Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging

Article abstract of DOI:10.1002/asia.201500314

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other.

Full text of DOI:10.1002/asia.201500314

DOI: 10.1002/asia.201500314
Full Paper
Fluorescent Probes
Two-Photon Probes for Lysosomes and Mitochondria:
Simultaneous Detection of Lysosomes and Mitochondria in Live
Tissues by Dual-Color Two-Photon Microscopy Imaging
Chang Su Lim,[a] Seung Taek Hong,[b] Seong Shick Ryu,[b] Dong Eun Kang,*[a] and
Bong Rae Cho*[a]
Abstract: Novel two-photon (TP) probes were developed for
lysosomes (PLT-yellow) and mitochondria (BMT-blue and
PMT-yellow). These probes emitted strong TP-excited fluo-
rescence in cells at widely separated wavelength regions
and displayed high organelle selectivity, good cell permea-
bility, low cytotoxicity, and pH insensitivity. The BMT-blue
and PLT-yellow probes could be utilized to detect lysosomes
and mitochondria simultaneously in live tissues by using
dual-color two-photon microscopy, with minimum interfer-
ence from each other.
Introduction
are the powerhouses of cells and the major source of reactive
oxygen species.[6,7] Their roles in biology can be elucidated by
monitoring their activities in live cells and tissues by TPM. This
requires organelle-specific TP probes. The most common
method for confirming the occurrence of a biological activity
in these organelles is the co-localization experiment, which
uses cells co-labeled with fluorescent probes for the specific
targets and organelles. These experiments can be performed
in the TP mode by using TP probes for specific organelles and
targets, which emit TP excited fluorescence (TPEF) at widely
separated wavelength regions. About 10 TP probes have been
reported for these organelles to date.[8] However, TP probes
that can be used for the above-mentioned applications are
rare.
Real time imaging using small molecule fluorescent probes is
a vital tool for the elucidation of multiple phenomena in living
systems. A number of fluorescent probes have been developed
over the past decades to visualize different biological process-
es, such as changes in pH value and metal ion concentrations,
enzymatic reactions, and the activities of organelles such as ly-
sosomes, mitochondria, DNA, and plasma membrane.[1] Howev-
er, most of these probes have been optimized for one-photon
microscopy (OPM), which requires a relatively short excitation
wavelength (l<500 nm). This confers qualities such as a shal-
low penetration depth (<100 nm), autofluorescence, and pho-
tobleaching to the probes, limiting their application to tissue
imaging. An attractive approach to overcoming these prob-
lems is the use of two-photon microscopy (TPM), which em-
ploys two near-infrared photons for the excitation.[2] Compared
to OPM, TPM detects biologically relevant species at greater
depths within an intact tissue, with higher spatial resolution,
and for a longer period of time, using appropriate two-photon
(TP) probes.[2,3]
We have recently developed TP probes for lysosomes (BLT-
blue) and mitochondria (FMT-green), which emit TPEF at 458
and 540 nm, respectively, in RAW 264.7 cells (mouse leukemic
monocyte macrophage cell; Scheme 1).[8e,m] These probes facili-
tated the visualization of autophagy,[9–11] a self-degradative pro-
cess that facilitates the removal of old, damaged, or surplus
mitochondria, by dual-color TPM imaging. The versatility of
TPM can be increased (which facilitates organelle monitoring
and assists in the performance of co-localization experiments)
Lysosomes are acidic organelles (pH 4.5–5.5) capable of
breaking down proteins and waste materials;[4,5] mitochondria
[a] Dr. C. S. Lim,+ Dr. D. E. Kang, Prof. Dr. B. R. Cho
Department of Chemistry
Korea University
1-Anamdong, Seoul 136-701 (Republic of Korea)
E-mail: dongeun@korea.ac.kr
chobr@korea.ac.kr
[b] S. T. Hong,+ S. S. Ryu
KU-KIST Graduate School of Converging Science and Technology
Korea University
1-Anamdong, Seoul 136-701 (Republic of Korea)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201500314.
Scheme 1. The structures of BLT-blue, BMT-blue, FMT-green, PLT-yellow, and
PMT-yellow.
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by developing TP probes for each organelle, which emit TPEF
at widely separated wavelength regions. Therefore, we have
developed novel TP probes for lysosomes (PLT-yellow) and mi-
tochondria (BMT-blue and PMT-yellow; Scheme 1), taking into
consideration the following requirements: 1) high selectivity
for each organelle; 2) a widely separated TPEF spectrum in the
cells, for the simultaneous detection of both organelles; 3) a
significant TP action cross-section (Fdmax; F: fluorescence
quantum yield; dmax: maximum TP cross section) to obtain
bright TPM images at low probe concentrations; 4) low molec-
ular weight to facilitate the staining; 5) low cytotoxicity, high
photo stability, and pH independency.
Scheme 2. a) 1) TFA, CH2Cl2, rt; 2) H2NCH2CH2PPh3+BrÀ, EDCI (1-ethyl-3-(3-di-
methylaminopropyl)carbodiimide), DMAP (4-dimethylaminopyridine), CH2Cl2,
rt.
+
BMT-blue was designed by introducing a PPh3 group in
place of the NMe2 group in BLT-blue, with the expectation that
the positively charged ion would facilitate its location in the
mitochondria (Scheme 1). PMT-yellow and PLY-yellow were de-
rived from bis-1,4-(p-dibutylaminostyryl)pyrazine (Pyr-2D),
which showed a long emission maximum (lfl =518 nm) and
a large Fdmax value (1250 GM at 770 nm) in toluene.[12] Since
the lfl is expected to be red-shifted and the F value is expect-
ed to decrease with changes in the solvent (to water), one of
the amino groups was substituted with a more weakly electron
donating alkoxy group, to modulate the lfl and F values. In
addition, the C=C bonds employed as the conjugation bridge
between the aryl groups and the pyrazine moiety were en-
closed in heterocyclic rings to improve photostability. A triphe-
nylphosphonium ion and a tertiary amino group were intro-
duced as the mitochondrial and lysosomal targeting moieties,
respectively (Scheme 1).
Scheme 3. a) CH2=CHCO2Et, (CF3)2CHOH, 608C; b) POCl3, DMF; c) AlCl3,
CH2Cl2, reflux; d) K2CO3, DMF, 1008C; e) NaBH4, THF/MeOH (9/1), reflux;
f) DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), PPh3, Bu4N+BrÀ, CH2Cl2,
rt; g) 5, K2CO3, DMF, 1008C; h) KOH, EtOH/THF/H2O (1/1/1), rt;
i) H2N(CH2)3NMe2 or H2NCH2CH2PPh3+BrÀ, EDCI, DMAP, CH2Cl2, rt.
compounds 5 and 10. Hydrolysis of compound 11 resulted in
the production of compound 12 at a yield of 78%. PLT-yellow
and PMT-yellow were obtained at yields of 58% and 65%, re-
spectively, by coupling compound 12 with 3-(dimethylamino)-
1-propylamine and (2-aminoethyl)triphenylphosphonium bro-
mide, respectively.
The photophysical properties of the new as well as the exist-
ing probes (BLT-blue and FMT-green) were evaluated under
the same conditions to determine the best combination of TP
probes for dual-color imaging. Herein, we report that BLT-blue
and PLT-yellow, as well as BMT-blue and PMT-yellow, are blue-
and yellow-emitting TP probes for lysosomes and mitochon-
dria, respectively, which meet all of the requirements outlined
above. Moreover, BMT-blue and PLT-yellow allowed the simul-
taneous detection of lysosomes and mitochondria in live tissue
via dual-color TPM imaging.
Spectroscopic Properties
BMT-blue, PLT-yellow, and PMT-yellow displayed absorption
maxima (lmax) at 355, 440, and 444 nm, with molar extinction
coefficients (e) of 18200, 24800, and 22800 cmÀ1 mÀ1, respec-
tively, in the spectroscopic studies conducted in water
(Table S1 in the Supporting Information). BMT-blue emitted
a strong fluorescence at 469 nm, with a fluorescence quantum
yield (F) of 0.85 (Table S1 in the Supporting Information). On
the other hand, the fluorescence emitted by PLT-yellow and
PMT-yellow was too weak for the accurate measurement of
emission maxima (lfl).
Results and Discussion
Synthesis
BMT-blue was obtained at a yield of 73% by hydrolyzing com-
pound 1,[13] followed by coupling with 2-(aminoethyl)triphenyl-
phosphonium ion (Scheme 2).
The solubility of BMT-blue in water was 10 mm (Figure S2 in
the Supporting Information), as measured by the fluorescence
method.[14] The solubilities of PLT-yellow and PMT-yellow were
measured using the absorption method because of their negli-
gible fluorescence in water. The water solubilities of PLT-yellow
and PMT-yellow were greater than 12 mm. Therefore, all devel-
oped probes showed sufficient water solubility to stain cells.
The solubility data measured by the absorption method were
observed to be slightly overestimated compared to that mea-
sured using the fluorescence method, as the visibility of aggre-
gation quenching was greater in the emission spectra.
The synthesis of PMT-yellow and PLT-yellow is summarized
in Scheme 3. Compound
3 was quantitatively obtained
through the reaction of compound 2 with ethyl acrylate. For-
mylation of compound 3, followed by demethylation, led to
the production of compound 5 up to an overall yield of 49%.
Compound 8 was obtained at a yield of 78% by reacting com-
pound 6 with compound 7. The reduction of compound 8, fol-
lowed by bromination, resulted in the production of com-
pound 10 at an overall yield of 42%. Compound 11 was ob-
tained at a yield of 78% as a result of the reaction between
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BMT-blue, PLT-yellow, and PMT-yellow displayed lmax of 359,
446, and 453 nm, and lfl of 432, 534, and 555 nm, respectively
(Table S1 in the Supporting Information) in 1,4-dioxane, in
which all probes emitted strong fluorescence. The larger
Stokes shifts observed for PLT-yellow and PMT-yellow indicate
the greater efficiency of intramolecular charge transfer (ICT)
between the donors and acceptors. In addition, all probes
showed gradual red shifts in lfl in polar solvents. Again, the ef-
fects were greater and more pronounced for PLT-yellow and
PMT-yellow (Figure S1 and Table S1 in the Supporting Informa-
tion). The larger solvatochromic effects observed for these
probes provided additional support for the enhanced ICT.
HeLa cells labeled with BLT-blue, BMT-blue, FMT-green, PLT-
yellow, and PMT-yellow demonstrated lfl values of 458, 458,
535, 572, and 562 nm, respectively (Figure 1), when 750 nm TP
excitation was used in a scanning lambda mode. The values
were similar to those measured in EtOH/H2O (2/1), EtOH/H2O
(2/1), EtOH/H2O (4/1), 1,4-dioxane/H2O (20/1), and 1,4-dioxane/
H2O (180/1; Figure S1 in the Supporting Information), indicat-
ing that these mixed solvents adequately represent the polari-
ty of the respective probes’organelle environments.
Table 1. Spectroscopic properties.
[a]
[c]
[d]
Compound
Solvent
lmax/lfl
[nm]
F[b]
dmax
Fdeff
[GM]
[GM]
BLT-blue[e]
BMT-blue[f]
EtOH/H2O (2/1)
EtOH/H2O (2/1)
1,4-dioxane/H2O
(20/1)
354/451
355/455
1.00
1.00
68
54
2120
1860
PLT-yellow[f]
447/572
0.21
550
2110
1,4-dioxane/H2O
(180/1)
EtOH/H2O (4/1)
PMT-yellow[f]
FMT-green[e]
451/564
372/523
0.43
0.65
507
123
1950
1480
[a] lmax of one-photon absorption and emission spectra. [b] Fluorescence
quantum yield. [c] Two-photon cross section in 10À50 cm4 s/photon (GM),
Æ15%. [d] Effective two-photon action cross section measured in HeLa
cells. [e] Ref. [8e,m]. [f] This work.
Although the Fdmax value is a good measure of TP bright-
ness in solution, it cannot be applied to the cellular environ-
ment because of the differences in the degree of staining
among the probes. The TPEF intensities emitted from the or-
ganelles in the probe-labeled cells were measured (in the delta
T-dish in the TPM set up) in order to provide a quantitative
measure of the TP brightness in the cells. The average of the
TPEF intensities measured at five regions of interest (ROI)
chosen in the bright domain was used in the calculation (Fig-
ure S3 in the Supporting Information). The effective Fdmax
(Fdeff) values were estimated by comparing the average TPEF
intensities with that of Rhodamine 6G in MeOH, measured
under the same condition (see the Experimental Section for
details). The Fdeff values of the BLT-blue, BMT-blue, FMT-green,
PLT-yellow, and PMT-yellow probes in HeLa cells (estimated by
this method) were 2120, 1860, 1480, 2110, and 1950 GM, re-
spectively (Figure 2b). A comparison of the Fdmax and Fdeff
values revealed the intravesicular concentrations of probes to
be in a range of 10–35 mm. The values are undoubtedly overes-
timated, and probably more so for BLT-blue and PLT-yellow, as
TPEF intensities were measured from the bright spots, and be-
cause of the compact distribution of lysosomes compared to
mitochondria in the ROI (see large dotted circles in Figure S3
in the Supporting Information). Despite this, a 10–35-fold
Figure 1. Normalized two-photon excited fluorescence spectra of HeLa cells
labeled with 1 mm of the BLT-blue (open circle), BMT-blue (closed circle),
FMT-green (open square), PLT-yellow (open triangle), and PMT-yellow (closed
triangle) probes. The cells were excited at a wavelength of 750 nm.
Two-Photon Brightness
The TP cross-section (d) was de-
termined by the fluorescence
method,[15] using Rhodamine 6G
(in MeOH) as the reference.[16]
The dmax values of BLT-blue, BMT-
blue, PLT-yellow, PMT-yellow,
and FMT-green (measured in the
model solvents) were 68, 54,
550, 507, and 123 GM, respec-
tively (Figure 2a and Table 1).
The larger dmax values for PLT-
yellow and PMT-yellow com-
Figure 2. a) Two-photon excitation spectra of BMT-blue, BLT-blue, FMT-green, PMT-yellow, and PLT-yellow in EtOH/
H2O (2/1), EtOH/H2O (2/1), EtOH/H2O (4/1), 1,4-dioxane/H2O (20/1), and 1,4-dioxane/H2O (180/1), respectively. b) Ef-
fective two-photon action cross-sections of the BMT-blue, BLT-blue, FMT-green, PMT-yellow, and PLT-yellow
probes in HeLa cells.
pared to those of the other
probes can be attributed to the
enhanced ICT.[17]
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higher concentration of probes in each organelle compared to
that in the staining medium could be explained by the uni-
form distribution of probes in the matrix inside the mitochon-
drial membrane or hydrolytic enzyme mixture inside the lyso-
somes, which facilitates TPEF emission without the formation
of aggregates. Interestingly, the Fdeff values of BLT-blue and
BMT-blue were similar to those shown by other probes despite
much smaller dmax values (Table 1). This outcome can be attrib-
uted to the smaller molecular size, which enhances the stain-
ing ability, thereby enriching the probe content in the organ-
elles.
other. The BLT-blue and PLT-yellow probes could be used in
co-localization experiments using TP probes for specific targets
and lysosomes, depending on the emission wavelength of the
latter. BMT-blue and PMT-yellow could also be used for such
experiments in the mitochondria.
Utility of PLT-Yellow as a TP Probe for Lysosomes
Initially, we attempted to assess whether PLT-yellow detects
lysosomes in live cells. For this experiment, HeLa cells were co-
labeled with PLT-yellow and LTB, well-known OP probes for ly-
sosomes.[1d] The result of this analysis showed that the TPM
image of PLT-yellow overlapped with the OPM image of LTB
(Figure 3Aa–c). The Pearson’s co-localization coefficient (A),
which correlates the intensity distribution between the chan-
nels,[18] was calculated using the AutoQuant X2 software. The A
value of PLT-yellow with LTB was 0.87. The TPM images of the
cells co-labeled with PLT-yellow and BLT-blue showed a nearly
perfect overlap, with an A value of 0.92 (Figure 3Ad–f).
These results confirmed the utility of PLT-yellow as a TP
probe for the lysosome. We noted that the co-localization
result between the two TPM images is superior to that be-
tween the OPM and TPM images. Similar results were reported
and attributed to the following two factors: 1) the two TPM
images are obtained simultaneously in the same xy plane,
while the OPM and TPM images were obtained one after the
other, allowing for lysosome movement; 2) the OPM image re-
veals the lysosomal distribution in the entire depth of the
cells.[8d] These results underline the importance of using two
TP probes for co-localization experiments.
Detection Windows
The detection windows of the probes were determined ensur-
ing good separation of the emission bands and similar emis-
sion intensities from the two probes under comparison. For
the co-localization experiments with probe-labeled cells, the
emission intensities were collected at wavelengths of 400–
450 nm (Ch1, BMT-blue, BLT-blue, and LTB (Lysotracker blue)),
475–525 nm (MTG (Mitotracker green)), 550–650 nm (Ch2, PLT-
yellow, PMT-yellow, and FMT-green), and 600–700 nm (MTR
(Mitotracker red); Figure S4 in the Supporting Information).
Under these conditions, the TPEF intensities of PLT-yellow,
PMT-yellow, and FMT-green contributed to the intensities of
BLT-blue and BMT-blue in Ch1 by 0%, 1%, and 10%, respec-
tively; the TPEF intensities of BLT-blue and BMT-blue contribut-
ed towards the intensities of PLT-yellow, PMT-yellow, and FMT-
green in Ch2 by 4%, 4%, and 6%, respectively. Therefore, the
TPEF of BMT-blue/PLY-yellow and BLT-blue/PMTY-yellow pairs
can be determined in the probe-labeled cells with minimum
interference from each other.
Utility of BMT-Blue and PMT-Yellow as TP Probes for Mito-
chondria
We then evaluated the applicability of BMT-blue and PMT-
yellow for the detection of mitochondria in live cells. The TPM
image of the cells co-labeled with BMT-blue and MTR, an OP
probe for mitochondria,[1d] overlapped with the OPM image of
mitochondria with an A value of 0.79 (Figure 3Ba–c). In addi-
tion, the two TPM images of the cells co-labeled with BMT-
blue and FMT-green demonstrated a superior overlap, with an
A value of 0.91 (Figure 3Bd–f). A similar result was observed
for the cells co-labeled with PMT-yellow and MTG, another OP
probe for mitochondria,[1d] with an A value of 0.75 (Fig-
ure 3Ca–c). Moreover, the co-localization experiment of the
cells co-labeled with BMT-blue and PMT-yellow (detected by
TPM) showed a nearly perfect overlap, with an A value of 0.93
(Figure 3Cd–f). A superior co-localization was evident between
the two TPM images. These results established the utilities of
BMT-blue and PMT-yellow as TP probes for mitochondria.
Cytotoxicity, Photostability, and pH Dependence
The assays performed using a CCK-8 kit revealed that BMT-
blue, PLT-yellow, and PMT-yellow exhibited low cytotoxicity
(Figure S5 in the Supporting Information). In addition, the TPEF
intensity at a given spot on the HeLa cells labeled with BMT-
blue, PLT-yellow, and PMT-yellow remained nearly the same
after continuous irradiation with the femtosecond-pulse for
1 h, indicating high photostabilities of these probes (Figure S6
in the Supporting Information). Furthermore, the TPEF intensi-
ties measured from each probe in the EtOH/buffer (1/1) solu-
tion remained nearly the same at pH 4–9 (Figure S7 in the Sup-
porting Information). These results indicate that BMT-blue, PLT-
yellow, and PMT-yellow can be applied to the detection of
target organelles using TPM, with minimum interference from
cytotoxicity, photostability, and pH value.
Dual-Color Imaging of Autophagy by TPM
TP Probes for Dual-Color Imaging
We also evaluated the applicability of BMT-blue and PLT-yellow
in the visualization of autophagy, a self-degradative process
important for balancing the energy sources in response to nu-
trient intake,[9–11] by dual-color TPM imaging. Autophagy is
known to proceed via an autophagosome, which is formed by
the enclosure of a portion of the cytoplasm (including mito-
The combined results reveal that the BMT-blue/PLT-yellow or
BLT-blue/PMT-yellow probe pairs could be applied to the si-
multaneous detection of mitochondria and lysosomes in
a living system by TPM, as they emit strong TPEF in the cells
that can be detected with minimum interference from each
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Figure 3. A) a) OPM and b,d,e) TPM images of HeLa cells co-labeled with LTB (a,b) and PLT-yellow and BLT-blue and PLT-yellow (d,e). c,f) Merged images. The
OPM image was collected at l=380–450 nm (LTB; a), and TPM images were collected at l=550–650 nm (PLT-yellow; b,e); l=400–450 nm (BLT-blue; d). Cells
were labeled with 1 mm of the probes. The wavelengths for one- and two-photon excitation were l=373 and 750 nm, respectively. Cells shown are represen-
tative images from replicate experiments (n=5). Scale bar 30 mm. B) a,d,e) TPM and b) OPM images of HeLa cells co-labeled with BMT-blue and MTR (a,b)
and BMT-blue and FMT-green (d,e). c,f) Merged images. The TPM image was collected at l=400–450 nm (BMT-blue; a,d); l=550–650 nm (FMT-green; e),
and the OPM image was collected at l=600–700 nm (MTR; b). Cells were labeled with 1 mm of the probes. The wavelengths for one- and two-photon excita-
tion were l=543 and 750 nm, respectively. Cells shown are representative images from replicate experiments (n=5). Scale bar 30 mm. C) a) OPM and
b,d,e) TPM images of HeLa cells co-labeled with MTG and PMT-yellow (a,b), and BMT-blue and PMT-yellow (d,e). c, f) Merged images. The OPM image was
collected at l=475–525 nm (MTG; a) and TPM images were collected at l=550–650 nm (PMT-yellow; b,e); l=400–450 nm (BMT-blue; d), respectively. Cells
were labeled with 2 mm MTG and 1 mm each of BMT-blue and PMT-yellow. The wavelengths for one- and two-photon excitation were l=488 and 750 nm, re-
spectively. Cells shown are representative images from replicate experiments (n=5). Scale bar 30 mm. D) a,b) TPM images of untreated HeLa cells co-labeled
with BMT-blue and PLT-yellow, using the detection windows at 400–450 nm (BMT-blue; a) and 550–650 nm (PMT-yellow; b). c) Merged image of (a) and (b).
e) Merged image of the TPM images collected at the two detection windows 4 h after treatment of the cells with 6-hydroxydopamine (40 mm) d,f) TPEF inten-
sities of BMT-blue and PLT-yellow collected along the white dotted lines in (c) and (e), respectively. Cells were labeled with 1 mm of the probes. The excitation
wavelength was l=750 nm. Cells shown are representative images from replicate experiments (n=5). Scale bar 30 mm. E) Dual-color TPM images of a rat hip-
pocampal slice co-labeled with 10 mm each of BMT-blue and PLT-yellow, collected at a depth of approximately 120 mm. The images were taken at a–c) 20”,
d–f) 100”, and g–i) 500” magnifications. c,f,i) Merged images. a–c) The images in the white boxes indicate the pyramidal neuronal layer of the CA3 region.
The TPM images were collected at l=400–450 (BMT-blue; a,d,g) and l=550–650 nm (PLT-yellow; d,e,h). g–i) Enlarged images. The green and red spots
within the whited dotted circles show the mitochondria and lysosomes, respectively, in a cell within the tissue. They showed little overlap with an A value of
0.16. The wavelength for two-photon excitation was l=750 nm. Scale bars: (c) 150, (f) 30, and (i) 6 mm.
chondria) by a phagophore or isolation membrane.[19] The
outer membrane of an autophagosome is fused with the en-
dosome, and subsequently with a lysosome, and the contents
are degraded.
the two organelles were monitored before and after cell stimu-
lation. The TPM images of the untreated cells clearly revealed
the mitochondria (green spots; Figure 3Da) and lysosomes
(red spots; Figure 3Db). In addition, the TPEF intensities col-
lected from the untreated cells along the dotted line showed
a partial overlap, with an A value of 0.25 (Figure 3Dc, d). Upon
Autophagy was investigated by co-labeling HeLa cells with
BMT-blue and PLT-yellow; the TPEF intensities emitted from
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treatment with 6-hydroxydopamine (40 mm), a pharmacologic
agent that induces autophagy,[19] the TPEF intensity at 400–
450 nm (BMT-blue) decreased, while that at 550–650 nm (PLT-
yellow) increased, with time (Figure 3De, f, and Figure S8 in
the Supporting Information); this result was consistent with
the activation of lysosomes and breakdown of mitochondria
during autophagy. Moreover, the A value of the red and green
domains collected from the treated cells along the dotted line
increased from 0.25 to 0.31, 0.42, and 0.67, after 0, 2, 3, and
4 h, respectively (Figure 3De, f, and Figure S8 in the Support-
ing Information). After 4 h, the TPEF intensity of the red
domain was always stronger than that shown by the green
domain (Figure 3Df). This outcome is consistent with the
fusion between autophagosomes and lysosomes, accompanied
by the breakdown of mitochondria during autophagy, al-
though the increase in lysosomal level upon 6-hydroxydopa-
mine stimulation might have contributed to the enhanced
TPEF intensity of the red domain.[21] Therefore, the BMT-blue
and PLT-yellow probes can be applied for the monitoring of
autophagy in live cells by dual-color TPM imaging, using
a single excitation source.
lysosomes and mitochondria deep inside a live tissue by dual-
color TPM imaging. These results suggested that the two pairs
of TP probes, BLT-blue/PLT-yellow and BMT-blue/PMT-yellow,
could find useful applications in the monitoring of organelles
and in the co-localization experiments in the two organelles.
Experimental Section
Synthesis
The BLT-blue and FMT-green probes were prepared in a previous
experiment.[8e] Compounds 1, 2, 6, and 7 were prepared as per the
methods detailed in literature.[13,22–24] PLT-yellow, PMT-yellow, and
BMT-blue were synthesized as described below.
BMT-Blue
CF3CO2H (1 mL) was added to a 0.24 mmol solution of compound
1 (100 mg) in CH2Cl2 (4 mL); the mixture was stirred for 12 h under
Ar. The solvent was removed in vacuo. The crude product was
used for the next step, without further purification. A mixture of
the crude product, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimi-
de·HCl (68 mg, 0.36 mmol), and 4-dimethylaminopyridine (2.9 mg,
0.024 mmol) in CH2Cl2 (10 mL) was stirred for 20 min. (2-aminoe-
thyl)triphenylphosphonium bromide (0.71 g, 2.1 mmol) was added
to this mixture, and stirred for 12 h under Ar. The product was ex-
tracted with ethyl acetate and dried over MgSO4. The solvent was
removed in vacuo. The product was purified by column chroma-
tography (silica gel), using CH2Cl2/MeOH (19/1) as the eluent to
Simultaneous Detection of Lysosomes and Mitochondria in
Live Tissues by Dual-Color TPM Imaging
The utility of the BMT-blue and PLT-yellow probes in tissue
imaging was further investigated. The bright-field image of
a fresh rat hippocampus section, obtained from a postnatal
two-week old rat, incubated with BMT-blue and PLT-yellow
(10 mm) for 1 h at 378C, revealed the presence of the CA3
region (Figure S9 in the Supporting Information).
1
obtain a pale yellow solid. Yield 120 mg (73%); H NMR (500 MHz,
CDCl3): d=9.02 (1H, t, J=5.9 Hz), 8.55 (1H, s), 8.10 (1H, dd, J=8.8,
1.5 Hz), 7.82–7.65 (17H, m), 7.45 (1H, d, J=8.8 Hz), 7.26 (1H, d, J=
2.4 Hz), 7.15 (1H, dd, J=8.8, 2.4 Hz), 6.93 (1H, d, J=2.4 Hz), 6.92
(1H, dd, J=8.8, 2.4 Hz), 4.10 (2H, s), 3.88 (3H, s), 3.88–3.84 (2H, m),
3.75–3.68 (2H, m), 3.30 ppm (3H, s); 13C NMR (100 MHz, CDCl3): d=
170.1, 164.4, 156.9, 148.3, 145.0, 142.8, 136.2, 135.0, 133.2, 133.1,
130.4, 130.2, 129.8, 127.4, 126.6, 125.8, 123.8, 119.8, 117.8, 116.6,
116.3, 112.5, 110.1, 105.8, 102.4, 56.3, 55.6, 40.4, 33.0, 22.0 ppm;
HRMS(FAB+): m/z calcd for [C41H37N3O3P+]: 650.2567, found:
650.2567.
The TPM images obtained at depths of 90–165 mm revealed
an even distribution of the two organelles along the xy plane
in its entire depth (Figure S9 in the Supporting Information).
The dual-channel TPM images and the merged image showed
lysosomes and mitochondria in the same region at a depth of
120 mm (Figure 3E). Moreover, the images taken at a higher
magnification clearly resolved the distribution of lysosomes
and mitochondria in the pyramidal neuron layer of the CA3
region (Figure 3Eg–i). In addition, the two images in the white
dotted circles showed little overlap, with an A value of 0.16
(Figure 3Eg–i). These results demonstrated the ability of BMT-
blue and PLT-yellow to detect mitochondria and lysosomes
(with minimum interference from each other) in live tissues at
depths of 90–165 mm, using TPM.
Compound 3
One gram of compound 2 (7.3 mmol) was added to ethyl acrylate
(2.2 g, 22 mmol) in (CF3)2CHOH (8 mL). The mixture was stirred at
608C for 2 h. The resulting mixture was concentrated in vacuo in
order to obtain the pure product as a white solid. Yield: 1.7 g
1
(quantitative); H NMR (500 MHz, CDCl3): d=7.15 (1H, t, J=8.2 Hz),
6.37 (1H, dd, J=8.2, 2.3 Hz), 6.31 (1H, dd, J=8.2, 2.3 Hz), 6.28 (1H,
d, J=2.3 Hz), 4.14 (2H, q, J=7.1 Hz), 3.80 (3H, s), 3.67 (2H, t, J=
7.1 Hz), 2.94 (3H, s), 2.57 (2H, t, J=7.1 Hz), 1.26 ppm (3H, t, J=
7.1 Hz); 13C NMR (100 MHz, CDCl3): d=172.1, 160.6, 149.8, 129.7,
105.3, 101.2, 98.8, 60.3, 54.8, 48.4, 38.1, 31.6, 14.0 ppm; HRMS(EI):
m/z calcd for [C13H19NO3+]: 237.1365, found: 237.1364.
Conclusions
In this study, we have developed new TP probes for lysosomes
(PLT-yellow) and mitochondria (BMT-blue and PMT-yellow) that
could be excited by 750 nm femtosecond laser pulses, showed
significant TP action cross-sections, could be easily loaded into
the cell and tissue, could emit strong TPEF in the cells, and
could visualize lysosomes and mitochondria in live cells and
tissues for a long period of time by using TPM, with minimum
interference from cytotoxicity and pH value. BMT-blue and PLT-
yellow could be applied for the simultaneous visualization of
Compound 4
POCl3 (0.97 g, 6.3 mmol) was added drop-wise to DMF (5 mL) at
08C, and stirred for 30 min. One gram of compound 3 (4.2 mmol)
in DMF (5 mL) was added to this mixture; the resulting mixture
was stirred for 3 h at room temperature. The solution was neutral-
ized with 1n NaOH, and stirred overnight. The residue was extract-
ed with CH2Cl2. The combined organic layer was dried over MgSO4
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and concentrated in vacuo. The crude product was purified by
column chromatography (silica gel), using hexane/EtOAc (3/1) as
the eluent to obtain a white solid. Yield: 0.59 g (53%); 1H NMR
(500 MHz, CDCl3): d=10.17 (1H, s), 7.73 (1H, d, J=8.8 Hz), 6.32
(1H, dd, J=8.8, 2.2 Hz), 6.10 (1H, d, J=2.2 Hz), 4.14 (2H, q, J=
7.1 Hz), 3.91 (3H, s), 3.77 (2H, t, J=7.1 Hz), 3.08 (3H, s), 2.62 (2H, t,
J=7.1 Hz), 1.26 ppm (3H, t, J=7.1 Hz); 13C NMR (100 MHz, CDCl3):
d=186.9, 171.3, 163.5, 154.3, 130.0, 114.5, 104.1, 92.8, 60.4, 54.9,
47.8, 38.2, 31.8, 13.8 ppm; HRMS(FAB+): m/z calcd for [C14H19NO4 +
H+]: 266.1387, found: 266.1386.
69.4, 67.6, 62.9, 58.8 ppm; HRMS(FAB+): m/z calcd for
[C18H20N2O5+H+]: 345.1444, found: 345.1445.
Compound 10
A solution of compound 9 (0.30 g, 0.74 mmol) in CH2Cl2 (5 mL) was
added to a solution containing DDQ (0.33 g, 1.5 mmol), PPh3
(0.39 g, 1.5 mmol), and Bu4N+BrÀ (0.47 g, 1.5 mmol) in CH2Cl2
(10 mL) through a cannula. The mixture was stirred for 1 h under
Ar. The resulting mixture was concentrated in vacuo. The crude
product was purified by silica gel column chromatography, using
hexane/EtOAc (3/1) as the eluent to obtain a yellow solid. Yield:
Compound 5
1
0.19 g (64%); H NMR (500 MHz, CDCl3): d=9.02 (1H, d, J=1.5 Hz),
A mixture of compound 4 (0.50 g, 1.9 mmol) and AlCl3 (1.5 g,
11 mmol) in CH2Cl2 (10 mL) was refluxed for 2 h under Ar. The re-
sulting mixture was quenched with NH4Cl and extracted with
CH2Cl2. The combined organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by silica gel
column chromatography, using hexane/EtOAc (3/1) as the eluent
to obtain a white solid. Yield: 0.45 g; (98%); 1H NMR (500 MHz,
CDCl3): d=11.56 (1H, d, J=1.5 Hz), 9.51 (1H, d, J=1.5 Hz), 7.28
(1H, d, J=8.8 Hz), 6.29 (1H, dd, J=8.8, 2.4 Hz), 6.07 (1H, d, J=
2.4 Hz), 4.12 (2H, q, J=7.1 Hz), 3.71 (2H, t, J=7.1 Hz), 3.03 (3H, s),
2.59 (2H, t, J=7.1 Hz), 1.24 ppm (3H, t, J=7.1 Hz); 13C NMR
(100 MHz, CDCl3): d=192.0, 170.9, 163.6, 154.4, 134.8, 111.4, 104.2,
96.8, 60.3, 47.5, 38.1, 31.6, 13.7 ppm; HRMS(FAB+): m/z calcd for
[C13H17NO4 +H+]: 252.1230, found: 252.1230.
8.67 (1H, d, J=1.5 Hz), 7.51 (1H, d, J=8.8 Hz), 7.45 (1H, d, J=
0.7 Hz), 7.11 (1H, d, J=2.2 Hz), 6.95 (1H, dd, J=8.8, 2.2 Hz), 4.60
(2H, s), 4.24–4.20 (2H, m), 3.94–3.90 (2H, m), 3.77–3.73 (2H, m),
3.62–3.58 (2H, m), 3.41 ppm (3H, s); 13C NMR (100 MHz, CDCl3): d=
158.3, 156.5, 151.3, 150.1, 143.8, 143.8, 139.9, 121.9, 121.5, 113.5,
107.2, 96.4, 71.7, 70.6, 69.5, 67.7, 58.9, 30.3 ppm; HRMS(FAB+): m/z
calcd for [C18H19BrN2O4+H+]: 407.0600, found: 407.0601.
Compound 11
A mixture containing compounds 5 (0.12 g, 0.49 mmol) and 10
(0.20 g, 0.49 mmol), and K2CO3 (0.34 g, 2.5 mmol) in DMF (3 mL)
was stirred for 8 h at 1008C under Ar. The resulting mixture was fil-
tered, and the filtrate concentrated in vacuo. The crude product
was purified by silica gel column chromatography, using hexane/
EtOAc (1/1) as the eluent to obtain an orange solid. Yield: 1.22 g
Compound 8
1
(78%); H NMR (500 MHz, CDCl3): d=9.05 (2H, m), 7.53 (1H, d, J=
A mixture containing compounds 6 (1.0 g, 4.2 mmol) and 7 (0.96 g,
4.2 mmol), and K2CO3 (2.9 g, 21 mmol) in DMF (10 mL) was stirred
for 12 h at 1008C under Ar. The resulting mixture was filtered, and
the filtrate was concentrated in vacuo. The crude product was puri-
fied by column chromatography (silica gel), using hexane/EtOAc
(1/1) as the eluent to obtain an orange solid. Yield: 1.22 g (78%);
1H NMR (500 MHz, CDCl3): d=9.28 (1H, d, J=1.5 Hz), 9.16 (1H, d,
J=1.5 Hz), 7.62 (1H, d, J=0.7 Hz), 7.55 (1H, d, J=8.8 Hz), 7.14 (1H,
d, J=2.2 Hz), 6.98 (1H, dd, J=8.8, 2.2 Hz), 4.25–4.21 (2H, m), 4.07
(3H, s), 3.95–3.3.91 (2H, m), 3.78–3.74 (2H, m), 3.63–3.59 (2H, m),
3.41 ppm (3H, s); 13C NMR (100 MHz, CDCl3): d=164.3, 159.2, 157.2,
151.0, 147.0, 145.9, 140.3, 140.0, 122.6, 121.6, 114.2, 109.8, 96.5,
72.1, 70.9, 69.7, 68.0, 59.2, 53.1 ppm; HRMS(FAB+): m/z calcd for
[C19H20N2O6+H+]: 373.1394, found: 373.1394.
8.8 Hz), 7.50 (1H, d, J=8.8 Hz), 7.43 (1H, d, J=0.7 Hz), 7.42 (1H, d,
J=0.7 Hz), 7.15 (1H, d, J=2.2 Hz), 6.96 (1H, dd, J=8.8, 2.2 Hz),
6.90 (1H, d, J=2.2 Hz), 6.80 (1H, dd, J=8.8, 2.2 Hz), 4.26–4.22 (2H,
m), 4.16 (2H, q, J=7.1 Hz), 3.95–3.91 (2H, m), 3.81–3.75 (4H, m),
3.63–3.60 (2H, m), 3.42 (3H, s), 3.05 (3H, s), 2.63 (2H, t, J=7.1 Hz),
1.27 ppm (3H, t, J=7.1 Hz); 13C NMR (100 MHz, CDCl3): d=172.0,
158.2, 157.8, 156.5, 152.1, 150.5, 148.2, 143.0, 142.0, 140.1, 140.0,
121.9, 121.8, 118.6, 113.3, 110.5, 107.2, 106.9, 106.2, 96.6, 94.4, 71.9,
70.7, 69.6, 67.8, 60.6, 59.0, 48.9, 38.7, 31.8, 14.1 ppm; HRMS(FAB+):
m/z calcd for [C31H33N3O7+H+]: 560.2391, found: 560.2391.
Compound 12
Compound 11 (0.10 g, 0.18 mmol) was added to a solution of KOH
in EtOH/THF/H2O (1/1/1), and stirred for 6 h. The solution was
evaporated until the volume was reduced by half. Dilute HCl (aq)
was added slowly to this solution until a pH value of 3–4 was at-
tained. The product precipitated as an orange solid. The product
was collected, washed with distilled water, and purified by silica
gel column chromatography, using CH2Cl2/MeOH (20/1) as the
eluent to obtain an orange solid. Yield: 42 mg (44%); 1H NMR
(500 MHz, [D6]DMSO): d=8.95–8.92 (2H, m), 7.53 (1H, s), 7.50 (1H,
d, J=8.8 Hz), 7.46 (1H, s), 7.41 (1H, d, J=8.8 Hz), 7.20 (1H, d, J=
2.2 Hz), 6.85 (1H, dd, J=8.8, 2.2 Hz), 6.81 (1H, d, J=2.2 Hz), 6.72
(1H, dd, J=8.8, 2.2 Hz), 4.09–4.05 (2H, m), 3.70–3.66 (2H, m), 3.58
(2H, t, J=7.1 Hz), 3.53–3.50 (2H, m), 3.40–3.37 (2H, m), 3.17 (3H,
s), 2.87 (3H, s), 2.44–2.40 ppm (2H, m). 13C NMR (100 MHz,
[D6]DMSO): d=174.0, 158.7, 158.1, 156.8, 152.3, 150.5, 149.1, 143.2,
142.2, 140.7, 140.4, 122.9, 122.1, 118.4, 114.0, 111.3, 108.1, 107.3,
97.2, 94.5, 72.0, 70.4, 69.5, 68.4, 58.8, 49.0, 39.0, 32.0 ppm;
HRMS(FAB+): m/z calcd for [C29H29N3O7+H+]: 532.2078, found:
532.2078.
Compound 9
A mixture of compound 8 (1.00 g, 2.7 mmol) and NaBH4 (0.76 g,
20 mmol) in THF (20 mL) was heated at reflux for 30 min. MeOH
(1 mL) was added drop-wise, and the mixture was stirred for 1 h at
708C. The reaction was cooled to room temperature and quenched
with saturated NH4Cl solution (15 mL). The organic layer was sepa-
rated, and the aqueous phase was extracted with CH2Cl2 (2”
30 mL). The combined organic layer was dried over MgSO4 and
concentrated to obtain the product. The residue was purified by
column chromatography (silica gel), using hexane/EtOAc (1/1) as
the eluent to obtain a yellow solid. Yield: 0.60 g (65%); 1H NMR
(500 MHz, CDCl3): d=8.97 (1H, d, J=1.5 Hz), 8.61 (1H, d, J=
1.5 Hz), 7.48 (1H, d, J=8.8 Hz), 7.39 (1H, d, J=0.7 Hz), 7.07 (1H, d,
J=2.2 Hz), 6.93 (1H, dd, J=8.8, 2.2 Hz), 4.86 (2H, s), 4.22–4.18 (2H,
m), 3.93–3.89 (2H, m), 3.77–3.73 (2H, m), 3.62–3.58 (2H, m),
3.41 ppm (3H, s); 13C NMR (100 MHz, CDCl3): d=157.9, 156.1, 153.3,
151.4, 143.2, 142.0, 139.0, 121.7, 121.5, 113.1, 106.2, 96.2, 71.6, 70.4,
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measuring the absorbance by the same method described above.
In both cases, the maximum concentration in the linear region was
considered to be the solubility.
PLT-Yellow
A mixture containing compound 12 (50 mg, 0.094 mmol), 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide·HCl (27 mg, 1.4 mmol),
and 4-dimethylaminopyridine (1.1 mg, 9.4 mmol) in CH2Cl2 (3 mL)
was stirred for 20 min. 3-(Dimethylamino)-1-propylamine (12 mg,
0.11 mmol) was added to this mixture and stirred for 12 h under
N2. The resulting mixture was extracted with CH2Cl2 and dried over
MgSO4. The solvent was removed in vacuo. The product was puri-
fied by column chromatography, using CH2Cl2/MeOH (10/1) as the
eluent to obtain an orange solid. Yield: 31 mg (58%); 1H NMR
(500 MHz, CDCl3): d=9.04–9.01 (2H, m), 7.53 (1H, d, J=8.8 Hz),
7.49 (1H, d, J=8.8 Hz), 7.43 (1H, s), 7.42 (1H, s), 7.16–7.12 (2H, m),
6.96 (1H, dd, J=8.8, 2.2 Hz), 6.89 (1H, d, J=2.2 Hz), 6.81 (1H, dd,
J=8.8, 2.2 Hz), 4.25–4.21 (2H, m), 3.95–3.91 (2H, m), 3.82–3.77 (4H,
m), 3.63–3.59 (2H, m), 3.42 (3H, s), 3.37–3.31 (2H, m), 3.05 (3H, s),
2.46 (2H, t, J=7.1 Hz), 2.31 (2H, t, J=7.1 Hz), 2.18 (6H, s), 1.66–
1.62 ppm (2H, m); 13C NMR (100 MHz, CDCl3): d=171.0, 152.3,
157.9, 156.6, 152.1, 150.4, 148.3, 143.0, 142.1, 140.3, 140.0, 122.0,
121.8, 118.5, 113.4, 110.6, 107.9, 107.0, 106.2, 96.7, 94.2, 71.9, 70.8,
69.6, 67.9, 59.1, 58.6, 49.7, 45.3, 39.5, 39.0, 34.0, 25.9 ppm;
HRMS(FAB+): m/z calcd. for [C34H41N5O6+H+]: 616.3130, found:
616.3129.
Measurement of the Two-Photon Cross Section
The two-photon cross section (d) was determined using the previ-
ously described femtosecond (fs) fluorescence measurement tech-
nique.[14] BMT-blue, BLT-blue, FMT-green, PMT-yellow, and PLT-
yellow (3.0”10À6 m) were dissolved in EtOH/H2O (2/1), EtOH/H2O
(2/1), EtOH/H2O (4/1), dioxane/H2O (180/1), and dioxane/H2O (20/
1), respectively. The two-photon induced fluorescence intensity
was measured at 740–940 nm by using Rhodamine 6G, whose
two-photon property has been well characterized.[15] The intensities
of the two-photon induced fluorescence spectra emitted by the
reference and the sample (at the same excitation wavelength)
were determined. The TPA cross section was calculated using the
formula d=dr(SsFrfrcr)/(SrFsfscs): where the subscripts s and r rep-
resent the sample and reference molecules, S denotes the intensity
of the signal collected by a CCD detector, F is the fluorescence
quantum yield, f is the overall fluorescence collection efficiency of
the experimental apparatus, c denotes the number density of the
molecules in solution, and dr refers to the TPA cross section of the
reference molecule.
PMT-Yellow
The effective TP action (Fdeff) was estimated by comparing the
TPEF intensities of each organelle with that of Rhodamine 6G in
MeOH (5.0 mm, 1.0 mL) in the delta T-dish in the TPM set up. The
average values of TPEF intensities measured in five chosen regions
of interest (ROI) in the bright domains were used in this calcula-
PMT-yellow was synthesized from compound 12 and (2-aminoe-
thyl)triphenylphosphonium bromide by using the same procedure
as that described for PLT-yellow. Yield: 55 mg (65%); 1H NMR
(500 MHz, CDCl3): d=8.99 (1H, d, J=1.5 Hz), 8.97 (1H, d, J=
1.5 Hz), 7.80–7.64 (16H, m), 7.52 (1H, d, J=8.8 Hz), 7.41 (1H, d, J=
0.7 Hz), 7.35 (1H, d, J=8.8 Hz), 7.26 (1H, d, J=0.7 Hz), 7.13 (1H, d,
J=2.2 Hz), 6.95 (1H, dd, J=8.8, 2.2 Hz), 6.92 (1H, d, J=2.2 Hz),
6.85 (1H, dd, J=8.8, 2.2 Hz), 4.24–4.20 (2H, m), 3.94–3.90 (2H, m),
3.77–3.69 (4H, m), 3.66–3.58 (4H, m), 3.54–3.47 (2H, m), 3.41 (3H,
s), 3.07 (3H, s), 2.51 ppm (2H, t, J=7.1 Hz); 13C NMR (100 MHz,
CDCl3): d=172.3, 158.2, 157.8, 156.5, 152.1, 150.1, 148.9, 143.0,
141.9, 140.1, 139.9, 135.1, 133.4, 133.3, 130.5, 130.4, 121.9, 121.8,
118.0, 117.9, 117.1, 113.4, 110.8, 107.0, 96.6, 94.1, 71.8, 70.6, 69.6,
67.8, 59.0, 49.2, 38.2, 33.7, 33.0, 22.5 ppm; HRMS(FAB+): m/z calcd.
for [C49H48N4O6P+]: 819.3306, found: 819.3306.
tion. The Fdeff values were calculated using the equation Fdeff
=
Fdref(Iprobe/Iref), where Fdref denotes the Fd value of Rhodamine 6G
in MeOH, and Iprobe, and Iref represent the TPEF intensities of the
probes in the organelle and Rhodamine 6G in MeOH, respectively.
Detection Window
The detection windows of the probes were identified such that the
emission bands were well separated and the emission intensities
from the two probes under comparison were nearly identical. The
emission intensities for the co-localization experiments using the
probe-labeled cells were 400–450 nm (Ch1, BMT-blue, BLT-blue,
and LTB), 475–525 nm (MTG), 550–650 nm (Ch2, PLT-yellow, PMT-
yellow, and FMT-green,), and 600–700 nm (MTR), respectively (Fig-
ure S4 in the Supporting Information).
Spectroscopic Measurements
Absorption spectra were recorded on a Hewlett–Packard 8453
diode array spectrophotometer; the fluorescence spectra were ob-
tained using an Aminco-Bowman series 2 luminescence spectrome-
ter with a 1 cm standard quartz cell. The fluorescence quantum
yield was determined using Coumarin 307 and Rhodamine B as the
references, using a method detailed in a previous study.[24] The
spectral data obtained under various conditions are summarized in
Figure S1 and Table S1 in the Supporting Information.
Cell Viability
The possible effect of the probe on the viability of cultured HeLa
cells was determined using the CCK-8 kit (Cell Counting Kit-8; Do-
jindo Laboratories, Tokyo, Japan), according to the manufacturer
protocols. The results are displayed in Figure S5 in the Supporting
Information.
Water Solubility
A small quantity of dye was dissolved in dimethyl sulfoxide
(DMSO) in order to prepare the stock solutions (1.0”10À3 m). The
solution was diluted to (6.0”10À3–6.0”10À5)m and added to a cuv-
ette containing 3.0 mL H2O, using a micro syringe. The concentra-
tion of DMSO in H2O was maintained at 0.2% in all cases.[13] The
fluorescence intensity was observed to be linear at low concentra-
tions of the dye, and showed a downward curvature at higher con-
centrations of the dye (Figure S2 in the Supporting Information).
The solubilities of PLT-yellow and PMT-yellow were determined by
Photostability
The photostabilities of the BMT-blue, PLT-yellow, and PMT-yellow
probes were determined by monitoring the changes in TPEF inten-
sity with time, at three designated positions of probe-labeled
(1 mm) HeLa cells chosen without bias. In all cases, the TPEF inten-
sity remained unaltered for 1 h, indicating high photostabilities of
the probes (Figure S6 in the Supporting Information).
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pH Dependence
Keywords: fluorescent probes · imaging agents · lysosomes ·
mitochondria · two photon
The fluorescence intensities of BMT-blue, PMT-yellow, and PLT-
yellow were measured in EtOH/universal buffer solution (0.1m
citric acid, 0.1m KH2PO4, 0.1m Na2B4O7, 0.1m Tris, 0.1m KCl) under
varying pH conditions. All probes were observed to be pH-insensi-
tive over the pH range 4.0–9.0 (Figure S7 in the Supporting Infor-
mation).
[1] a) C. Bucci, P. Thomsen, P. Nicoziani, J. McCarthy, B. van Deurs, Mol. Biol.
Cell 2000, 11, 476–480; b) P. Bernardi, V. Petronilli, F. D. Lisa, M. Forte,
Trends Biochem. Sci. 2001, 26, 112–117; c) J. Zhang, R. E. Campbell, A. Y.
Ting, R. Y. Tsien, Nat. Rev. Mol. Cell Biol. 2002, 3, 906–918; d) A Guide to
Fluorescent Probes and Labeling Technologies, 10th ed. (Ed.: R. P. Haug-
land), Molecular Probes, Eugene, 2005; e) B. N. G. Giepmans, S. R.
Adams, M. H. Ellisman, R. Y. Tsien, Science 2006, 312, 217–224; f) D. W.
Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4, 168–175; g) H.
Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y. Urano, Chem. Rev. 2010,
110, 2620–2640; h) J. Han, K. Burgess, Chem. Rev. 2010, 110, 2709–
2728; i) D. T. Quang, J. S. Kim, Chem. Rev. 2010, 110, 6280–6301.
[2] F. Helmchen, W. Denk, Nat. Methods 2005, 2, 932–940.
[3] H. M. Kim, B. R. Cho, Acc. Chem. Res. 2009, 42, 863–872.
[4] J. P. Luzio, P. R. Pryor, N. A. Bright, Nat. Rev. Mol. Cell Biol. 2007, 8, 622–
632.
[5] G. Kroemer, M. Jaattela, Nat. Rev. Cancer 2005, 5, 886–897.
[6] D. D. Newmeyer, Cell 2003, 112, 481–490.
Cell Culture
HeLa human cervical carcinoma cells (ATCC, Manassas, VA, USA)
were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wel-
Gene Inc, Seoul, Korea) supplemented with 10% fetal bovine
serum (FBS; WelGene), penicillin (100 unitsmLÀ1), and streptomycin
(100 mgmLÀ1). All cells were maintained in a humidified atmos-
phere of 5/95 (v/v) CO2/air at 378C. The cells were passed and
plated on glass-bottomed dishes (MatTek Corp., Ashland, MA, USA)
two days prior to imaging. For the labeling experiment, the
growth medium was removed and replaced with DMEM without
FBS. The cells were incubated with 1 mm BMT-blue, PLT-yellow, and
PMT-yellow for 20 min at 378C, and washed three times with
DMEM without FBS.
[7] D. C. Chan, Cell 2006, 125, 1241–1252.
[8] a) S. Yao, H. Y. Ahn, X. Wang, J. Fu, E. W. Van Stryland, D. J. Hagan, K. D.
Belfield, J. Org. Chem. 2010, 75, 3965–3974; b) X. Wang, D. M. Nguyen,
C. O. Yanez, L. Rodriguez, H. Y. Ahn, M. V. Bondar, K. D. Belfield, J. Am.
Chem. Soc. 2010, 132, 12237–12239; c) C. D. Andrade, C. O. Yanez, M. A.
Qaddoura, X. Wang, C. L. Arnett, S. A. Coombs, J. Yu, R. Bassiouni, M. V.
Bondar, K. D. Belfield, J. Fluoresc. 2011, 21, 1223–1230; d) J. H. Son, C. S.
Lim, J. H. Han, I. A. Danish, H. M. Kim, B. R. Cho, J. Org. Chem. 2011, 76,
8113–8116; e) J. H. Han, S. K. Park, C. S. Lim, M. K. Park, H. J. Kim, H. M.
Kim, B. R. Cho, Chem. Eur. J. 2012, 18, 15246–15249; f) W. Yang, P. S.
Chan, M. S. Chan, K. F. Li, P. K. Lo, N. K. Mak, K. W. Cheah, M. S. Wong,
Chem. Commun. 2013, 49, 3428–3430; g) A. R. Sarkar, C. H. Heo, H. W.
Lee, K. H. Park, Y. H. Suh, H. M. Kim, Anal. Chem. 2014, 86, 5638–5641;
h) F. Miao, W. Zhang, Y. Sun, R. Zhang, Y. Liu, F. Guo, G. Song, M. Tian, X.
Yu, Biosens. Bioelectron. 2014, 55, 423–429; i) M. Tominaga, S. Mochida,
H. Sugihara, K. Satomi, H. Moritomo, A. Fuji, A. Tomoyuki, Y. Suzuki, J.
Kawamata, Chem. Lett. 2014, 43, 1490–1492; j) B. Wang, Y. Wang, H.
Wu, X. Song, X. Guo, D. Zhang, X. Ma, M, Tan, RSC Adv. 2014, 4,
49960–49963; k) Y. Dai, B.-K. Lv, X.-F. Zhang, Y. Xiao, Chin. Chem. Lett.
2014, 25, 1001–1005; l) A. L. Capodilupo, V. Vergaro, E. Fabiano, M. De
Giorgi, F. Baldassarre, A. Cardone, A. Maggiore, V. Maiorano, D. Sanvitto,
G. Gigli, J. Mater. Chem. B 2015, 3, 3315–3323; m) J. H. Han, S. K. Park,
C. S. Lim, M. K. Park, H. J. Kim, H. M. Kim, B. R. Cho, Chem. Eur. J. 2015,
21, 7324.
[9] a) M. C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Nat. Rev. Mol. Cell
Biol. 2007, 8, 741–752; b) N. Mizushima, Genes Dev. 2007, 21, 2861–
2873; c) B. Levine, G. Kroemer, Cell 2008, 132, 27–42.
[10] a) M. G. Gutierrez, S. S. Master, S. B. Singh, G. A. Taylor, M. I. Colombo, V.
Deretic, Cell 2004, 119, 753–766; b) S. B. Singh, A. S. Davis, G. A. Taylor,
V. Deretic, Science 2006, 313, 1438–1441.
[11] a) S. Rodriguez-Enriquez, I. Kim, R. T. Currin, J. J. Lemasters, Autophagy
2006, 2, 39–46; b) I. Kim, S. Rodriguez-Enriquez, J. J. Lemasters, Arch.
Biochem. Biophys. 2007, 462, 245–253.
[12] a) M. Rumi, S. J. K. Pond, T. Meyer-Friedrichsen, Q. Zhang, M. Bishop, Y.
Zhang, S. Barlow, S. R. Marder, J. W. Perry, J. Phys. Chem. C 2008, 112,
8061–8071; b) A. I. Ciuciu, D. Firmansyah, V. Hugues, M. Blanchard-
Desce, D. T. Gryko, L. Flamigni, J. Mater. Chem. C 2014, 2, 4552–4565.
[13] K. Rathore, C. S. Lim, Y. Lee, B. R. Cho, Org. Biomol. Chem. 2014, 12,
3406–3412.
Preparation and Staining of Fresh Rat Hippocampal and Hy-
pothalamic Slices
Slices were prepared from the hippocampus and hypothalamus of
a 2 day-old rat (SD). Coronal slices were cut into 400 mm-thick
slices by using a vibrating-blade microtome and stored 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 20 mm ACCu2 in ACSF
bubbled with 95% O2 and 5% CO2, for 30 min at 378C. Slices were
then washed thrice with ACSF and transferred to glass-bottomed
dishes (MatTek Corp.). The slices were observed under a spectral
confocal multiphoton microscope.
Two-Photon Fluorescence Microscopy
Two-photon fluorescence microscope images of probe-labeled
HeLa cells and tissues were obtained by spectral confocal and mul-
tiphoton microscopes (Leica TCS SP2; Leica Camera, Solms, Germa-
ny), using the 100” oil objective with a numerical aperture (NA) of
1.30, and a 20” dry objective with a numerical aperture (NA) of
0.50. The two-photon fluorescence microscope images were ob-
tained using a DM IRE2 microscope (Leica Camera), which excited
the probes using a mode-locked titanium-sapphire laser source
(Chameleon, 90 MHz, 200 fs; Coherent Inc., Santa Clara, CA, USA)
set at a wavelength and output power of 750 nm and 1305 mW,
respectively, which corresponded to a power of 6.26”108 mWcmÀ2
(”20) and 3.80”109 mWcmÀ2 (”100) at the focal plane. Images
were obtained at the 400–450 nm (channel 2) and 550–650 nm
(channel 2) ranges by using internal PMTs to collect the signals in
an 8-bit unsigned 512”512 pixel format, at a scan speed of
400 Hz.
[14] H. M. Kim, H. J. Choo, S. Y. Jung, Y. G. Ko, W. H. Park, S. J. Jeon, C. H. Kim,
T. Joo, B. R. Cho, ChemBioChem 2007, 8, 553–559.
[15] S. K. Lee, W. J. Yang, J. J. Choi, C. H. Kim, S. J. Jeon, B. R. Cho, Org. Lett.
2005, 7, 323–326.
Acknowledgements
[16] N. S. Makarov, M. Drobizhev, A. Rebane, Opt. Express 2008, 16, 4029–
4047.
[17] H. M. Kim, B. R. Cho, Chem. Commun. 2009, 153–164.
[18] J. Adler, I. Parmryd, Cytometry Part A 2010, 77, 733–742.
[19] D. Glick, S. Barth, K. F. Macleod, J. Pathol. 2010, 221, 3–12.
This research was supported by the National Research Founda-
tion of the of Korea (NRF), grant funded by the Korea govern-
ment (MEST)
(NRF-2015R1A2A2A01004052,
NRF-
2013R1A6A3A04058351)
Chem. Asian J. 2015, 10, 2240 – 2249
www.chemasianj.org
2248
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[20] a) R. K. Dagda, J. Zhu, S. M. Kulich, C. T. Chu, Autophagy 2008, 4, 770–
782; b) C. T. Chu, E. D. Plowey, R. K. Dagda, R. W. Hickey, S. J. Cherra III,
R. S. B. Clark, Methods Enzymol. 2009, 453, 217–249.
[23] M. G. Gichinga, S. Striegler, Tetrahedron 2009, 65, 4917–4922.
[24] A. Aditya, T. Kodadek, ACS Comb. Sci. 2012, 14, 164–169.
[21] J. Yoon, S. H. Bang, J. S. Park, S. T. Chang, Y. H. Kim, J. Kim, Appl Biochem
Biotechnol. 2011, 163, 1002–1011.
[22] T. Hirayama, S. Iyoshi, M. Taki, Y. Maedab, Y. Yamamoto, Org. Biomol.
Chem. 2007, 5, 2040–2045.
Manuscript received: April 30, 2015
Accepted Article published: June 9, 2015
Final Article published: July 14, 2015
Chem. Asian J. 2015, 10, 2240 – 2249
www.chemasianj.org
2249
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim