A two-photon fluorescent probe for near-membrane calcium ions in live cells and tissues

Article abstract of DOI:10.1039/b911337a

A two-photon fluorescent probe (ACaL) is reported that can be excited by 780 nm fs pulses, shows high photostability and negligible toxicity, and can visualize near-membrane Ca²⁺ in live cells and deep inside live tissues by two-photon microsco

Full text of DOI:10.1039/b911337a

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A two-photon fluorescent probe for near-membrane calcium ions in live
cells and tissuesw
a
b
Palathurai Subramaniam Mohan,z Chang Su Lim,z Yu Shun Tian,b Won Young Roh,b
Jun Han Leeb and Bong Rae Cho*b
Received (in Cambridge, UK) 10th June 2009, Accepted 1st July 2009
First published as an Advance Article on the web 29th July 2009
DOI: 10.1039/b911337a
A two-photon fluorescent probe (ACaL) is reported that can be
excited by 780 nm fs pulses, shows high photostability and
negligible toxicity, and can visualize near-membrane Ca2+ in
live cells and deep inside live tissues by two-photon microscopy.
cells and tissues at 4100 mm depth by TPM. Our strategy was
to link the 5-methyl derivative of O,O0-bis(2-aminophenyl)-
ethyleneglycol-N,N,N0,N0-tetraacetic acid (BAPTA-5-Me)
with 2-dimethylamino-6-laurylnaphthalene (laurdan) as the
TP chromophore. It was expected that ACaL would be
predominantly located in the plasma membrane with the
orientation of the dodecanoyl group in the membrane interior
and the BAPTA-5-Me moiety lying near the membrane
surface and thereby able to trap the Ca2+ extruded from
the cytoplasm, causing fluorescence. It is to be noted that
BAPTA-5-Me is a well known Ca2+ chelator that has been
widely employed in various one-photon fluorescent probes
such as Fura-2, Fluo-3, Rod-2,8 as well as in a TP probe
(ACa2),7 whereas laurdan is a TP polarity probe that has been
used to detect the lipid rafts in the cell membrane.9
Calcium is a versatile intracellular signal messenger controlling
numerous cellular functions; it plays vital roles in many cellular
processes such as fertilization, cell death, sensory transduction,
muscle contraction, motility, exocytosis, and fluid secretion.1
Intracellular free Ca2+ levels ([Ca2+]i) are maintained by the
coordinated actions of various calcium pumps, ion channels,
and calcium-buffering proteins. When stimulated with agonists,
cytosolic Ca2+ levels are rapidly elevated either by Ca2+-influx
from the extracellular space or by Ca2+-release from internal
Ca2+ stores such as endoplasmic reticulum (ER). In resting
cells, which must maintain low [Ca2+]i, the excess Ca2+ must be
extruded to the outside via plasma membrane Ca2+ ATPase
(PMCA) or transported back into the ER through sarcoplasmic/
endoplasmic reticulum Ca2+ ATPase (SERCA).2
To investigate the changes in near-membrane Ca2+ con-
centration and the translocation of Ca2+ across the plasma
membrane, C18-Fura-2 and Calcium Green C18 have been
developed.3 However, use of these probes with one-photon
microscopy (OPM) results in blurred cell images. Moreover,
tissue imaging was not possible due to the short excitation
wavelength (o500 nm). To visualize the near-membrane Ca2+
deep inside live tissues, it is crucial to use two-photon microscopy
(TPM). TPM, which employs two lower energy, near-infrared
photons as the excitation source, has the advantages of increased
penetration depth (4500 mm), localized excitation, and pro-
longed observation time.4–6 Recently, we reported a series of
two-photon (TP) probes for calcium ion (ACa1–ACa3)7 that can
visualize the calcium waves in live cells and tissues at 4100 mm
depth by TPM for a long period of time with minimum
interference from tissue preparation artifacts, self-absorption,
auto-fluorescence, photobleaching, and photodamage.
The preparation of ACaL is described in the ESI.w The
absorption and emission spectra of ACaL showed gradual red
shifts with the solvent polarity in the order 1,4-dioxane
o DMF o EtOH o EtOH–H2O (1 : 1) (Fig. S1 and Table S1,
ESIw). The effect was greater for the emission (54 nm) than
absorption spectra (13 nm), thus indicating the utility of this
compound as a polarity probe.
When Ca2+ was added to ACaL in 3-(N-morpholino)-
propanesulfonic acid (MOPS) buffer solution (30 mM, 100 mM
KCl, 10 mM EGTA, pH 7.2), the fluorescence intensity
increased dramatically as a function of metal ion concentra-
tion (Fig. 1(a)), probably due to the blocking of the photo-
induced electron transfer (PeT) process by the complexation
with the metal ions. A nearly identical result was observed in
the two-photon process (Fig. S2a in ESIw). The fluorescence
enhancement factors [(F ꢀ Fmin)/Fmin] of ACaL determined
for the one- and two-photon processes were 12 and 10,
respectively, in the presence of 39 mM Ca2+ (Table 1 and
Fig. S2a in ESIw).10 The values are similar to that (14) reported
for Calcium-Green.3a Moreover, linear Hill plots determined
for Ca2+ binding with slopes of 1.0 indicated 1 : 1 complexa-
tion between ACaL and Ca2+ (Fig. S2c in ESIw).11
As an extension to these studies, we have now developed a
novel TP probe for near-membrane Ca2+ (ACaL; Scheme 1)
that is capable of imaging the near-membrane Ca2+ in live
ACaL showed a modest response toward Zn2+ and Mn2+
,
a much weaker response toward Mg2+, and no response
a On leave from Department of Chemistry, Bharathiar University,
Coimbatore-641046, India
b Department of Chemistry, Korea University, 1-Anamdong, Seoul,
Korea. E-mail: chobr@korea.ac.kr; Fax: (+82) 2-3290-3544;
Tel: (+82) 2-3290-3129
w Electronic supplementary information (ESI) available: Experimental
details for the synthesis, photophysical studies, and cell and tissue
imaging. See DOI: 10.1039/b911337a
z These two authors contributed equally to this work.
Scheme 1 The structure of ACaL.
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Chem. Commun., 2009, 5365–5367 | 5365

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values measured in the vesicles would better reflect its binding
ability in the cell membrane compared with that measured in
the MOPS buffer; ACaL can detect near-membrane Ca2+ in
the submicromolar range.
The TP action spectra of the Ca2+ complex with ACaL in
buffer solution indicated a Fd value of 90 GM at 780 nm for
ACaL–Ca2+, 2.5-fold larger than those of Calcium-Green-Ca2+
and Fura-2-Ca2+ (Fig. 1(b) and Table 1). Since the TP action
cross section (Fd) of C18-Fura-2 and Calcium Green C18 are
expected to be similar to those of Calcium-Green-Ca2+ and
Fura-2-Ca2+, TPM images for samples stained with ACaL
would be much brighter than those stained with the existing
probes.
Fig. 1 (a) One-photon fluorescence spectra of 2 mM ACaL in MOPS
buffer in the presence of free Ca2+ (0–39 mM). (b) Two-photon action
spectra of ACaL (K), Fura-2 (’) and Calcium-Green (&) in MOPS
buffer in the presence of 39 mM free Ca2+
.
The pseudo-colored TPM image of cultured ROS cells [rat
osteoblast-like osteosarcoma cells (ROS 17/2.8)] labeled with
5 mM ACaL clearly reveals the Ca2+ distribution in the
plasma membrane. The TPEF spectrum from the plasma
membrane (red curve) was unsymmetrical and could be fitted
to two Gaussian functions with peak maxima at 435 nm (blue
curve) and 475 nm (green curve), respectively (Fig. 2(b)).
Moreover, the spectrum is very similar to that measured in
the raft mixture with similar peak maxima of the dissected
Gaussian functions (Fig. S5 in ESIw). Hence, the TPM
image can most reasonably be attributed to the AcaL–Ca2+
complexes associated with lo and ld domains in the plasma
membrane. Further, the TPM images collected at 360–460
and 500–620 nm ranges are almost the same except for the
brightness (Fig. 2(c) and (d)). This indicates that Ca2+ is
almost evenly distributed in both domains. Therefore, we have
detected the near-membrane Ca2+ with ACaL by TPM by
using the detection window at 360–620 nm.
toward Fe2+, Cu2+, Co2+, Ba2+, and Sr2+ (Fig. S3a in ESIw).
As the intracellular free-ion concentration of Mn2+ is negligible,
this probe can detect the intracellular Ca2+ concentration
in the regions in which the chelatable Zn2+ concentration
is much lower than KdTP(Ca2+). Furthermore, it was
pH-insensitive at pH 46.5 (Fig. S3b in ESIw).
To assess whether ACaL can detect near-membrane Ca2+
,
OP
the Kd
values were determined in vesicles composed of
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/40 mol%
cholesterol (CHL), 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC) and DOPC–sphingomyelin–CHL (1 : 1 : 1, raft
mixture). It is well established that the cell membrane is
composed of liquid-ordered (lo) and liquid-disordered (ld)
domains, and DPPC/CHL, DOPC, and raft mixture are good
models for the lo and ld domains and the cell membrane,
respectively.9,12 The KdOP values measured in these vesicles are
0.082 ꢂ 0.008, 0.11 ꢂ 0.02 and 0.097 ꢂ 0.010 mM, respectively
(see footnotes for Table 1 and Fig. S4b, S4e and S4h in
ESIw).10 The values are B2-fold larger than that measured
To demonstrate the utility of this probe, we have monitored
TPEF intensity of the ACaL-labeled ROS cells after addition
of parathyroid hormone (PTH), a hormone that stimulates the
cells to release free Ca2+ ([Ca2+]i) from intracellular stores.13
Because [Ca2+]i must be restored to the basal level, the excess
[Ca2+]i must be extruded from the cell via PMCA and/or
transported to ER through SERCA.2,8 Hence, the near-
membrane Ca2+ concentration is expected to increase upon
treatment with PTH and then decrease with time. Indeed, the
TPEF intensities in the cell membrane began to rise at
50–100 s after addition of PTH (100 nM), reached at the
maxima after 400–500 s, and then decreased to the basal
levels after B1200 s. When PTH was added after treating
the cells for 10 min with thapsigarin (5 mM), an inhibitor that
blocks the re-entry of Ca2+ into intracellular store formed by
OP
in MOPS buffer and comparable to Kd = (0.062 ꢂ 0.007)
mM reported for Calcium Green C18 in DOPC.3a The larger
OP
Kd value of ACaL than that of ACa2 can be attributed to
the lauryl group, which would stabilize the ACaL–Ca2+
complex by inducing a more hydrophobic environment
around BAPTA. Moreover, the emission spectra of the
ACaL–Ca2+ complex show lmax at 435 and 462 nm in
DPPC/CHL and DOPC, whereas that from the raft mixture
shows lmax at 439 nm with a shoulder, which could be fitted
to two Gaussian functions centered at 432 and 468 nm,
respectively (Fig. S5 in ESIw). This indicates that the emission
from the AcaL–Ca2+ complex can reasonably reflect both of
OP
the lo and ld domains in the raft mixture. Therefore, the Kd
Table 1 Photophysical data for ACaL, Calcium-green and Fura-2a
Fc
KdOP/Kd
lmax
Fdf
fl b
TP d
(2) e
Compd.
lmax(1)/lmax
ACaL
ACaL + Ca2+
Calcium Green C18 + Ca2+
C18-Fura-2 + Ca2+
369/500
372/502
494/523j
335/505l
0.0037
0.043
0.75j
0.49l
ndg
780
800k
ndgh
90
0.045i/0.041
0.17j/ndg
0.14l/ndg
37k
780m
36m
a
b
All data were measured in 30 mM MOPS buffer in the absence and presence (39 mM) of free Ca2+
.
lmax of the one-photon absorption and
emission spectra in nm. Fluorescence quantum yield. The uncertainty is ꢂ10%. Dissociation constants for Ca2+ in mM measured by
c
d
one- (KdOP) and two-photon (KdTP) processes. The uncertainty is ꢂ12%. lmax of the two-photon excitation spectra in nm. The two-photon
e
f
action cross section in 10ꢀ50 cm4 s photonꢀ1 (GM). The uncertainty is ꢂ15%. Not determined. TPEF intensity was too weak to measure the
g
h
i
OP
TP action cross section accurately. Kd values measured in DPPC/CHL, DOPC, and raft mixture are 0.082 ꢂ 0.008, 0.11 ꢂ 0.02, and
j
k
l
m
0.097 ꢂ 0.010 mM, respectively. Ref. 3a Measured by using Calcium Green. Ref. 3b Measured by using Fura-2.
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Fig. 4 Images of a fresh rat hippocampal slice labeled with 10 mM
ACaL. (a) Bright-field images showing the CA1 and CA3 regions as
well as dentate gyrus (DG) upon 10ꢃ magnification. White dotted
lines indicate the pyramidal neuron layers. (b) TPM images of the red
labeled region at a depth of B120 mm by 100ꢃ magnification. (c) TPM
images of the same region after addition of 200 mM EGTA to (b).
Images were collected at 360–620 nm upon excitation at 780 nm with fs
pulses. Scale bar, 300 (a) and 30 (b, c) mm, respectively.
Fig. 2 Pseudo-colored TPM images of ACaL-labeled (5 mM) ROS
cells collected at 360–620 nm (a), 360–460 nm (c), and 500–620 nm (d).
(b) Two-photon excited fluorescence spectrum from the plasma
membrane (red curve). The sky blue and pink curves represent the
dissected Gaussian functions. The excitation wavelength was 780 nm.
Cells shown are representative images from replicate experiments
(n = 5). Scale bar, 30 mm.
37 1C. The bright field image reveals the CA1 and CA3 regions
as well as the dentate gyrus (DG, Fig. 4(a)). The TPM image
obtained at a higher magnification clearly reveals Ca2+
distribution in the cell membrane at 120 mm depth in live
tissue (Fig. 4(b)). When EGTA was added to the imaging
solution, the TPEF intensity decreased (Fig. 4(c)). These
findings demonstrate that ACaL is capable of detecting
membrane Ca2+ at 120 mm depth in live tissues using TPM.
To conclude, we have developed a TP probe (ACaL) that
14
endoplasmic reticulum (ER),
the TPEF intensities were
more enhanced and the decay rates were slower (Fig. 3(d)).
Further, the TPEF intensity decreased upon treatment with
ethylene glycol tetraacetic acid (EGTA), a membrane-permeable
Ca2+ chelator that can effectively remove Ca2+ (Fig. S6 in
ESIw). It is to be noted that the TPM image shown in Fig. 3 is
much clearer and brighter than the OPM image obtained with
shows 10-fold TPEF enhancement in response to Ca2+
,
TP
dissociation constants (Kd
)
of (0.041
ꢂ
0.005) mM,
pH-insensitive in the biologically relevant pH, and emit
2.5-fold stronger TPEF than Calcium-Green and Fura-2 upon
complexation with Ca2+. Better than the currently available
probes, this probe can visualize near-membrane Ca2+ in live
cells for more than 1500 s and living tissues at 120 mm depth
without interference from other metal ions.
Calcium Green C18 after addition of external 1.2 mM Ca2+
.
Hence, ACaL is far superior to the existing probes and is
clearly capable of detecting near-membrane Ca2+ in live cells
for longer than 1500 s.
We further investigated the utility of this probe in tissue
imaging. TPM images were obtained from a part of fresh rat
hippocampal slice incubated with 10 mM ACaL for 30 min at
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Fig. 3 Pseudo-colored TPM images of ROS cells labeled with ACaL
(5 mM) in the absence (a) and presence of thapsigargin (5 mM) (b).
(c, d) Time courses of TPEF at each designated position for samples a
(c) and b (b) after stimulation with PTH (100 nM). Images were
collected at 360–620 nm with 1.6 s intervals using an excitation
wavelength at 780 nm. Scale bar, 30 mm.
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Chem. Commun., 2009, 5365–5367 | 5367