Sodium-ion-selective two-photon fluorescent probe for in vivo imaging

Article abstract of DOI:10.1002/anie.200904835

Chemical Equation Presented Two photons are better than one: A two-photon probe (ANa1, see picture) shows strong twophoton fluorescence enhancement In response to Na⁺ and a dissociation constant (K _d^l) of (26±2) mM in the cell. It can be easily loaded into the cells and can selectively detect intracellular free Na ⁺ in live cells and living tissues at a depth of 100-200 μm for a long period of time.

Full text of DOI:10.1002/anie.200904835

Communications
DOI: 10.1002/anie.200904835
Fluorescent Probes
Sodium-Ion-Selective Two-Photon Fluorescent Probe for In Vivo
Imaging**
Mi Kyung Kim, Chang Su Lim, Jong Tae Hong, Ji Hee Han, Hye-Young Jang,
Hwan Myung Kim,* and Bong Rae Cho*
In memory of Chi Sun Hahn
The sodium ion plays critical roles in many physiological and
pathological processes such as nerve conduction and muscle
and heart contraction; it modulates the electrolyte level,
cation transport, and cell volume.[1,2] In the mammalian body,
most Na+ ions reside in the extracellular compartment, with
much less Na+ inside cells. The concentration of intracellular
free Na+ ([Na+]i) is in the range of 5–30 mm, whereas that of
extracellular Na+ is several times higher (greater than
100 mm).[1] The influx of Na+ into a resting cell is regulated
by Na+/Ca2+ exchanger (NCX), Na+/H+ exchanger (NHE),
and Na+ channels, whereas the efflux is mediated by the
activity of Na+/K+-ATPase, which transports three Na+ ions
out of the cell and takes two K+ ions into the cell.[1,2]
To understand these functions, a number of one-photon
(OP) fluorescent probes have been developed, with SBFI and
Sodium Green (SG) being commercially available
(Scheme 1).[3–5] A disadvantage of these probes is that they
are bulky and difficult to load into live cells.[4c,d,6] The cell
loading could be increased by reducing the molecular weight
(MW).[5] The second problem, which is common for most OP
probes,[7] is their short excitation wavelength (below 500 nm).
This requirement limits their application in tissue imaging
Scheme 1. The structures of ANa1, SBFI, and Sodium Green (SG).
owing to the shallow penetration depth (less than 100 mm),
photobleaching, and cellular autofluorescence. An ideal
solution to this problem is two-photon microscopy (TPM),
which utilizes two near-infrared photons of lower energy for
the excitation. Using TPM, intact tissue can be imaged for a
long period of time with minimum interference from tissue
preparation artifacts that can extend more than 70 mm into
the tissue slice.[8] However, no two-photon (TP) probe for Na+
that is capable of imaging [Na+]i deep inside (more than
100 mm) living tissues has been reported to date.
To address both of these problems, we designed a TP
probe (ANa1) derived from 2-acetyl-6-(dimethylamino)naph-
thalene (acedan) as the reporter and 1,7-diaza-15-crown-5 as
the Na+ ion receptor (Scheme 1) by considering the following
requirements: 1) small MW for cell permeability; 2) high
selectivity for Na+ ions; 3) significant TP cross section for
bright TPM images; 4) large spectral shifts in different
environments to distinguish between the cytosolic and
membrane-bound probes; and 5) high photostability. We
adopted acedan from our previous work on various TP
probes[9] and 1,7-diaza-15-crown-5 from the work of Tsien
and Minta.[3] Herein, we report that ANa1 is capable of
detecting intracellular free Na+ ions in live cells and tissues at
depths greater than 100 mm for long periods of time without
mistargeting and photobleaching problems.
ANa1 was prepared in 47% yield by the coupling reaction
between 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naph-
thalene (A)[9] and N-(2-methoxyphenyl)-N’-(4-amino-2-
methoxyphenyl)-1,7-diaza-15-crown-5 (B; see the Supporting
Information).[10] The water solubility of ANa1 was approx-
imately 2 mm, which was sufficient to stain the cells (Figure S2,
Supporting Information). The fluorescent spectra of ANa1
showed gradual red shifts with the solvent polarity (ENT ) in the
[*] M. K. Kim,[+] J. T. Hong, Prof. H. Y. Jang, Prof. H. M. Kim
Division of Energy Systems Research, Ajou University
Suwon, 443-749 (Korea)
Fax: (+82)31-219-1615
E-mail: kimhm@ajou.ac.kr
C. S. Lim,[+] J. H. Han, 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 NRF grants (No. 2009-0065783 and
No. R0A-2007-000-20027-0).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904835.
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Angew. Chem. Int. Ed. 2010, 49, 364 –367

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order 1,4-dioxane < DMF < EtOH < H2O (Figure S1 and
Table S1, Supporting Information). The large solvatochromic
shifts with increasing solvent polarity indicated the utility of
ANa1 as an environment-sensitive probe.
was reported for SG (Table S2, Supporting Information).[5]
Moreover, the Kd value of ANa1 for K+ is (280 Æ 15) mm, thus
indicating that the selectivity of ANa1 for Na+ over K+ is
higher than that of SBFI and comparable to that of SG
(Table S2, Supporting Information). We also measured the
When small increments of Na+ ions were added to ANa1
in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer
solution (10 mm, pH 7.0) while maintaining the total concen-
tration of Na+ and K+ at 135 mm to mimic physiological
condition,[3,5] OP- and TP-excited fluorescence intensity
increased dramatically (Figure 1a,b), probably owing to the
blocking of photoinduced electron transfer processes by the
complexation of the metal ions. Similar results were observed
with K+, except that the changes were smaller (Figure S4,
Supporting Information). The fluorescence enhancement
factor [(FÀFmin)/Fmin] of ANa1 for OP and TP processes
was 8 in the presence of 135 mm Na+, a value nearly identical
to that of SG (Table S2, Supporting Information).[5] Further-
more, Benesi–Hildebrand plots for Na+ and K+ binding
showed a good linear relationship, indicating 1:1 complex-
ation between probe and cations (Figure S3 and S4, Support-
ing Information).[9a]
i
dissociation constant (Kd) in ionophore-treated astrocyte
cells by two-photon spectroscopy (Supporting Informa-
tion).[11] The Kd value of (26 Æ 2) mm is in reasonable
i
agreement with that measured in MOPS buffer (Figure 1b).
This value allows quantitative measurement of [Na+]i by using
i
[Na+]i = Kd [(FÀFmin)/(FmaxÀF)], where Fmin, Fmax, and F are
the two-photon excited fluorescence (TPEF) intensities in the
absence and presence of excess Na+ and the observed TPEF
intensity, respectively.[12] Further, the TPEF intensity at a
given spot on the ANa1-labeled HeLa cells did not show any
decay after continuous irradiation with femtosecond pulses
for 60 min, thus indicating its high photostability (Figure S8,
Supporting Information).
ANa1 showed a strong response toward Na+, much
weaker responses toward K+, Li+, Ca2+, and no response
toward Mg2+, Zn2+, Fe2+, Cu2+, Cu+, Mn2+, Co2+, Ni2+
(Figure 1d). Therefore, this probe can selectively detect
[Na+]i with minimal interference from other biologically
relevant cations. Moreover, ANa1 is insensitive to the
pH value in the biologically relevant pH range (Figure S5,
Supporting Information). The combined results reveal that
ANa1 can detect [Na+]i for a long period of time with
minimum interference from
The dissociation constants (Kd) were calculated from the
fluorescence titration curves (Figure S3, Supporting Informa-
tion). The Kd value of ANa1 for Na+ measured in the absence
and presence of K+ ([Na+] + [K+] = 135 mm) are (8.0 Æ 0.5)
and (20 Æ 1) mm, respectively.[3] The values are well within the
range of [Na+]i in the live cells (see above). A similar value
pH value and other metal
ions.
The TP action cross sec-
tion (dF) of the ANa1-Na+
complex in buffer solutions
indicated a value of 95 GM at
780 nm, which is three- to
fivefold larger than those of
SG and SBFI (Figure 1c and
Table S2, Supporting Infor-
mation). Moreover, the TPM
image of HeLa cells was much
brighter when stained with
ANa1 than with the commer-
cial probes (Figure S6, Sup-
porting Information), pre-
sumably owing to the larger
dF value and increased cell
loading.
The TPM images of cul-
tured astrocytes labeled with
1 mm ANa1 showed very weak
TPEF at 360–460 nm and
strong TPEF at 500–620 nm
(Figure S7, Supporting Infor-
mation). For comparison, the
Figure 1. a) One-photon fluorescence spectra of 1 mm ANa1 (10 mm MOPS, [Na+]+[K+]=135 mm, pH 7.0)
in the presence of free Na+ (0–135 mm). b) Two-photon fluorescence titration of ANa1 with Na+ in MOPS
TPEF spectra from the
intense domains in the TPM
images of the cells labeled
&
buffer ( ) and in astrocytes (&). The excitation wavelength was 780 nm. c) Two-photon action spectra of
+
~
&
*
ANa1 ( ), SG ( ), and SBFI ( ) in the presence of 135 mm free Na . d) The relative fluorescence intensity
of ANa1 in the presence of 200 mm for K+, 5 mm for Li+, Ca2+, Mg2+; 100 mm for Zn2+, Fe2+, Cu2+, Cu+,
Mn2+, Co2+, Ni2+ (empty bars) and subsequent addition of 100 mm of Na+ (filled bars).
with
acedan-derived
TP
probes for Mg2+ (AMg1)[9a]
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Communications
and Ca2+ (ACa1)[9b] could be dissected into two Gaussian
functions with emission maxima at approximately 440 and
500 nm, respectively, which had been attributed to probes
associated with membrane and cytosol. In contrast, the
fluorescence spectra of the two domains with different
intensities in the ANa1-labeled astrocytes are almost the
same (Figure S7b, Supporting Information). Moreover, they
are nearly identical to that of ANa1 in the buffer solution with
fl
max
l
ꢀ 500 nm. Hence, ANa1 appears to be predominantly
located in the cytosol, probably owing to its low MW.
Nevertheless, we have collected TP fluorescence data in the
500–620 nm range to absolutely rule out possible interference
from the membrane-bound probes.
To demonstrate the utility of this probe, we monitored TP
fluorescence intensity of the ANa1-labeled astrocytes after
addition of ouabain, a steroid hormone that inhibits Na+/K+
ATPase and increases the cytosolic free Na+ concentra-
tion.[11b,13] The TP fluorescence intensity increased gradually
with time and reached a maximum value after 1800 s
(Figure 2a–c). The resting [Na+]i in the astrocytes calculated
by [Na+]i = Kd [(FÀFmin)/(FmaxÀF)] is (10 Æ 2.1) mm. It began
i
to rise after treatment with ouabain (1 mm) and reached a
maximum value of (50 Æ 5.0) mm after 1800 s (Figure 2c),
which is in good agreement with the reported value.[11b] We
next monitored glutamate-evoked [Na+]i response of the
ANa1-labeled astrocytes. Glutamate is a well-known excita-
tory neurotransmitter in the brain that is co-transported with
three Na+ ions into the astrocytes, thereby inducing [Na+]i
elevation.[11b,14] When 100 mm glutamate was added, the [Na+]i
began to rise in about 10 s, reached the peak value of (30 Æ
3.2) mm after 60 s, and then decreased to the basal level within
2.5 min, which concurs with literature results.[11b] Hence,
ANa1 is clearly capable of monitoring the change in [Na+]i
in live cells for a long period time.
To further investigate the utility of this probe in deep-
tissue imaging, TPM images were obtained from part of a
fresh rat hippocampal slice incubated with 20 mm ANa1 for
40 min at 378C. The slice of a 14 day old rat was too big to
show with one image, so two TPM images were obtained in
each plane and combined. The bright-field image of a part of a
rat hippocampal slice shows the CA1–CA3 regions as well as
the dentate gyrus (DG; Figure 3a). The TPM images revealed
Na+ distribution in the same region at 100–200 mm depth
(Figure S9, Supporting Information), but each image repre-
sents the distribution exclusively in the given plane. As the
structure of the brain tissue is known to be inhomogeneous in
its entire depth, we accumulated 25 TPM images at different
depths to visualize the distribution of the Na+ ions (Fig-
ure 3b).
Figure 2. a,b,d,e) TPM images of 1 mm ANa1-labeled astrocytes before
(a, 0 s) and after (b, 1800 s) addition of 1 mm ouabain to the imaging
solution and before (d, 0 s) and after (e, 260 s) addition of 100 mm
glutamate to the imaging solution. The TPM images were obtained by
collecting the TPEF at 500–620 nm upon excitation at 780 nm with a
femtosecond pulse. c,f) The TPEF intensity collected at 500–620 nm as
a function of time. Scale bars 30 mm. Cells shown are representative
images from replicate experiments (n=5).
The accumulated TPM images show that Na+ is more
abundant in the pyramidal neuron layers of the CA1–CA3
regions than in the DG. The image taken at a higher
magnification clearly resolved Na+ distributions in the
pyramidal neuron layer of the CA3 region (Figure 3c).
These results demonstrate that ANa1 is capable of detecting
the [Na+]i at 100–200 mm depth in live tissue using TPM.
In conclusion, we have developed a TP probe (ANa1) that
shows strong TP fluorescence enhancement in response to
cell. The probe can be easily loaded into the cells and emits
three- to fivefold larger TPEF intensity than SG and SBFI
upon complexation with Na+. Unlike the previously available
probes, this novel probe can selectively detect [Na+]i in live
Na+ and a dissociation constant (Kd ) of (26 Æ 2) mm in the
i
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(Eds.: J. C. Skou, J. G. Norby, A. B. Maunsbach, M. Esmann,
A. R. Liss), Wiley, New York, 1998.
[2] a) E. Murphy, D. A. Eisner, Circ. Res. 2009, 104, 292; b) D. M.
Bers, W. H. Barry, S. Despa, Cardiovasc. Res. 2003, 57, 897.
[3] A. Minta, R. Y. Tsien, J. Biol. Chem. 1989, 264, 19449.
[4] a) A. P. de Silva, H. Q. Gunaratne, T. Gunnlaugsson, A. J.
Huxley, C. P. McCoy, J. T. Rademacher, T. E. Rice, Chem. Rev.
1997, 97, 1515; b) T. Gunnlaugsson, M. Nieuwenhuyzen, L.
Richard, V. Thoss, J. Chem. Soc. Perkin Trans. 2 2002, 141;
c) V. V. Martin, A. Rothe, Z. Diwu, K. R. Gee, Bioorg. Med.
Chem. Lett. 2004, 14, 5313; d) V. V. Martin, A. Rothe, K. R. Gee,
Bioorg. Med. Chem. Lett. 2005, 15, 1851; e) S. Kenmoku, Y.
Urano, K. Kanda, H. Kojima, K. Kikuchi, T. Nagano, Tetrahe-
dron 2004, 60, 11067.
[5] A Guide to Fluorescent Probes and Labeling Technologies, 10th
ed. (Ed.: R. P. Haugland), Molecular Probes, Eugene, OR, 2005.
[6] G. P. Amorino, M. H. Fox, Cytometry 1995, 21, 248.
[7] D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4,
168.
[8] a) W. R. Zipfel, R. M. Williams, W. W. Webb, Nat. Biotechnol.
2003, 21, 1369; b) F. Helmchen, W. Denk, Nat. Methods 2005, 2,
932; c) R. M. Williams, W. R. Zipfel, W. W. Webb, Curr. Opin.
Chem. Biol. 2001, 5, 603.
[9] a) H. M. Kim, C. Jung, B. R. Kim, S.-Y. Jung, J. H. Hong, Y.-G.
Ko, K. J. Lee, B. R. Cho, Angew. Chem. 2007, 119, 3530; Angew.
Chem. Int. Ed. 2007, 46, 3460; b) H. M. Kim, B. R. Kim, J. H.
Hong, J.-S. Park, K. J. Lee, B. R. Cho, Angew. Chem. 2007, 119,
7589; Angew. Chem. Int. Ed. 2007, 46, 7445; c) H. M. Kim, M. S.
Seo, M. J. An, J. H. Hong, Y. S. Tian, J. H. Choi, O. Kwon, K. J.
Lee, B. R. Cho, Angew. Chem. 2008, 120, 5245; Angew. Chem.
Int. Ed. 2008, 47, 5167; d) H. M. Kim, B. R. Cho, Acc. Chem. Res.
2009, 42, 863.
Figure 3. Images of a rat hippocampal slice stained with 20 mm ANa1.
a) Bright-field image of the CA1–CA3 regions as well as the dentate
gyrus by 10ꢀ magnification. b) TPM image by 10ꢀ magnification. 25
TPM images were accumulated along the z direction at a depth of
approximately 100–200 mm. Scale bar 300 mm. c) Magnification at
100ꢀ in the pyramidal neuron layer of CA3 regions (red box in
Figure 3b) at a depth of approximately 120 mm. Scale bar 30 mm. The
TPM images were collected at 500–620 nm upon excitation at 780 nm
with a femtosecond pulse.
[10] T. Gunnlaugsson, H. Q. N. Gunaratne, M. Nieuwenhuyzen, J. P.
Leonard, J. Chem. Soc. Perkin Trans. 1 2002, 1954.
[11] a) C. R. Rose, B. R. Ransom, J. Physiol. 1996, 491, 291; b) J. Y.
Chatton, P. Marquet, P. J. Magistretti, Eur. J. Neurosci. 2000, 12,
3843.
[12] a) G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem. 1985,
260, 3440; b) H. M. Kim, P. R. Yang, M. S. Seo, J.-S. Yi, J. H.
Hong, S.-J. Jeon, Y.-G. Ko, K. J. Lee, B. R. Cho, J. Org. Chem.
2007, 72, 2088.
cells and living tissues at 100–200 mm depth for more than
60 min using TPM.
Received: August 29, 2009
Revised: October 28, 2009
Published online: December 8, 2009
Keywords: fluorescent probes · live tissue · sodium ·
.
[13] G. Scheiner-Bobis, W. Schoner, Nat. Med. 2001, 7, 1288.
[14] Y. Bernardinelli, P. J. Magistretti, J. Y. Chatton, Proc. Natl. Acad.
Sci. USA 2004, 101, 14937.
two-photon microscopy
[1] a) M. Burnier, Sodium in Health Disease, Informa Healthcare,
New York, 2008; b) The Na+, K+ Pump, Part B: Cellular Aspects
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