Dual-color imaging of sodium/calcium ion activities with twophoton fluorescent probes

Article abstract of DOI:10.1002/anie.201002907

A two-photon probe (BCaM) shows 14fold enhancement in two-photon emission fluorescence in response to Ca²⁺ and shows high sensitivity and selectivity for near-membrane Ca²⁺ ions (see picture). Combined with the known Na⁺ twophoton probe ANa1, BCaM allows simultaneous dual-color imaging of Ca²⁺/Na⁺ activities within live cells and in tissues at more than 100 μm depth for long time periods without photobleaching. (Figure Presented)

Full text of DOI:10.1002/anie.201002907

Communications
DOI: 10.1002/anie.201002907
Two-Photon Fluorescent Probes
Dual-Color Imaging of Sodium/Calcium Ion Activities with Two-
Photon Fluorescent Probes**
Hyung Joong Kim, Ji Hee Han, Mi Kyung Kim, Chang Su Lim, Hwan Myung Kim,* and
Bong Rae Cho*
Calcium ions act as a ubiquitous second messenger that
controls various functions in cells.[1,2] In mammallian cells, the
cytosolic free Ca2+ concentration ([Ca2+]c) is maintained at
about 0.1 mm by the coordinated actions of various calcium
ion channels and transporters,[1,2] whereas the extracellular
Ca2+ concentration is greater than 1 mm. The near-membrane
Ca2+ concentration ([Ca2+]m) is much higher than [Ca2+]c and
can reach values of greater than 100 mm upon activation.[2]
The domains with high [Ca2+]m are the key regions that
regulate physiological processes, such as exocytosis, ion-
channel activities, and sensory transductions.[2]
Na+/Ca2+ exchange is an important activity that is relevant
to Ca2+ homeostasis,[2,3] and the simultaneous detection of
Scheme 1. Structures of ACaL, BCaM, and ANa1.
Na+ and Ca2+ near the cell membrane is crucial to under-
standing this process. Two-photon microscopy (TPM), a
technique that utilizes two photons of lower energy for
excitation,[4] is an ideal tool to study Na+/Ca2+ exchange.
Combined with appropriate TP probes, TPM can visualize
biological events within live cells and deep inside intact
tissues (> 500 mm) for an extended periods of time.[5] Very
recently, we reported TP probes for [Ca2+]m (ACaL) and
that of ANa1. We have therefore designed a new TP probe for
[Ca2+]m (BCaM, Scheme 1) derived from 2-(2’-morpholino-2’-
oxoethoxy)-N,N-bis(hydroxycarbonylmethyl)aniline
(MOBHA) as the Ca2+ receptor and 6-(benzo[d]oxazol-2’-yl)-
2-(N,N-dimethylamino)naphthalene as the reporter. Herein,
we show that, by using BCaM and ANa1, we can simulta-
neously detect [Ca2+]m and [Na+]i in live cells and tissues at
depths of more than 100 mm for lengthy periods of time
without photobleaching problems.
intracellular free Na+ ([Na+]i) (ANa1) that can detect [Ca2+
]
m
and [Na+]i, respectively, in live cells and intact tissues by TPM
(Scheme 1).[6,7] However, the dissociation constant of ACaL
(Kd = 41 nm) was too small to detect [Ca2+
]
at the 100 mm
The preparation of BCaM is described in the Supporting
Information. The water solubility of BCaM was approxi-
mately 5 mm, which was sufficient to stain the cells (Support-
ing Information, Figure S1). The absorption and emission
spectra of BCaM showed gradual red shifts with increasing
solvent polarity (Supporting Information, Figure S2). The
shifts were greater for the fluorescence emission (40 nm) than
for the absorption spectra (5 nm), but not as sensitive as
ANa1 to the polarity of the environment (Dlfl = 40 vs
79 nm).[7] Moreover, the emission band of BCaM was well
separated from that of ANa1 (Figure 1d).
m
level. Moreover, simultaneous detection of the two ions was
not possible because the emission bands from the two probes
appeared in the same wavelength range. Overcoming these
problems requires the development of an efficient TP probe
for [Ca2+]m that shows Kd of about 100 mm and emits TP
excited fluorescence (TPEF) at a wavelength different from
[*] H. J. Kim,[+] M. K. Kim, Prof. H. M. Kim
Division of Energy Systems Research, Ajou University
Suwon, 443-749 (Korea)
When Ca2+ was added to BCaM in 3-(N-morpholino)pro-
panesulfonic acid (MOPS) buffer solution (30 mm, 100 mm
KCl, pH 7.2), the fluorescence intensity increased dramati-
cally as a function of metal ion concentration (Figure 1a),
which is probably due to the blocking of the photoinduced
electron transfer (PET) process by the complexation with the
metal ions. A nearly identical result was observed in the TP
process (Supporting Information, Figure S3). The fluores-
cence enhancement factors (FEF = (FÀFmin)/Fmin) of BCaM
determined for the one- and two-photon processes in the
presence of 2.5 mm Ca2+ was 13 and 14, respectively. It is
worth noting that the BCaM/Ca2+ complex shows the largest
fluorescent quantum yield (F = 0.98) reported to date among
Ca2+ ion probes (Supporting Information, Table S1). The
Fax: (+82)31-219-1615
E-mail: kimhm@ajou.ac.kr
J. H. Han,[+] C. S. Lim, Prof. B. R. Cho
Department of Chemistry, Korea University
1-Anamdong, Seoul, 136-701 (Korea)
Fax: (+82)2-3290-3544
E-mail: chobr@korea.ac.kr
[+] These two authors contributed equally to this work.
[**] This work was supported by the National Research Foundation
(NRF) grants funded by the Korean Government (No. 2009-0065783
and 2009-0083078) and Priority Research Centers Program through
the NRF funded by the Ministry of Education, Science, and
Technology (2009-0093826).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002907.
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the cells stained with BCaM would be much brighter than
those stained with the commercial probes.
To test whether BCaM can detect near-membrane Ca2+,
we have determined the TPEF spectra of BCaM in large
unilamellar vesicles (LUVs) composed of 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine/cholesterol (DPPC)/40 mol% cho-
lesterol (CHL), DOPC/sphingomyelin/CHL (1:1:1, raft mix-
ture), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)
in the presence of excess Ca2+. It is well-established that the
cell membrane is composed of liquid-ordered (lo) and liquid-
disordered (ld) domains, and DPPC/CHL, DOPC, and the raft
mixture are good models for the lo domain, the ld domain, and
the cell membrane, respectively.[9,10] The KdOP value measured
in the raft mixture was 81 Æ 4 mm, a value slightly smaller than
that measured in the MOPS buffer (Figure 1b; Supporting
Information, Figure S5). This difference can be attributed to
the more hydrophobic environment within the vesicles, which
would stabilize the BCaM/Ca2+ complex and shift the
equilibrium toward the formation of the complex. Moreover,
the emission spectrum of the BCaM/Ca2+ complex showed
lmax values at 436 and 452 nm in DPPC/CHL and DOPC,
respectively, whereas that in the raft mixture exhibited lmax at
450 nm, which could be fitted to two Gaussian functions
centered at 436 and 467 nm, respectively (Supporting Infor-
mation, Figure S6a); the emission from the BCaM/Ca2+
complex in the raft mixture can reasonably reflect those
from the lo and ld domains. Thus, BCaM is suitable for the
detection of Ca2+ in the model membrane.
Figure 1. a) One-photon fluorescence spectra of 1 mm BCaM (30 mm
MOPS, 100 mm KCl, pH 7.2) in the presence of free Ca2+ (0–2.5 mm).
b) One-photon fluorescence titration of BCaM in LUVs composed of
&
raft mixture ( ) and two-photon fluorescence titration in HeLa cells
( ) in the presence of various concentrations of free Ca2+ ions (0–
*
~
&
2.5 mm). c) Two-photon action spectra of BCaM ( ), Fura-2 ( ), and
Calcium Green ( ) in the presence of excess free Ca2+ ions.
!
*
d) Normalized emission spectra of BCaM ( ) and ANa1 (&) in HeLa
cells.
The pseudo-colored TPM image of HeLa cells labeled
with 0.5 mm BCaM clearly revealed the Ca2+ distribution in
the plasma membrane (Figure 2a). It is worth noting that
dissociation constants (KdOP and KdTP) of BCaM for the one-
and two-photon processes were calculated from the fluores-
cence titration curves (Supporting Information, Fig-
ure S3).[6,7] The titration curves fitted well with a 1:1 binding
model and the Hill plots were linear with a slope of 1.0,
indicating 1:1 complexation between the probe and Ca2+
(Supporting Information, Figure S3).[6,7] The Kd and Kd
OP
TP
values for Ca2+ are 90 Æ 2 mm and 89 Æ 3 mm, respectively,
which are well within the range of [Ca2+]m in the live cells. We
i
also measured the dissociation constant Kd in digitonin-
i
treated HeLa cells by TPM. The value of Kd = 78 Æ 5 mm is
almost the same as that measured in the raft mixture and is
well within the range of [Ca2+]m in live cells. A similar value
was reported for Calcium Green FlAsH (CaGF).[8] This result
confirms that BCaM is capable of detecting [Ca2+]m in live
cells.
Figure 2. a) Pseudo colored two-photon microscopy (TPM) images of
HeLa cells labeled with 0.5 mm BCaM. Scale bar: 20 mm. b) Time
course of two-photon excited fluorescence (TPEF) at the position
marked with a dotted line in (a) after stimulation with 100 mm
histamine in nominally calcium-ion-free buffer, followed by addition of
2 mm CaCl2 to the imaging solution. TPEF was collected at 390–
450 nm upon excitation at 780 nm.
BCaM showed a weak response toward Mg2+ at 2 mm, and
to Zn2+ and Mn2+ at 100 mm, and no response toward Fe2+,
Cu2+, and Co2+ at 100 mm (Supporting Information, Fig-
ure S4a). Therefore, this probe can selectively detect near-
membrane Ca2+ ions with minimal interference from other
biologically relevant cations. Moreover, BCaM is pH-insensi-
tive in the biologically relevant pH range (Supporting
Information, Figure S4b).
The TP action spectra of BCaM in MOPS buffer
containing excess Ca2+ indicated a Fd value of 150 GM at
780 nm, a value that exceeded those of Calcium Green/Ca2+
and Fura-2/Ca2+ by a factor of three to five (Figure 1c;
Supporting Information, Table S1).[6] Thus, TPM images of
BCaM can exclusively stain the plasma membrane despite the
absence of the long-chain alkyl group, which is common in
most membrane TP probes such as CL.[10] The image was
bright, which is probably due to the large TP action cross-
section. Also, there were very bright domains (red spots) in
addition to the less bright ones, indicating the existence of
Ca2+-rich domains in the plasma membrane. Moreover, the
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TPEF spectrum from the plasma membrane showed a lmax at
430 nm with a shoulder that could be fitted to two Gaussian
functions centered at 430 and 460 nm, respectively, as
measured in the raft mixture (Supporting Information, Fig-
ure S6a,b). Thus, the TPM image can most reasonably be
attributed to the BCaM/Ca2+ complexes associated with lo and
ld domains in the plasma membrane. Moreover, the TPEF
intensity in four individual BCaM-labeled HeLa cells chosen
without bias remained nearly the same for about 1 h,
indicating high photostability (Supporting Information, Fig-
ure S7). The combined results confirm that BCaM can detect
[Ca2+]m in live cells for a long period of time. Further, the
HeLa cells labeled with BCaM and ANa1 emitted bright
TPEF at 390–450 (Ch1) and 500–560 nm (Ch2), respectively
(Supporting Information, Figure S8). Therefore, we have
selectively detected [Ca2+]m and [Na+]i by using the detection
windows of 390–450 (Ch1) and 500–560 nm (Ch2), respec-
tively.
To demonstrate the utility of this probe in cell imaging, we
monitored TPEF intensity of HeLa cells labeled with BCaM
after addition of histamine, an agonist that stimulates the cells
to release [Ca2+]c from intracellular stores, such as endoplas-
mic reticulum (ER).[11] We expected that the excess [Ca2+]c
liberated by histamine would be extruded from the cell by the
cell membrane to maintain the Ca2+ homeostasis, thereby
increasing the [Ca2+]m.[11] Indeed, the TPEF intensities in the
cell membrane began to rise after addition of histamine
(100 mm), reached the peak value after 30 s, and returned to
the baseline level after 150 s. When the cells were treated with
2 mm CaCl2, the [Ca2+]m increased immediately, reached a
maximum after 50 s, and then decreased to the baseline level
after 6.5 min, which concurred with literature results (Fig-
ure 2b).[11] Therefore, BCaM is clearly capable of monitoring
Figure 3. a–e) Dual-channel TPM images of HeLa cells co-labeled with
BCaM and ANa1 collected at a) 390–450 nm (BCaM, Ch1) and b) 500–
560 nm (ANa1, Ch2). c) Merged image of (a) and (b); d) enlargement
of the white box in (c). e) Time course of TPEF at designated positions
(1) and (2) in (d) after stimulation with 100 mm histamine in nominally
calcium-ion-free buffer. f–j) Images of a rat hippocampal slice co-
stained with BCaM and ANa1. f) Bright-field image of the CA1-CA3
regions and the dentate gyrus (DG) at tenfold magnification. g) 25
TPM images collected at Ch1 and Ch2 in (f) along the z direction at
the depths of approximately 100–200 mm were accumulated and then
merged. h–j) TPM images of CA3 regions collected at h) Ch1 and
i) Ch2 at a depth of about 100 mm at tenfold magnification. j) Merged
image of (h) and (i). Excitation wavelength: 780 nm. Scale bars: 30 mm
(a,h) and 300 mm (g).
the change in [Ca2+
] in live cells over a long time period.
m
We then investigated Na+/Ca2+ exchange with BCaM in
live cells. It is well-established that [Ca2+]c is strictly main-
tained by many cellular functions, including the Na+/Ca2+
exchangers (NCX), which transport one Ca2+ out of the cell
and take three Na+ into the cell.[2,3] To visualize such activity,
we have monitored TPM images of HeLa cells co-labeled
with BCaM and ANa1 (Figure 3a–e). When the cells were
treated with histamine (100 mm), the TPEF intensities in the
plasma membrane (Figure 3e, position 1, green curve) and
cytoplasm (Figure 3e, position 2, red curve) increased sharply
until they reached the peak intensities and then decreased depth, we accumulated 25 TPM images at depths of 100–
slowly to the baseline level. The rates were faster in the 200 mm to visualize the distributions of the Ca2+ and Na+ ions
plasma membrane than in the cytoplasm, indicating that Na+ (Figure 3g; Supporting Information, Figure S9).
influx occurred after Ca2+ efflux through the NCX in the
The TPM images collected from Ch1 and Ch2 revealed
plasma membrane.[12] Therefore, BCaM, in combination with the Ca2+ and Na+ distributions, which did not merge (Fig-
ANa1, is clearly capable of monitoring Na+/Ca2+ exchange. ure 3g–j; Supporting Information, Figure S9b–d). This out-
We further investigated the utility of this probe in tissue come confirms that BCaM and ANa1 can independently
imaging. TPM images were obtained from a slice of fresh rat detect [Ca2+]m and [Na+]i with minimum interference from
hippocampal tissue incubated with 10 mm BCaM and 20 mm each other. The images taken at a higher magnification at a
ANa1 for 30 min at 378C. The slice from the brain of a 14- tissue depth of about 100 mm clearly revealed Na+/Ca2+
day-old rat was too large to show with one image, so two distribution in the pyramidal neuron layer composed of cell
images were obtained in each plane and combined. The bodies in the CA3 region (Figure 3h–j). Thus, dual-channel
bright-field image revealed the CA1 and CA3 regions and imaging of [Ca2+]m and [Na+]i is clearly possible at a 100–
also the dentate gyrus (DG; Figure 3 f). As the structure of 200 mm depth in live tissues by TPM using BCaM and ANa1
the brain tissue is known to be inhomogeneous in its entire as the probes.
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selectivity for [Ca2+]m, and TPEF emission that was three
times stronger than Calcium Green and Fura-2 upon com-
plexation with Ca2+. BCaM, which is superior to currently
available probes, in combination with ANa1 allows dual-color
imaging of the activities of near membrane Ca2+ and cytosolic
free Na+ ions in live cells and tissues at depths of over 100 mm
for long periods of time without photobleaching artifacts.
Received: May 14, 2010
Published online: August 16, 2010
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Keywords: calcium · fluorescence spectroscopy ·
imaging agents · live tissue · two-photon microscopy
.
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