2.0 Hz, 1H), 6.88 (d, J=8.8 Hz, 1H), 6.82 (d, J=9.0 Hz,
1H), 6.77 (d, J=1.6 Hz, 1H), 6.76 (d, J=2.0 Hz, 1H), 6.67 (dd, J=9.0,
2.0 Hz, 1H), 4.37 (s, 2H), 4.32 (t, 2H), 4.26 (t, 2H), 4.02 (s, 4H), 3.96 (s,
4H), 2.62 (s, 3H), 2.42 ppm (s, 3H); 13C NMR (100 MHz, CD3OD): d=
199.2, 174.8, 150.7, 149.4, 138.6, 138.0, 136.3, 134.1, 133.1, 130.9, 130.6,
130.1, 126.1, 125.8, 125.7, 124.0, 123.9, 121.5, 120.2, 119.4, 118.8, 118.2,
114.2, 112.7, 67.2, 66.9, 55.2, 55.0, 46.7, 25.3, 25.2, 19.8 ppm; IR (KBr): n˜
General methods: Synthetic reagents were purchased from Sigma-Al-
drich and TCI, and used without further purification. All solvents were
from Riedel-de Han and Sigma-Aldrich and were distilled prior to use.
NMR spectra were obtained on a Varian AS400 MHz spectrophotometer
1
operating at 283 K and both H and 13C NMR spectra were referenced to
internal probe standards. IR spectra were obtained by using a Nicolet
380 FTIR instrument, and samples were prepared as KBr pellets. Ele-
mental analyses were performed on a Carlo Erba 1108 element analyzer.
Absorption spectra were recorded on a Hewlett-Packard 8453 diode
array spectrophotometer, and fluorescence spectra were obtained with
Amico-Bowman series 2 luminescence spectrometer with a 1 cm standard
quartz cell. The fluorescence quantum yield was determined by using
Coumarin 307 as the reference by the literature method.[21]
=
3350, 2915, 1750, 1662 cmꢁ1
; elemental analysis calcd (%) for
C36H37N3O11: C 62.87, H 5.42, N 6.11; found: C 62.64, H 5.90, N 6.02.
ACa2-AM: A mixture of ACa2 (0.12 g, 0.17 mmol), bromomethyl acetate
(0.21 g, 1.4 mmol), and Et3N (0.14 g, 1.4 mmol) in CHCl3 (5 mL) was
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Fluorescent Probes for Imaging in Live Tissue
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Figure 7. a,b) Pseudocolored TPM images of an acute rat hypothalamic
slice stained with 10 mm ACa2-AM taken after 40 (a) and 1460 s (b).
Figure 6. a) Pseudocolored TPM images of ACa2-AM-labeled astrocytes
taken after 20 s. b,c) Time courses of the calcium waves in different loca-
tions showing the spontaneous intracellular (b) and intercellular (c) Ca2+
propagation. All images were collected at 500–620 nm with 1.6 s intervals
and the excitation wavelength was 780 nm with femtosecond pulses. Scale
bar: 30 mm.
Magnification at 100” shows the hypothalamic area at
a depth of
ꢂ120 mm. c) Spontaneous Ca2+ transients recorded in cells (1–3). The
TPEF images were collected at 500–620 nm with 1.6 s intervals and the
excitation wavelength was 780 nm with femtosecond pulses. Scale bar:
30 mm.
stirred under N2 for 24 h. The solvent was removed in vacuo and the
crude product was purified by column chromatography by using ethyl
acetate/hexane (3:1) as the eluent. It was further purified by recrystalliza-
tion from MeOH. Yield: 85 mg (51%); m.p. 788C; 1H NMR (400 MHz,
CDCl3): d=8.30 (d, J=1.6 Hz, 1H), 7.91 (dd, J=8.8, 1.6 Hz, 1H), 7.72
(d, J=8.6 Hz, 1H), 7.59 (d, J=9.0 Hz, 1H), 6.95 (m, 3H), 6.75 (m, 5H),
5.61 (s, 4H), 5.56 (s, 4H), 4.65 (brs, 1H), 4.36 (s, 2H), 4.27 (s, 4H), 4.20
(s, 4H), 4.15 (s, 4H), 2.67 (s, 3H), 2.27 (s, 3H), 2.07 (s, 6H), 2.05 ppm (s,
6H); 13C NMR (100 MHz, CDCl3): d=198.1, 170.5, 170.4, 169.8, 169.7,
150.9, 150.6, 148.4, 138.2, 136.3, 135.4, 133.4, 133.1, 131.2, 131.1, 130.6,
130.4, 126.3, 125.0, 122.2, 121.1, 119.8, 118.8, 114.5, 114.4, 113.0, 104.0,
79.5, 79.4, 79.3, 67.4, 67.1, 53.6, 53.5, 47.9, 26.7, 22.9, 20.9 ppm; IR (KBr):
Chem. Eur. J. 2008, 14, 2075 – 2083
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B. R. Cho et al.
n˜
=
3345, 1750, 1710, 1662 cmꢁ1; elemental analysis calcd (%) for
probe and Ca2+, respectively. The total probe and metal-ion concentra-
tions are defined as [L]0 =[L]+[LM] and [M]0 =[M]+[LM], respectively.
With [L]0 and [M]0, the value of Kd is given by [Eq. (2)]:
C48H53N3O19: C 59.07, H 5.47, N 4.31; found: C 58.80, H 5.78, N 4.12.
ACa3: Compound C (0.80 g, 1.08 mmol) was dissolved in MeCN (30 mL)
and proton sponge (0.46 g, 2.15 mmol), NaI (0.032 g, 0.22 mmol), and
CH3I (0.46 g, 3.24 mmol) were added. The reaction mixture was refluxed
for 12 h under N2. The solvent was removed in vacuo and the product
was purified by column chromatography by using chloroform/ethyl ace-
tate/hexane (3:1:1) as the eluent. Yield: 0.73 g (90%); m.p. 828C;
1H NMR (400 MHz, CDCl3): d=8.30 (d, J=1.6 Hz, 1H), 7.91 (dd, J=
8.6, 1.8 Hz, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.60 (d, J=9.0 Hz, 1H), 7.16
(dd, J=9.0, 2.0 Hz, 1H), 6.90 (d, J=2.0 Hz, 1H), 6.66 (m, 6H), 4.60 (s,
2H), 4.20 (s, 4H), 4.13 (s, 4H), 4.08 (s, 4H), 3.55 (s, 6H), 3.51 (s, 6H),
3.14 (s, 3H), 2.67 (s, 3H), 2.25 ppm (s, 3H); 13C NMR (100 MHz, CDCl3):
d=198.0, 172.3, 172.2, 150.8, 150.4, 138.5, 137.9, 136.9, 132.4, 132.2, 131.0,
130.6, 126.4, 125.4, 124.8, 121.9, 119.7, 119.3, 119.1, 116.6, 114.2, 111.4,
105.6, 67.2, 67.0, 58.7, 56.3, 53.6, 53.5, 51.8, 51.7, 39.0, 26.7, 21.2,
18.7 ppm; IR (KBr): n˜ = 1756, 1662 cmꢁ1; elemental analysis calcd (%)
for C41H47N3O11: C 64.98, H 6.25, N 5.54; found: C 65.04, H 6.19, N 5.59.
2
½LMŠ ꢁð½LŠ0 þ ½MŠ0 þ KdÞ½LMŠ þ ½LŠ ½MŠ
0
0
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð2Þ
2
ð½LŠ0 þ ½MŠ0 þ KdÞꢁ ð½LŠ0 þ ½MŠ0 þ KdÞ ꢁ4½LŠ ½MŠ
0
0
¼ 0, ½LMŠ ¼
2
or [Eq. (3)]:
ðFꢁFmin
Þ
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
2
ð½LŠ0 þ ½MŠ0 þ KdÞꢁ ð½LŠ0 þ ½MŠ0 þ KdÞ ꢁ4½LŠ ½MŠ
0
0
¼
ðFmaxꢁFminÞ
2½LŠ
0
ð3Þ
in which F is the observed fluorescence intensity, Fmin is the minimum
fluorescence intensity, and Fmax is the maximum fluorescence intensity.
The Kd value that best fits the titration curve (Figure 2d and Figure S3c
in the Supporting Information) with Equation (3) was calculated by using
the Excel program as reported.[18]
To determine KTdP for the TP process, the TPEF spectra were obtained
with a DM IRE2 microscope (Leica) by using the xyl mode at a scan
speed of 800 Hz. They were excited by a mode-locked titanium–sapphire
laser source (Coherent Chameleon, 90 MHz, 200 fs) set at a wavelength
of 780 nm and output power 1180 mW, which corresponded to approxi-
mately 10 mW average power in the focal plane. The TPEF titration
curves (Figure 2d and Figure S3c in the Supporting Information) were
obtained and fitted to Equation (3).
This ester (0.50 g, 0.66 mmol) was hydrolyzed by the method described
above. Yield: 0.24 g (52%); m.p. 1418C; 1H NMR (400 MHz, CD3OD):
d=8.35 (d, J=1.8 Hz, 1H), 7.82 (dd, J=8.8, 1.6 Hz, 1H), 7.79 (d, J=
8.8 Hz, 1H), 7.59 (d, J=9.0 Hz, 1H), 7.26 (dd, J=9.0, 2.0 Hz, 1H), 6.98
(d, J=2.0 Hz, 1H), 6.82 (m, 6H), 4.67 (s, 2H), 4.25 (m, 4H), 3.96 (s,
4H), 3.90 (s, 4H), 3.16 (s, 3H), 2.63 (s, 3H), 2.24 ppm (s, 3H); 13C NMR
(100 MHz, CD3OD): d=199.0, 172.6, 172.5, 150.4, 150.2, 138.4, 138.1,
137.6, 135.3, 134.2, 130.8, 130.7, 125.1, 124.7, 121.3, 119.4, 119.1, 118.7,
118.4, 116.2, 114.3, 112.7, 112.1, 105.8, 67.3, 67.0, 58.3, 56.3, 54.5, 51.6,
39.2, 25.3, 19.0 ppm; IR (KBr): n˜ = 2915, 1748, 1663 cmꢁ1; elemental
analysis calcd (%) for C37H39N3O11: C 63.33, H 5.60, N 5.99; found: C
63.22, H 5.75, N 5.91.
Measurement of a two-photon cross-section: The TP cross-section (d)
was determined by using the femtosecond fluorescence measurement
technique as described previously.[24] ACa1–ACa3 and OG1 were dis-
solved in MOPS buffer (30 mm; 100 mm KCl, 10 mm EGTA, pH 7.2) at
concentrations of 1.0”10ꢁ5 m and then the TP-induced fluorescence in-
tensity was measured at 740–940 nm by using fluorescein (8.0”10ꢁ5 m,
pH 11) as the reference, the TP property of which has been well charac-
terized in the literature.[25] The intensities of the TP-induced fluorescence
spectra of the reference and sample emitted at the same excitation wave-
length were determined. The TPA cross-section was calculated according
to Equation (4):
ACa3-AM: This compound was prepared by using the procedure de-
scribed for ACa2-AM. Yield: 54%; m.p. 1028C; 1H NMR (400 MHz,
CDCl3): d=8.30 (d, J=1.6 Hz, 1H), 7.92 (dd, J=8.6, 1.6 Hz, 1H), 7.77
(d, J=8.8 Hz, 1H), 7.62 (d, J=9.0 Hz, 1H), 7.17 (dd, J=9.0, 2.0 Hz, 1H),
6.93 (d, J=2.0 Hz, 1H), 6.74 (m, 6H), 5.60 (s, 4H), 5.58 (s, 4H), 4.60 (s,
2H), 4.22 (s, 4H), 4.17 (s, 4H), 4.14 (s, 4H), 3.15 (s, 3H), 2.67 (s, 3H),
2.26 (s, 3H), 2.05 (s, 6H), 2.04 ppm (s, 6H); d=198.0, 171.1, 170.8, 169.7,
169.6, 150.5, 150.2, 148.7, 138.6, 136.3, 135.4, 133.3, 133.2, 131.7, 131.6,
130.8, 130.5, 126.5, 125.2, 122.7, 121.1, 119.8, 118.8, 114.7, 114.6, 113.2,
104.0, 79.7, 79.5, 79.4, 68.4, 68.1, 54.6, 54.5, 47.9, 42.8, 26.7, 23.2,
21.0 ppm; IR (KBr): n˜ = 1750, 1710, 1660 cmꢁ1; elemental analysis calcd
(%) for C49H55N3O19: C 59.45, H 5.60, N 4.24; found: C 59.42, H 5.54, N
4.30.
SSFrꢀrcr
SrFSꢀScS
ð4Þ
d ¼
dr
Determination of apparent dissociation constants: A series of calibration
solutions containing various [Ca2+] was prepared by mixing two solutions
(solution A, containing 10 mm K2EGTA, and solution B, containing
10 mm CaEGTA) in various ratios.[3,23] Both solutions contained ACa1–
ACa3 (1 mm), KCl (100 mm), and MOPS (30 mm), and were adjusted to
pH 7.2.
Cell culture: Astrocytes were taken from cerebral cortices of one-day-old
rats (Sprague–Dawley; SD). Cerebral cortices were dissociated in Hankꢀs
balanced salt solution (Gibco BRL, Gaithersburg, MD, USA), containing
papain (3 UmLꢁ1; Worthington Biochemical Corporation, NJ, USA) and
plated in 75 mm flasks. To prepare a purified astrocyte culture, the flasks
were shaken for 6 h on a shaker at 378C and the floating cells that were
displaced into the medium were removed. Astrocytes were passaged with
To determine the Kd values for Ca2+ complexes with ACa1–ACa3, the
fluorescence spectrum was recorded with solution A (2.0 mL; 0 mm free
Ca2+) at 208C. Then this solution (203 mL) was discarded and replaced
by solution B (203 mL; 39 mm free Ca2+), and the spectrum was recorded.
5 min exposure to 0.25% trypsin, replated onto polyCAHTRE(UGN d-lysine)-coated
glass coverslips at 50–100 cells per mm2, and maintained in Dulbeccoꢀs
modified Eagleꢀs medium (DMEM; Gibco) supplemented with penicillin/
streptomycin and 10% fetal bovine serum (FBS; Gibco) in a CO2 incuba-
tor at 378C. After 7–15 days in vitro, the astrocytes were washed three
times with serum-free medium, and then incubated with ACa2-AM
(2 mm) in serum-free medium for 20 min at 378C. The cells were washed
three times with phosphate-buffered saline (PBS; Gibco) and then
imaged after further incubation in colorless serum-free medium for
15 min.
This brought the CaEGTA concentration to 1.00 mm and [Ca2+
]
free
to
about 0.017 mm with no change in the concentration of the probe or of
the total EGTA. [Ca2+
]
was calculated from the Kd of EGTA for Ca2+
free
(150.5 nm) by using Equation (1).[3,23]
½CaEGTAŠ
½Ca2þ
Š
free
¼ KEd GTA
Â
ð1Þ
½K2EGTAŠ
Two-photon fluorescence microscopy: TP fluorescence microscopy
images of ACa2-AM-labeled astrocytes and tissues were obtained with
spectral confocal and multiphoton microscopes (Leica TCS SP2) with a
”100 oil objective and numerical aperture (NA)=1.30. The images were
obtained with a DM IRE2 microscope (Leica) by exciting the probes
with a mode-locked titanium–sapphire laser source (Coherent Chame-
leon, 90 MHz, 200 fs) set at a wavelength of 780 nm and output power
1180 mW, which corresponded to approximately 10 mW average power in
Further iterations attained 0.038, 0.065, 0.101, 0.150, 0.230, 0.350, 0.601,
0.800, 1.00, 1.30, 2.50, 5.30, 10.0, and 20.0 mm free Ca2+ by successively
discarding 223, 251, 285, 327, 421, 479, 667, 420, 350, 412, 905, 1028, 926,
and 992 mL of solution A and replacing each with an equal volume of sol-
ution B.
When a 1:1 metal–ligand complex is formed between probe and Ca2+
,
one can describe the equilibrium as follows, in which L and M represent
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Fluorescent Probes for Imaging in Live Tissue
FULL PAPER
the focal plane. To obtain images in the ranges 360–620, 360–460, and
500–620 nm, internal photomultiplier tubes were used to collect the sig-
nals in 8-bit unsigned 512”512 pixels at a scan speed of 400 Hz.
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378C. The cells were washed with PBS three times, fixed with formalde-
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by time-correlated single-photon counting by using an SPC830 acquisi-
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Preparation and staining of acute rat hippocampal and hypothalamic
slices: Slices were prepared from the hippocampi and the hypothalmi of
two-day-old rats (SD). Coronal slices were cut into 400-mm-thick slices
by using a vibrating-blade microtome in artificial cerebrospinal fluid
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This work was supported by a Korea Science and Engineering Founda-
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Received: September 13, 2007
Published online: January 3, 2008
Chem. Eur. J. 2008, 14, 2075 – 2083
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2083