B. R. Cho et al.
FULL PAPERS
148.4, 145.5, 140.4, 138.5, 138.1, 133.6, 130.6, 130.2, 130.0, 129.7, 125.9,
125.7, 124.1, 120.2, 118.8, 118.0, 115.2, 112.8, 108.1, 103.2, 67.7, 66.7, 54.4,
46.9, 46.7, 37.9, 35.3, 33.8, 31.9, 29.6, 29.3, 26.9, 25.7, 24.9, 22.6 ,13.3 ppm.
HR-MS calculated for [M+H]+: 859.3767; found: 859.3765.
for 5 min at 258C. The cells were washed three times with phosphate buf-
fered saline (PBS; Gibco) without Ca2+ and Mg2+ and then imaged.
Two-Photon Fluorescence Microscopy
Two-photon fluorescence microscopy images of ACaLN-labeled cells and
tissues were obtained with spectral confocal and multiphoton micro-
scopes (Leica TCS SP2) with a ꢁ100 (NA=1.30 OIL) and ꢁ10 (NA=
0.30 DRY) objective lens, respectively. The two-photon fluorescence mi-
croscopy images were obtained with a DM IRE2 Microscope (Leica) by
exciting the probes with a mode-locked titanium-sapphire laser source
(Coherent Chameleon, 90 MHz, 200 fs) set at wavelength of 750 nm and
output power of 1348 mW, which corresponded to approximately 10 mW
average power in the focal plane. To obtain images in the 360~460 nm
and 500~620 nm ranges, internal PMTs were used to collect the signals
in 8 bit unsigned 512ꢁ512 pixels at 400 Hz scan speed.
Spectroscopic Measurements
Absorption spectra were recorded on a Hewlett–Packard 8453 diode
array spectrophotometer and fluorescence spectra were obtained with
Aminco-Bowman series 2 luminescence spectrometer using a 1 cm path-
length standard quartz cell. The fluorescence quantum yield was deter-
mined by using Coumarin 307 as the reference using the literature
method.[13]
Solubility of ACaLN in MOPS Buffer
A small amount of dye was dissolved in DMSO to prepare the stock sol-
Detection window
utions (1.0ꢁ10ꢁ3 m). The solution was diluted to 6.0ꢁ10ꢁ3 ~6.0ꢁ10ꢁ5
m
The TPEF spectrum from the ACaLN-labeled cells was unsymmetrical
and could be fitted to two Gaussian functions with peak maxima at
434 nm (blue curve) and 475 nm (green curve), respectively (Figure S6b
in the Supporting Information). The spectra are very similar to those
measured in DPPC/CHL and DOPC, which are good models for the lo
and ld domains, respectively (Figure S5 in the Supporting Information).
Moreover, the TPM images collected in the 360–460 and 500–620 nm
ranges are nearly the same except for the brightness (Figure S6c, d in the
Supporting Information). Therefore, we have detected near-membrane
Ca2+ with ACaLN by using the detection window at 360–620 nm.
and added to a cuvette containing MOPS buffer (3.0 mL, pH 7.2) using a
micro syringe. In all cases, the concentration of DMSO in H2O was main-
tained to be 0.2%.[3] The plots of absorbance against the dye concentra-
tion were linear at low concentration and showed downward curvature at
higher concentrations (Figure S1 in the Supporting Information). The
maximum concentration in the linear region was taken as the solubility.
The solubility of ACaLN in MOPS buffer was greater than 15 mm.
Determination of Apparent Dissociation Constants
A series of solutions containing various [Ca2+] were prepared in the pres-
ence of 3 mm ACaLN in 30 mm MOPS buffer and they were adjusted to
pH 7.2. The apparent dissociation constant (Kd) was determined using
Preparation and Staining of Fresh Rat Hippocampal Slices
All experiments were performed in accordance with the guidelines estab-
lished by the Committee of Animal Research Policy of Korea University
College of Medicine. Slices were prepared from the hippocampi and the
hypothalmi of 2 day-old rats (SD). Coronal slices were cut into 400 mm-
thick using a vibrating-blade microtome in artificial cerebrospinal fluid
(ACSF); 138.6 mm NaCl, 3.5 mm KCl, 21 mm NaHCO3, 0.6 mm NaH2PO4,
9.9 mm d-glucose, 1 mm CaCl2, and 3 mm MgCl2. Slices were incubated
with 30 mm ACaLN (or BCaM) in ACSF bubbled with 95% O2 and 5%
CO2 for 30 min at 378C. Slices were then washed three times with ACSF
and transferred to glass-bottomed dishes (MatTek) and observed in a
spectral confocal multiphoton microscope. After washing three times
with ACSF, the slices were transferred to glass bottomed dishes
(MatTek) and observed under a spectral confocal multiphoton micro-
scope. To observe the effect of EDTA, a solution of EDTA in MOPS
buffer (200 mm) was added to this sample and the TPM image was ob-
tained.
the following equation: FꢁFmin =[Ca2+](FmaxꢁFmin)/
[Ca2+]), where F
ACHUTGTNRENNUG(Kd+ACHTUNGTRENNUGN
is the observed fluorescence, Fmax is the fluorescence for the Ca2+
-
ACaLN complex, and Fmin is the fluorescence for the free ACaLN. The
Kd value that best fits the titration curve (Figures 2 and S3 in the Sup-
porting Information) using the equation was calculated by using the
TP
Excel program as previously reported.[9,10] In order to determine the Kd
for the two-photon process, the TPEF intensity was recorded in the range
of 400–620 nm with a CCD detector excited by a mode-locked titanium-
sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at a
wavelength of 750 nm and output power 1348 mW, which corresponded
to approximately 200 mW average power in the focal plane.
Measurement of Two-Photon Cross Section
The two-photon cross section (d) was determined by using the femtosec-
ond (fs) fluorescence measurement technique as described previously.[14]
ACaLN was dissolved in 30 mm MOPS buffer (pH 7.2) at concentrations
of 5.0ꢁ10ꢁ6 m and then the two-photon induced fluorescence intensity
was measured at 740–900 nm using rhodamine 6G as the reference, the
two-photon property of which has been well characterized in the litera-
ture.[15] The intensities of the two-photon induced fluorescence spectra of
the reference and sample emitted at the same excitation wavelength
were determined. The TPA cross section was calculated by using d=dr-
Acknowledgements
This work was supported by the National Research Foundation (NRF)
grants funded by the Korean Government (No. 2010-0018921) and Priori-
ty Research Centers Program through the NRF funded by the Ministry
of Education, Science and Technology (2010-0020209). C. S. Lim, M. Y.
Kang, J. H. Han, and I. A. Danish were supported by BK21 scholarship.
ACHTUNGTRENNUNG(SsFrfrcr)/ACHTUNGTRENNUNG(SrFsfscs): where the subscripts s and r stand for the sample
and reference molecules, respectively. The intensity of the signal collect-
ed by a CCD detector was denoted as S. F is the fluorescence quantum
yield. f is the overall fluorescence collection efficiency of the experimen-
tal apparatus. The number density of the molecules in solution was de-
noted as c. dr is the TPA cross section of the reference molecule. The TP
action cross section of ACaLN was 20 GM at 750 nm.
[1] E. Carafoli, Physiol. Rev. 1991, 71, 129–153.
Bootman, M. J. Berridge, P. Lipp, Nature 1998, 395, 645–648;
[3] a) P. S. Mohan, C. S. Lim, Y. S. Tian, W. Y. Roh, J. H. Lee, B. R.
Y. S. Tian, W. Y. Rho, B. R. Cho, Bull. Korean Chem. Soc. 2010, 31,
599–605; c) H. J. Kim, J. H. Han, M. J. Kim, C. S. Lim, H. M. Kim,
Cell Culture and Imaging
HT22 clonal mouse hippocampal cells were cultured in Dulbecco’s Modi-
fied Eagle’s Medium (DMEM, WelGene) supplemented with heat-inacti-
vated 10% fetal bovine serum (FBS, WelGene), penicillin
(100 unitsmLꢁ1), and streptomycin (100 mgmLꢁ1). All cells were kept in a
humidified atmosphere of 5:95 (v/v) of CO2/air at 378C. Two days before
imaging, the cells were passed and plated on glass-bottomed dishes
(MatTek). For labeling, the growth medium was removed and replaced
with DMEM without FBS. The cells were incubated with ACaLN (3 mm)
2032
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Chem. Asian J. 2011, 6, 2028 – 2033