Cyclometalated Iridium(III) Complexes as Two-Photon Phosphorescent Probes for Specific Mitochondrial Dynamics Tracking in Living Cells

Article abstract of DOI:10.1002/chem.201501882

Five cyclometalated iridium(III) complexes with 2-phenylimidazo[4,5-f][1,10]phenanthroline derivatives (IrL1-IrL5) were synthesized and developed to image and track mitochondria in living cells under two-photon (750 nm) excitation, with two-photon absorpt

Full text of DOI:10.1002/chem.201501882

DOI: 10.1002/chem.201501882
Full Paper
&
Imaging Agents
Cyclometalated Iridium(III) Complexes as Two-Photon
Phosphorescent Probes for Specific Mitochondrial Dynamics
Tracking in Living Cells
Chengzhi Jin, Jiangping Liu, Yu Chen, Leli Zeng, Ruilin Guan, Cheng Ouyang, Liangnian Ji,
and Hui Chao*[a]
Chem. Eur. J. 2015, 21, 12000 – 12010
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Abstract: Five cyclometalated iridium(III) complexes with 2-
phenylimidazo[4,5-f][1,10]phenanthroline derivatives (IrL1–
IrL5) were synthesized and developed to image and track
mitochondria in living cells under two-photon (750 nm) exci-
tation, with two-photon absorption cross-sections of 48.8–
65.5 GM at 750 nm. Confocal microscopy and inductive cou-
pled plasma-mass spectrometry (ICP-MS) demonstrated that
these complexes selectively accumulate in mitochondria
within 5 min, without needing additional reagents for mem-
brane permeabilization, or replacement of the culture
medium. In addition, photobleaching experiments and lumi-
nescence measurements confirmed the photostability of
these complexes under continuous laser irradiation and
physiological pH resistance. Moreover, results using 3D mul-
ticellular spheroids demonstrate the proficiency of these
two-photon luminescent complexes in deep penetration
imaging. Two-photon excitation using such novel complexes
of iridium(III) for exclusive visualization of mitochondria in
living cells may substantially enhance practical applications
of bioimaging and tracking.
talated cationic IrIII complexes as labels and sensors began late
compared to research on the other transition metal com-
plexes,[11] they have been shown to have excellent photostabil-
ity, good permeability of cell membranes and favorable quan-
tum efficiency, which makes them attractive alternatives to flu-
orescent organic dyes for bioimaging and labelling.[9,12] Howev-
er, regrettably, for many IrIII complexes, their one-photon ab-
sorbing (OPA) wavelength often lies in the bioincompatible
ultraviolet region.[13] Compared to OPA, excitation with the
two-photon absorbing (TPA) wavelength in the near-infrared
region has several advantages, including a high confined exci-
tation capacity and intrinsic three-dimension resolution, as well
as the ability to image at greater tissue depth, with reduced
photodamage and background fluorescence.[14] The 3D multi-
cellular spheroid model is an alternative approach to analysis
in living animals. This model provides a convenient way to ad-
equately reflect the microenvironment of cells in vivo and
better mimics the complex environment of animal tissue than
do other models.[15] The penetration depth of two-photon lu-
minescence can be easily tested in this model due to its in-
creased depth relative to a cell monolayer.
In previous research, we found that some IrIII complexes pos-
sess high specificity for mitochondria and superior photostabil-
ity, and can be used for mitochondrial imaging and track-
ing.[9,12c,16] More importantly, IrIII complexes have been devel-
oped as two-photon mitochondrial probes.[16d,e] In the present
work, to obtain specific novel complexes for mitochondrial
two-photon phosphorescent probes, a series of 2-phenylimida-
zo-[4,5-][1,10]phenanthroline derivatives that exhibited excel-
lent light-emitting efficiency were synthesized and conjugated
to iridium(III). The cellular location of these IrIII complexes was
determined using one-photon microscopy (OPM) and two-
photon microscopy (TPM) imaging with MitoTracker Green FM
(MTG) co-staining and inductively coupled plasma mass spec-
trometry (ICP-MS). We investigated the cytotoxicity and cellular
uptake mechanism of all five IrIII complexes (IrL1–IrL5), as well
as the phosphorescence stability of IrL1–IrL5 under photo-
bleaching and pH fluctuation. Finally, using the 3D multicellular
spheroid approach, we tracked and observed mitochondrial
morphology at increasing imaging depths.
Introduction
Mitochondria, a membrane-bound organelle found in most eu-
karyotic cells, play a vital role in a variety of cellular processes,
such as energy production, apoptosis regulation, central me-
tabolism, calcium modulation and redox signaling.[1] In addi-
tion, defects in mitochondrial function are directly related to
aging, cancer, and neurodegenerative disorders including Alz-
heimer’s, Huntington’s, and Parkinson’s diseases.[2] These phys-
iological and pathological processes involve subcellular level
mitochondrial dynamics, including microtubule-based mito-
chondrial motility, mitochondrial fusion and fission and mi-
tophagy-induced mitochondrial clearance.[3] Designing probes
to image and track mitochondria is crucial for linking changes
in cell morphology to cellular functions.[4] In luminescence bio-
imaging, luminescent probes are used to label specific biomol-
ecules or subcellular organelles.[5] To obtain high-resolution
images at the subcellular level, organic organelle probes have
been synthesized and commercialized, such as 4,6-diamidino-
2-phenylindole (DAPI), rhodamine 123 and MitoTracker
Green.[6] However, most of the commercially available dyes
suffer from notable shortcomings, including low water solubili-
ty, high toxicity to living cells, small Stokes shifts and poor
photostability.[7] These organic dyes may also cause extensive
cellular damage and unwanted background signals due to the
ultraviolet radiation required for their excitation.[8] In addition,
conventional organic dyes can be easily washed out when
stained cells lose their mitochondrial potential, limiting their
application for observing mitochondrial dynamics.[9]
Compared to organic dyes, metal-based emissive dyes are
interesting alternatives because they display many superior
physicochemical properties for bioimaging, such as large
Stokes shifts (hundreds of nm), long luminescence lifetimes
(100 ns to 1 ms), and enhanced photostabilities (lower photo-
bleaching).[10] Although research on phosphorescent cyclome-
[a] C. Jin, J. Liu, Dr. Y. Chen, L. Zeng, R. Guan, C. Ouyang, Prof. L. Ji,
Prof. Dr. H. Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry and Chemical Engineering, Sun Yat-Sen University
Guangzhou 510275 (P. R. China)
E-mail: ceschh@mail.sysu.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501882.
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Results and Discussion
Table 1. Crystal data and structure refinement for IrL2 and IrL5.
Identification code
IrL2
IrL5
Synthesis and characterization
empirical formula
formula weight
T [K]
crystal system
space group
a [Š]
b [Š]
c [Š]
C50H36Cl7IrN6
1161.20
150.0
triclinic
¯
P1
C49H38ClIrN7
952.51
153(2)
triclinic
¯
P1
14.7899(6)
15.3662(7)
16.2713(9)
96.332(2)
116.5670(10)
113.0000(10)
2856.2(2)
A series of 2-phenylimidazo[4,5-f][1,10]phenanthro-
line derivatives (dpip=1,2-diphenyl-1H-imidazo[4,5-f]
[1,10]phenanthroline;
ptip=2-phenyl-1-p-tolyl-1H-
imidazo[4,5-f][1,10]phenanthroline; fpip=1-(4-fluoro-
phenyl)-2-phenyl-1H-imidazo[4,5-f][1,10]phenanthro-
line; mopip=1-(4-methoxyphenyl)-2-phenyl-1H-imi-
dazo[4,5-f][1,10]phenanthroline; and dmpip=N,N-di-
methyl-4-(2-phenyl-1H-imidazo[4,5-f][1,10]phenan-
throlin-1-yl)benzenamine) were synthesized using
a “one-pot” method by mixing benzaldehyde, 1,10-
phenanthroline-5,6-dione and substituted-aniline in
acetic acid/NH4Cl[16b,17] as the chosen ligand. We pre-
pared five IrIII complexes by mixing [Ir(ppy)2Cl]2 and
the corresponding ligands in aqueous CHCl3/CH3OH
(2:1) and refluxing for 12 h (Scheme 1). All IrIII com-
plexes were purified by column chromatography and
11.1667(4)
11.6133(4)
19.1520(6)
107.603(3)
91.508(3)
101.386(3)
2310.93(14)
2
1.669
9.662
1148.0
0.38”0.35”0.14
CuKa (l=1.54178 Š)
a [8]
b [8]
g [8]
V [Š3]
Z
2
1calcd [gcmÀ3
]
1.108
2.416
950.0
0.143”0.119”0.105
MoKa (l=0.71073 Š)
6.022 to 54.894
m [mmÀ1
F(000)
]
crystal size [mm3]
radiation
2V range for data collec- 8.098 to 133.752
tion [8]
independent reflections
8069 [Rint =0.0490,
sigma =0.0540]
data/restraints/parameters 8069/22/597
GoF on F2
1.065
final R indexes [I> =2s(I)] R1 =0.0421, wR2 =0.1055
final R indexes [all data] R1 =0.0468, wR2 =0.1104
12603 [Rint =0.0642,
Rsigma =0.0906]
12603/67/511
1.015
R1 =0.0492, wR2 =0.1142
R1 =0.0810, wR2 =0.1323
1
characterized by ES-MS, H and 13C NMR spectroscopy
R
(Figures S1–S10 in the Supporting Information).
Single crystals of the [Ir(ppy)2ptip]Cl complexes
(IrL2) and [Ir(ppy)2dmpip]Cl (IrL5) were obtained by
slowly evaporating the solvent at room temperature.
Absorption and emission spec-
troscopy
The absorption and emission
spectra of IrL1–IrL5 at 298 K in
PBS buffer are shown in Fig-
ures S11 and S12, respectively, in
the Supporting Information. The
photophysical data are summar-
ized in Table S1 in the Support-
ing Information. These com-
plexes displayed weak absorp-
Scheme 1. Synthesis of IrL1–IrL5: i) formaldehyde, p-substituted aniline, NH4Cl, acetic acid, 1358C, 14 h;
ii) [Ir(ppy)2Cl]2, CHCl3/CH3OH (v/v, 2:1), 658C, 12 h.
The crystal data and structural refinements are shown in
Table 1. Selected bond lengths and angles are listed in Table 2.
tion bands in the region of 330–400 nm, and stronger absorp-
tion bands in the region of 250–325 nm, which can be as-
signed to mixed singlet and triplet metal-to-ligand charge-
¯
The IrL2 and IrL5 complexes both crystallized in the P1 tri-
3
clinic space group. Ir1 iridium atoms within IrL2 had octahe-
dral distortions, with the largest deviation observed in the bite
angle (76.888) of the ptip ligand (Figure 1). The bite angles of
the IrL5 ligand (77.168) were slightly larger than that of the
ptip ligand. The lengths of the IrÀN bond in the cyclometalat-
ed ligands were 2.042 and 2.043 Š, respectively, which is short-
er than that in the main ligand (2.144 and 2.157 Š). A similar
observation was made for the IrL5 complex. The IrÀN distan-
ces in both IrL2 and IrL5 complexes are longer than those for
ancillary ppy ligands, possibly due to a high trans influence.
This result is congruent with those observed for [Ir(ppy)2-
(PhenSe)]+.[9] In the two IrIII complexes, the main ligands ptip
and dmpip have a tendency to form a large planar aromatic
area and have the potential for larger two-photon absorption
cross-sections.
transfers (1MLCT and MLCT) and intraligand transitions (CÀN li-
gands).[18] The emission spectra of IrL1–IrL5 exhibit maximum
peaks of approximately 595 nm with quantum yields (F) of
0.101–0.127, using [Ru(bpy)3]2+ as a standard (Table S1 in the
Supporting Information).[19] Also, the effect of oxygen on the
iridium luminescence was investigated. The results show that
the quantum yields approximately doubled in degassed MeOH
for all five complexes (Table S1 in the Supporting Informa-
tion).[20] The Stokes shifts of these values were approximately
210 nm. These large Stokes shifts offer advantages over com-
mercial organic cellular dyes, such as rhodamine 6G and
BIDOPY FL (Stokes shift=30 and 8 nm, respectively).[6]
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tions.[21] The cytotoxicity of the IrL1–IrL5 complexes was deter-
mined using an MTT assay after incubating HeLa cells with
IrL1–IrL5.[22] The time-dependent effects of IrL1–IrL5 on cell vi-
ability at 378C are shown in Figure 2. More than 90% of the
cells survived after incubation with these agents for 12 h.
These results suggest that all five IrIII complexes exhibit low
toxicities when imaging cells under the applied conditions
(200 nm of complex incubated for 5 min).
Table 2. Select bond lengths and angles for IrL2 and IrL5.
IrL2
bond lengths [Š]
IrL5
Ir1ÀN1
2.042(4)
2.043(4)
2.147(4)
2.157(4)
2.018(6)
2.003(5)
Ir1ÀN1
Ir1ÀN2
Ir1ÀN3
Ir1ÀN4
Ir1ÀC1
Ir1ÀC12
2.045(6)
2.010(5)
2.150(4)
2.140(4)
2.018(6)
1.997(6)
Ir1ÀN2
Ir1ÀN3
Ir1ÀN4
Ir1ÀC1
Ir1ÀC22
bond angles [8]
N1ÀIr1ÀN2
N1ÀIr1ÀN3
N1ÀIr1ÀN4
N2ÀIr1ÀN3
N2ÀIr1ÀN4
N3ÀIr1ÀN4
C1ÀIr1ÀN1
C1ÀIr1ÀN2
C1ÀIr1ÀN3
C1ÀIr1ÀN4
C22ÀIr1ÀN1
C22ÀIr1ÀN2
C22ÀIr1ÀN3
C22ÀIr1ÀN4
C22ÀIr1ÀC1
171.23(16)
88.84(15)
99.20(16)
98.21(16)
87.54(16)
76.88(14)
80.3(2)
93.51(19)
97.66(16)
174.54(15)
93.2(2)
N1ÀIr1ÀN3
N1ÀIr1ÀN4
N2ÀIr1ÀN1
N2ÀIr1ÀN3
N2ÀIr1ÀN4
N2ÀIr1ÀC1
N4ÀIr1ÀN3
C1ÀIr1ÀN1
C1ÀIr1ÀN3
C1ÀIr1ÀN4
C12ÀIr1ÀN1
C12ÀIr1ÀN2
C12ÀIr1ÀN3
C12ÀIr1ÀN4
C12ÀIr1ÀC1
174.7(2)
98.33(18)
87.8(2)
96.77(19)
173.60(19)
93.1(2)
77.16(16)
79.7(3)
97.42(18)
89.73(18)
96.4(3)
80.3(2)
80.7(2)
86.9(2)
96.83(18)
172.82(16)
174.14(18)
97.36(18)
88.10(19)
Figure 2. Viability of HeLa cells incubated with 0.2 mm IrL1–IrL5.
Cellular uptake and localization
To demonstrate that the specific localization of IrL1–IrL5 is in
the mitochondria, co-localization imaging experiments were
performed with these complexes using the commercially avail-
able mitochondrial dye, MitoTracker Green (MTG), to co-stain
the HeLa cells. Because some complexes, such as [Ru(dip)2-
(dppz)]2+, can experience luminescence quenching in the pres-
ence of serum, we removed the serum before adding our IrIII
complexes.[23] Interestingly, none of the studied complexes ex-
hibited a significant difference when HeLa cells were incubated
in media with or without fetal bovine serum (FBS). Incubation
of 200 nm of IrL1–IrL5 in DMEM with 10% FBS for 5 min was
followed by the removal of excess complex by washing with
PBS three times and incubation using 50 nm MTG for 30 min.
As shown in Figure 3, under these conditions, we observed ex-
cellent concordance between overlay images of the complexes
and MTG, which suggested that IrL1–IrL5 is selectively local-
ized within mitochondria. Pearson’s co-localization coeffi-
cients,[24] which measure the correlation of the intensity distri-
bution between the IrIII complexes and MTG signal, were 0.90,
0.92, 0.87, 0.85 and 0.86 for IrL1–IrL5, respectively. Because iri-
dium is not an endogenous component of cells, accurate cellu-
lar uptake levels of IrIII can be determined quantitatively using
ICP-MS.[25] We performed ICP-MS experiments for each IrL1–
IrL5 complex at a staining concentration of 200 nm. The ICP-
MS measurements (Figure S13 in the Supporting Information)
indicated that very high levels of IrL1–IrL5 accumulated in mi-
tochondria. For all five IrIII complexes, the ratios of mitochon-
drial Ir-to-whole cell Ir (nuclei+cytoplasm) were more than
80%, and the ratios for IrL1 and IrL2 surpassed 90% (91.3 and
91.1%, respectively). To rationally determine our incubation
Figure 1. The structures of complexes: a) IrL2, and b) IrL5. For clarity, the
solvent molecules, hydrogen atoms, and counteranions are omitted.
Cytotoxicity measurements
An ideal cellular probe for practical application should mini-
mally perturb living systems at the employed concentra-
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plex crossing the plasma membrane to localize in mitochon-
dria; the activity of several proteins that aid membrane cross-
ing may be inhibited when the incubation temperature is less
than 378C. As a general cell entry mechanism that involves re-
ceptor signaling (including signaling from receptor tyrosine
kinases and G protein-coupled receptors), endocytic mem-
brane trafficking is energy-dependent.[28] Thus, inhibitors of en-
docytosis (chloroquine and NH4Cl) were used to investigate
IrL2 entry into cells.[29] The confocal image revealed that endo-
cytosis inhibition does not block IrL2 from crossing the mem-
brane (Figure 4e and f). In addition, these results were con-
firmed by flow cytometry (Figure S16 in the Supporting Infor-
mation). Furthermore, entry by the endocytotic pathway is typ-
ically a slow process.[37,38] Time-dependent ICP-MS results indi-
cated that IrL2 uptake reached a maximum within 5 min
(Figure S14 in the Supporting Information). These results sug-
gest that the endocytotic pathway is not responsible for the
mitochondrial localization of IrL2. In summary, IrL2 crosses the
plasma membrane using non-endocytic energy-dependent
active transport. Considering the structural similarity of these
five IrIII complexes, the other complexes may share similar
mechanisms of transmembrane transport.
Figure 3. OPM and TPM confocal phosphorescence images and their over-
lays with bright-field images of living HeLa cells incubated with 200 nm of
IrL1–IrL5 in DMSO and DMEM with 10% FBS (pH 7.4, 1:999, v/v) for 5 min at
378C followed by 50 nm of MTR. Lane 1: OPM confocal phosphorescence
images of IrL1–IrL5; lane 2: confocal phosphorescence images of MTG;
lane 3: TPM confocal phosphorescence images of IrL1–IrL5; lane 4: overlay
of lane 1, lane 2, lane 3 and bright field; lane 4: the overlap coefficient of
columns lane 1 and lane 2, and Pearson’s co-localization coefficients are also
presented. Excitation wavelength: 405 nm (OPM for all IrIII complexes),
750 nm (TPM for all IrIII complexes), 488 nm (for MTG); emission filter:
595(Æ30) nm (for all IrIII complexes) and 520(Æ20) nm (for MTG). Scale bars:
20 mm.
time, time-dependent cellular uptake of IrL2 using real-time
imaging and ICP-MS were conducted, and the results (Fig-
ures S14 and S15 in the Supporting Information) demonstrate
that uptake of IrIII complexes reached saturation within 6 min.
The imaging results revealed that at a low concentration
(200 nm), all of these complexes quickly crossed the membrane
(within 5 min) and selectively accumulated in mitochondria
(above 80%, IrL1 and IrL2 above 90%).
Mechanisms of cellular uptake
Cellular uptake of small molecules can occur through energy-
independent (diffusion, passive diffusion) and energy-depen-
dent (endocytosis, active transport) pathways.[26] To explore the
mechanism of cellular entry, we selected IrL2 as an example to
investigate its cellular uptake at varying temperatures and dif-
ferent metabolic and endocytic levels.
Blockage of cellular uptake was observed using confocal lu-
minescence microscopy when the cell was incubated at 48C or
pretreated with metabolic inhibitors (2-deoxy-d-glucose and
oligomycin).[23,27] As shown in Figure 4a and d, the intracellular
luminescence was too weak to be visualized. In addition, when
the incubation temperature was set to 208C (Figure 4b), the
intracellular luminescence was recovered but remained weaker
than luminescence at 378C (Figure 4c). Based on this observa-
tion, energy seems to play a significant role in the IrL2 com-
Figure 4. Confocal luminescence image and bright-field images of living
HeLa cells incubated with 200 nm IrL2 in DMSO/PBS (pH 7.4, 1:999, v/v)
under different conditions. a) Cells incubated with 200 nm IrL2 at 48C for
5 min; scale bars: 100 mm. b) Cells incubated with 200 nm MitoIr7 at 208C
for 5 min. c) Cells incubated with 200 nm IrL2 at 378C for 5 min. d) Cells pre-
incubated with 50 mm 2-deoxy-d-glucose and 5 mm oligomycin in PBS for
1 h at 378C and then incubated with 200 nm IrL2 at 378C for 5 min.
e,f) Cells pretreated with endocytic inhibitors (chloroquine (50 mm) and
NH4Cl (50 mm), respectively) and then incubated with 200 nm IrL2 at 378C
for 15 min (lex =405 nm, lem =595(Æ30) nm). Scale bars: 20 mm.
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Two-photon absorption (TPA) cross-sections
demonstrated that the rising cell numbers and the presence of
ECM that accompany the formation of 3D multicellular sphe-
roids do not significantly impede the speed at which these IrIII
complexes cross the membrane. However, one noteworthy fea-
ture is that the inner core of the 3D multicellular spheroids
cannot take in the IrIII complexes even after extending the in-
cubation time to 8 h and increasing the concentration to 2 mm
(Figure S22 in the Supporting Information). This phenomenon
may be attributed to the mechanism of uptake, which involves
energy-dependent active transport; the necrotic cells in the
core are unable to perform this process.[33] Similar results were
observed when the spheroids were pretreated with IrL1, IrL3,
IrL4 and IrL5 (Figures S23–S26 in the Supporting Information).
In summary, the penetrating depth we observed for these IrIII
complexes highlights their value in 3D observation using two-
photon imaging.
The phosphorescence quantum yields of the five complexes
were 0.108–0.127. The TPA cross-sections of these complexes
were determined at the wavelength range of 720–860 nm in
CH3OH (500 mm). From the log–log plot of the emission inten-
sity against incident power, the linear regression slopes of
1.85Æ0.05, 2.04Æ0.06, 2.07Æ0.07, 2.04Æ0.07 and 2.05Æ0.04,
respectively, indicate a two-photon excitation process (Fig-
ures S17–S21 in the Supporting Information). The two-photon
absorption cross-section of the complexes were measured as
61.7,
65.5,
48.8,
49.7
and
52.0 GM
(1 GM=10”
50 cm4 sphotonÀ1), using rhodamine B as the standard (Fig-
ure 5).[30] As suggested by Furuta et al., TPA cross-section
values should be higher than 0.1 GM for optical imaging appli-
cations in live specimens. The values of our complexes not
only meet the requirements mentioned above but also exceed
those of some reported for mononuclear transition metal com-
plexes.[31]
Photobleaching and pH-dependent luminescent stability ex-
periments
Photostability is one of the most important criteria for devel-
oping fluorescent imaging agents.[34] The photostability of all
five IrIII complexes compared to those of the organic dyes
under continuous laser irradiation was determined using time-
dependent imaging of IrIII complexes and MTG co-stained HeLa
cells. The initial intensities from the first scans at a co-stained
cell concentration of 5 mm were normalized before calculating
decreases in percentage of fluorescence. As shown in Fig-
ure 7a, using IrL2 versus MTG as an example, during 20 scans
with a total irradiation time of approximately 5 min, almost no
significant luminescence was observed in the MTG image,
whereas no obvious change in the luminescence was observed
for IrL2. Statistically, as shown in Figure 7b, after 20 scans, the
fluorescence signal intensity of MTG decreased to approxi-
mately 33%. In contrast, the signal intensity of the IrIII com-
plexes merely slightly declined. The photobleaching properties
of IrL1–IrL5 (except IrL2) are listed in Figures S27–S30 in the
Supporting Information. For biological applications, dyes
should be usable within a particular pH range.[17] Therefore, we
investigated the effect of pH variation from 4 to 10 on the lu-
minescence of the IrIII complexes. The results indicate that the
luminescence of all IrIII complexes except IrL5 changed very
little when the pH was in the physiological range. This result
may be due to the blockage of the N atom in the imidazole
group of their structures. A mild decrease in luminescence was
observed for IrL5, which may be due to the presence of an
ÀN(CH3)2 group (Figure S31 in the Supporting Information).
Figure 5. Two-photon absorption cross-section of IrL1–IrL5 at different exci-
tation wavelength. The maximum TPA cross-sections value is 65.5 GM (IrL2)
at 750 nm.
One- and two-photon imaging of IrIII complexes in 3D multi-
cellular spheroids
There remain significant technical challenges to increasing the
imaging depth for microscopic study of living tumor spheroids.
3D multicellular spheroids contain an extensive extracellular
matrix (ECM) that differ in their relative amounts and in their
assembly in corresponding monolayer cultures.[32] Thus, in this
work, 3D multicellular spheroids were imaged under confocal
laser scanning microscopy to demonstrate the 3D distribution
and two-photon penetration of IrIII complexes. For IrL2, after
treatment with IrIII complexes, phosphorescence was observed
from the surface of the spheroids to a depth of approximately
90 mm for one-photon excitation and about 250 mm for two-
photon excitation. The phosphorescent images were captured
every 6.9 mm along the Z-axis. The spheroids exhibited much
stronger phosphorescence in deep layer cells using two-
photon excitation than one-photon excitation, indicating
deeper penetration of two-photon excitation light with an in-
cubation of 30 min (Figure 6). The results of the incubation
Tracking mitochondrial morphological changes
Mitochondria are the organelles in which cellular respiration
occurs. Mitochondria continuously oxidize substrates and
maintain a proton gradient across the lipid bilayer with a very
large membrane potential of approximately 180 mV.[35] Varia-
tion in the membrane potential of mitochondria in cells has
evolved as a read-out of mitochondrial functional status and
generates signals to activate pathways that repair or eliminate
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Figure 6. a) OPM (left) and TPM (right) images of 3D multicellular spheroids after incubation with IrL2 (500 nm) for 0.5 h. b) Z-stack images were taken every
approximately 7 mm section along the Z-axis of an intact spheroid. c) The merged images of Z-stack images of an intact spheroid. The wavelengths for one-
and two-photon excitation were set as 405 and 750 nm, respectively. The images were taken under 10” objective. Scale bars: 100 mm.
Figure 7. Photobleaching experiments of the IrIII complexes in HeLa cells. a) Time-dependent confocal imaging of IrL2/MTG co-stained HeLa cells. Time inter-
val: 15 s. Scale bar: 20 mm (lex =405 nm, lem =595(Æ30) nm). b) Quantitative photobleaching results indicate that IrL1–IrL5 exhibited robust emission intensi-
ty under continuous light irradiation.
defective mitochondria.[36] Therefore, tracking the mitochondri-
al morphological changes related to a variety of cellular func-
tions may provide insight into the conditions that are associat-
ed with apoptosis and degeneration.[37] However, commercial
organic dyes, such as MTG, will lose their specificity to mito-
chondria when cells are treated with the uncoupler 3-chloro-
phenylhydrazone (CCCP).[38] In contrast, IrL1–IrL5 have a high
tolerance to decreases in mitochondrial membrane potential.
Using IrL2 as an example, reticulum-like mitochondria were
gradually transformed into small and dispersed fragments
upon exposure to CCCP, as shown in Figure 8 (Video S1 in the
Supporting Information), consistent with the morphological
changes of mitochondria in the early stages of apoptosis.
These processes are involved in the remodeling of mitochon-
drial cristae and the mobilization of cytochrome c, which is
considered an irreversible process associated with the collapse
of the membrane potential.[39] Similar results were observed
when we used IrL1, IrL3, IrL4 and IrL5 to conduct this experi-
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analyzer. Electrospray mass spectra
were recorded using an LCQ
system (Finnigan MAT).
Materials and synthesis
All reactants and solvents were
purchased from commercial sour-
ces and used as received unless
otherwise stated. All procedures
involving IrCl3·xH2O were carried
out under an argon atmosphere.
Synthesis of ligands and the
corresponding iridium(III) com-
plexes
1,10-Phenanthroline-5,6-dione[40]
Figure 8. Phosphorescence images of CCCP (20 mm) treated living HeLa cells stained with IrL2 (200 nm) with in-
creasing scan time (the scan time is shown in the upper panel). The upper panel is the luminescence images of
IrL2. The middle panels are the bright-field images. The lower panels are the merged images. (lex =405 nm,
lem =595(Æ30) nm).
[41]
and [Ir(ppy)2Cl]2
were synthe-
sized according to previously pub-
lished methods.
1,2-Diphenyl-1H-imidazo[4,5-f]
[1,10]phenanthroline (dpip):
A
mixture of 1,10-phenanthroline-
ment (Figures S32–S35 in the Supporting Information), which
5,6-dione (0.525 g, 2.5 mmol), ammonium acetate (3.85 g,
50 mmol), benzaldehyde (0.265 g, 2.5 mmol), aniline (0.233 g,
2.5 mmol) and glacial acetic acid (10 cm3) was refluxed under
argon for 14 h. Then, the reaction mixture was cooled to room
temperature and poured into water (30 cm3). The solution was
neutralized with 25% NH3 aqueous solution and extracted with di-
chloromethane (20 mL”3). The organic phase was dried over
MgSO4, and the solvent was removed under vacuum. The crude
product was purified by column chromatography on silica with
CH2Cl2/ethanol (20:1, v/v) as the eluent. The resulting solid was re-
crystallized from chloroform/toluene to afford a white microcrystal.
Yield, 0.756 g, 81.3%; elemental analysis calcd (%) for C25H16N4: C
80.63, H 4.33, N 15.04; found: C 80.54, H 4.65, N 15.11; 1H NMR
(300 MHz, [D6]DMSO): d=9.07 (dd, J=4.2, 1.8 Hz, 1H), 9.00 (dd, J=
8.1, 1.8 Hz, 1H), 8.93 (dd, J=4.3, 1.6 Hz, 1H), 7.86 (dd, J=8.1,
4.2 Hz, 1H), 7.73 (ddd, J=15.6, 8.4, 4.5 Hz, 5H), 7.61–7.55 (m, 2H),
7.47 (dd, J=8.4, 4.2 Hz, 1H), 7.40–7.30 ppm (m, 4H); ES-MS
(CH2Cl2): m/z 373.1 [M+H]+, 767.4 [2M+Na]+.
is consistent with previous results from our laboratory with
similar complexes.[16c]
Conclusion
In conclusion, a new series of IrIII complexes (IrL1–IrL5) with
two-photon luminescence have been synthesized and utilized
as agents for mitochondrial imaging and tracking in monolayer
cells. The two-photon penetrating depth of the IrIII complexes
was evaluated using 3D multicellular spheroids. These com-
plexes have numerous advantages for two-photon imaging, in-
cluding rapid incubation time (5 min for monolayer cells and
30 min for 3D multicellular spheroids), appreciable photostabil-
ity, excellent pH resistance, low imaging concentration
(200 nm), low cytotoxicity at the imaging concentration, and
sufficient TPA cross-sections for imaging. Cellular uptake ex-
periments indicated that these IrIII complexes cross the mem-
brane using non-endocytosis active transport. Moreover, the
IrIII complexes have a high resistance to decreases in the mito-
chondrial membrane potential, making them suitable for track-
ing mitochondrial morphological changes during the early
stages of apoptosis, which highlights their potential for appli-
cations in biomedical research.
2-Phenyl-1-p-tolyl-1H-imidazo[4,5-f][1,10]phenanthroline (ptip):
This compound was synthesized in a manner identical to that de-
scribed for ligand dpip but using p-toluidine (0.268 g, 2.5 mmol) in-
stead of aniline. Yield: 0.737 g, 76.4%; elemental analysis calcd (%)
for C26H18N4: C 80.81, H 4.69, N 14.50; found: C 80.93, H 4.36, N
1
14.41; H NMR (300 MHz, CDCl3): d=9.24–9.10 (m, 2H), 9.03 (s, 1H),
7.75 (dd, J=8.1, 4.2 Hz, 1H), 7.66–7.52 (m, 4H), 7.44 (t, J=9.3 Hz,
3H), 7.35–7.26 (m, 4H), 1.45–1.43 ppm (m, 3H); ES-MS (CH2Cl2): m/z
387.1 [M+H]+, 795.4 [2M+Na]+.
1-(4-Fluorophenyl)-2-phenyl-1H-imidazo[4,5-f][1,10]phenanthro-
line (fpip): This compound was synthesized in a manner identical
to that described for ligand dpip but using 4-fluoroaniline (0.278 g,
2.5 mmol) instead of aniline. Yield: 0.801 g, 82.2%; elemental analy-
sis calcd (%) for C25H15FN4: C 72.60, H 3.87, N 14.35; found: C 72.67,
H 4.03, N 14.22; H NMR (300 MHz, DMSO): d=9.07 (s, 1H), 9.03–
8.85 (m, 2H), 7.84 (s, 3H), 7.62–7.45 (m, 5H), 7.39 ppm (s, 4H); ES-
MS (CH2Cl2): m/z 391.1 [M+H]+, 803.3 [2M+Na]+.
Experimental Section
Characterization
1H NMR spectra were recorded using a 300 MHz nuclear magnetic
resonance spectrometer (Varian, Mercury-Plus 300) and Bruker 500
nuclear magnetic resonance spectrometer. All chemical shifts are
reported relative to tetramethylsilane (TMS). The electronic absorp-
tion spectra were recorded using a Perkin–Elmer Lambda 850 UV/
Vis spectrometer. The emission spectra were recorded using
a Perkin–Elmer LS 55 luminescence spectrometer. Microanalysis (C,
H, and N) was carried out using an Elementar Vario EL elemental
1
1-(4-Methoxyphenyl)-2-phenyl-1H-imidazo[4,5-f][1,10]phenan-
throline (mopip): This compound was synthesized in a manner
identical to that described for ligand dpip but using 4-methoxyani-
line (0.308 g, 2.5 mmol) instead of aniline. Yield: 0.722 g, 71.2%; el-
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emental analysis calcd (%) for C26H18N4O: C 77.59, H 4.51, N 13.92;
found: C 72.66, H 4.37, N 13.98; H NMR (300 MHz, DMSO): d=8.99
(dd, J=22.2, 14.4 Hz, 3H), 7.84 (s, 1H), 7.74–7.55 (m, 4H), 7.51 (s,
1H), 7.41 (d, J=14.1 Hz, 4H), 7.20 (d, J=8.1 Hz, 2H), 3.88 ppm (s,
3H); ES-MS (CH2Cl2): m/z 403.1 [M+H]+, 827.3 [2M+Na]+.
149.78, 149.52, 148.87, 145.05, 144.71, 144.44, 139.23, 136.49,
133.33, 132.91, 131.67, 130.77, 130.52, 130.41, 129.83, 129.48,
129.05, 128.32, 128.27, 127.25, 126.48, 125.59, 124.37, 124.29,
122.89, 122.51, 120.54, 118.38, 118.20 ppm; ES-MS (CH3OH): m/z
891.25 [MÀClÀ]+.
1
[Ir(ppy2)mopip]Cl (IrL4): This compound was synthesized in
a manner identical to that described for [Ir(ppy2)pi]Cl but using
mopip (0.049 g, 0.015 mmol) instead of dpip. Yield: 0.1086 g,
77.2%; elemental analysis calcd (%) for C48H34N6OClIr: C 61.43, H
3.65, N 8.96; found: C 61.56, H 3.77, N 8.91; 1H NMR (500 MHz,
[D6]DMSO): d=9.30 (dd, J=8.5, 1.5 Hz, 1H), 8.29–8.21 (m, 3H), 8.13
(dd, J=8.5, 5.0 Hz, 1H), 8.08 (d, J=5.0 Hz, 1H), 7.94 (dd, J=14.0,
7.5 Hz, 2H), 7.88 (q, J=7.5 Hz, 2H), 7.80 (dd, J=8.5, 5.0 Hz, 1H),
7.69 (dd, J=6.0, 3.0 Hz, 2H), 7.62 (dd, J=21.0, 7.0 Hz, 3H), 7.50–
7.42 (m, 5H), 7.26–7.18 (m, 2H), 6.98 (dddt, J=15.0, 10.0, 7.5,
4.5 Hz, 6H), 6.28 (dd, J=14.0, 7.5 Hz, 2H), 3.88 ppm (s, 3H);
13C NMR (125 MHz, [D6]DMSO): d=171.91, 167.33, 161.06, 154.57,
151.03, 150.62, 149.67, 149.54, 148.74, 145.06, 144.70, 144.45,
139.22, 136.41, 132.88, 131.66, 130.75, 130.44, 130.25, 129.73,
129.36, 129.02, 128.47, 128.21, 127.07, 126.52, 125.58, 124.37,
124.30, 122.87, 122.64, 120.52, 116.27, 56.16 ppm; ES-MS (CH3OH):
m/z 903.25 [MÀClÀ]+.
[Ir(ppy2)dmpip]Cl (IrL5): This compound was synthesized in
a manner identical to that described for [Ir(ppy2)pi]Cl but using
dmpip (0.063 g, 0.015 mmol) instead of dpip. Yield: 0.0725 g,
57.6%; elemental analysis calcd (%) for C43H33N7ClIr: C 62.77, H
3.98, N 8.96; found: C 62.68, H 4.13, N 8.84; 1H NMR (500 MHz,
[D6]DMSO): d=9.29 (dd, J=8.0, 1.5 Hz, 1H), 8.23 (ddd, J=9.0, 6.0,
4.5 Hz, 3H), 8.12 (dd, J=8.3, 5.1 Hz, 1H), 8.07 (d, J=5.0 Hz, 1H),
7.94 (dd, J=14.0, 7.5 Hz, 2H), 7.88 (dd, J=14.5, 7.0 Hz, 2H), 7.81
(dd, J=8.5, 5.0 Hz, 1H), 7.71–7.65 (m, 3H), 7.51–7.41 (m, 7H), 7.08–
6.87 (m, 8H), 6.28 (dd, J=12.0, 7.5 Hz, 2H), 3.03 ppm (s, 6H);
13C NMR (125 MHz, [D6]DMSO): d=167.32, 154.69, 151.49, 151.07,
150.65, 149.57, 148.60, 145.05, 144.69, 144.46, 139.20, 136.30,
132.83, 131.66, 130.75, 130.33, 130.23, 129.90, 129.66, 129.44,
128.99, 128.68, 128.13, 126.95, 126.56, 125.57, 124.37, 124.26,
122.84, 122.77, 120.47, 113.01 ppm; ES-MS (CH3OH): m/z 916.30
[MÀClÀ]+.
N,N-Dimethyl-4-(2-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-
1-yl)benzenamine (dmpip): This compound was synthesized in
a manner identical to that described for ligand dpip but using
N1,N1-dimethylbenzene-1,4-diamine (0.341 g, 2.5 mmol) instead of
aniline. Yield: 0.582 g, 56.1%; elemental analysis calcd (%) for
C27H21N5: C 78.05, H 5.09, N 16.86; found: C 78.11, H 5.25, N 16.77;
1H NMR (300 MHz, CDCl3): d=9.20–9.07 (m, 2H), 9.03 (d, J=4.2 Hz,
1H), 7.73 (dd, J=8.3, 4.5 Hz, 1H), 7.64 (dd, J=16.5, 6.3 Hz, 3H),
7.31 (dd, J=10.2, 6.3 Hz, 6H), 6.82 (d, J=8.6 Hz, 2H), 3.11 ppm (s,
6H); ES-MS (CH2Cl2): m/z 416.1 [M+H]+, 853.3 [2M+Na]+.
[Ir(ppy2)dpip]Cl (IrL1): This complex was synthesized as described
below. A chloro-bridged dimer [Ir(ppy)2Cl]2 (0.088 g, 0.08 mmol)
and dpip (0.056 g, 0.015 mmol) were placed in a 100 mL round-
bottom flask with 40 mL of methanol and CH3Cl (1:1, v/v). The mix-
ture was heated at 658C for 12 h under argon. After the solution
cooled to room temperature, the solvent was removed under
vacuum to afford a bright yellow precipitate. The product was pu-
rified by column chromatography on alumina using acetonitrile/
ethanol (20:1, v/v) as the eluent. Yield: 0.0971 g, 71.3%; elemental
analysis calcd (%) for C47H32N6ClIr: C 62.14, H 3.55, N 9.25; found: C
62.27, H 3.75, N 9.17; 1H NMR (500 MHz, [D6]DMSO): d=9.31 (dd,
J=8.5, 1.4 Hz, 1H), 8.30–8.24 (m, 3H), 8.23 (d, J=3.5 Hz, 1H), 8.14
(dd, J=8.5, 5.0 Hz, 1H), 8.08 (d, J=4.5 Hz, 1H), 7.94 (dd, J=16.0,
8.0 Hz, 2H), 7.90–7.86 (m, 2H), 7.80–7.68 (m, 6H), 7.62 (d, J=
7.0 Hz, 2H), 7.53–7.38 (m, 7H), 7.09–6.99 (m, 4H), 6.97–6.91 (m,
2H), 6.28 ppm (dd, J=16.0, 7.5 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=171.90, 167.32, 154.30, 150.99, 150.59, 149.74,
149.56, 148.77, 145.11, 144.74, 144.45, 139.22, 137.06, 136.55,
132.93, 131.66, 131.59, 131.27, 130.75, 130.49, 130.12, 129.75,
129.55, 129.29, 129.00, 128.24, 127.01, 126.52, 125.58, 124.37,
124.29, 122.86, 122.53, 120.52 ppm; ES-MS (CH3OH): m/z 873.25
[MÀClÀ]+.
[Ir(ppy2)ptip]Cl (IrL2): This compound was synthesized in
a manner identical to that described for [Ir(ppy2)pi]Cl but using
ptip (0.058 g, 0.015 mmol) instead of dpip. Yield: 0.1036 g, 74.9%;
elemental analysis calcd (%) for C48H34N6ClIr: C 62.50, H 3.71, N
X-ray crystallography
X-ray diffraction measurements were performed using an Oxford-
1
9.11; found: C 62.63, H 3.92, N 9.05; H NMR (500 MHz, [D6]DMSO):
diffraction-Xcalibur-Nova diffractometer and
a Rigaku R-AXIS
d=9.30 (dd, J=8.5, 1.5 Hz, 1H), 8.29–8.21 (m, 3H), 8.14 (dd, J=8.5,
5.0 Hz, 1H), 8.08 (d, J=5.0 Hz, 1H), 7.94 (dd, J=15.0, 8.0 Hz, 2H),
7.88 (q, J=7.5 Hz, 2H), 7.78 (dd, J=8.5, 5.0 Hz, 1H), 7.64 (dd, J=
13.0, 5.5 Hz, 4H), 7.57–7.39 (m, 8H), 7.08–6.98 (m, 4H), 6.94 (dtd,
J=11.0, 7.5, 1.2 Hz, 2H), 6.27 (dd, J=13.0, 7.5 Hz, 2H), 2.48 ppm (s,
3H); 13C NMR (125 MHz, [D6]DMSO): d=167.32, 154.42, 151.02,
150.59, 149.70, 149.59, 148.73, 145.08, 144.72, 144.45, 141.28,
139.22, 136.51, 134.44, 132.91, 131.67, 130.75, 130.45, 130.14,
129.75, 129.64, 129.01, 128.95, 128.25, 127.01, 126.53, 125.58,
124.38, 124.30, 122.88, 122.59, 120.52, 21.47 ppm; ES-MS (CH3OH):
m/z 887.25 [MÀClÀ]+.
SPIDER image plate diffractometer with graphite-monochromated
CuKa (l=1.54178 Š) and MoKa radiation (l=0.71073 Š), respective-
ly. Absorption correction was applied using the SADABS pro-
gram.[42] The solution structures of IrL2 and IrL5 and full-matrix
least-squares refinement based on F2 were determined using the
SHELXL-2014 program incorporated in the OLEX2 program pack-
age.[43,44] All of the hydrogen atoms were included in the calculated
positions and refined with isotropic thermal parameters riding on
the positions of the parent atoms. The disordered chloroform and
toluene molecules in IrL5 were removed using the SQUEEZE rou-
tine in the PLATON software.[45] CCDC 1059823 (IrL2) and 1059824
(IrL5) contain the supplementary crystallographic data for this
paper. These data are provided free of charge by The Cambridge
Crystallographic Data Centre.
[Ir(ppy2)fpip]Cl (IrL3): This compound was synthesized in
a manner identical to that described for [Ir(ppy2)pi]Cl but using
fpip (0.059 g, 0.015 mmol) instead of dpip. Yield: 0.0895 g, 64.4%;
elemental analysis calcd (%) for C47H31N6FClIr: C 60.93, H 3.37, N
1
9.07; found: C 60.88, H 3.56, N 9.13; H NMR (500 MHz, [D6]DMSO):
Spectral analysis
d=9.31 (dd, J=8.5, 1.5 Hz, 1H), 8.29–8.22 (m, 3H), 8.14 (dd, J=8.5,
5.0 Hz, 1H), 8.10 (d, J=5.0 Hz, 1H), 7.98–7.85 (m, 6H), 7.81 (dd, J=
8.5, 5.0 Hz, 1H), 7.61 (d, J=7.5 Hz, 4H), 7.52–7.42 (m, 5H), 7.09–
6.92 (m, 6H), 6.28 ppm (dd, J=15.0, 7.5 Hz, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=167.34, 162.41, 154.53, 150.99, 150.59,
For physiological applications, the photophysical and sensing
properties of the IrL1–IrL5 complexes were investigated in DMSO/
PBS (10 mm, pH 7.4, v/v, 1:999) buffer solutions. Both the absorp-
tion and emission spectra were recorded in standard 3.0 cm quartz
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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cells. The mixture was equilibrated for 5 min prior to the measure-
ment. The excitation wavelength was 382 nm, and both slits were
set to 10 nm for the emission spectra.
coated transparent 96-well plates with 200 mL of DMEM containing
10% FBS (1% penicillin/streptomycin). The single cells generated
3D multicellular spheroids approximately 700 mm in diameter on
the fourth day at 378C in a 5% CO2 incubator with a humidified at-
mosphere. After formation, the spheroids were treated with
500 nm IrIII complexes for 0.5 h to ensure that the complexes pene-
trated throughout the spheroids. The spheroids were then imaged
using confocal laser scanning microscopy.
Determination of two-photon absorption cross-sections
The two-photon absorption spectra of probes determined over
a broad spectral region by the typical two-photon induced fluores-
cence (TPF) method relative to rhodamine B in methanol as stan-
dard. The two-photon fluorescence data were acquired using Opo-
letteTM 355 II (pulse width ꢀ100 fs, 80 MHz repetition rate, tuning
range 720–860 nm, Spectra Physics Inc., USA). Two-photon fluores-
cence measurements were performed in fluorometric quartz cuv-
ettes with IrL1–IrL5 at 5”10À4 m in methanol at 298 K. The experi-
mental fluorescence excitation and detection conditions were con-
ducted with negligible reabsorption processes, which can affect
TPA measurements. The quadratic dependence of two-photon in-
duced fluorescence intensity on the excitation power was verified
for an excitation wavelength of 750 nm. The two-photon absorp-
tion cross-section of the probes was calculated at each wavelength
according to Equation (1) in which I is the integrated fluorescence
intensity, A is the integrated absorbance intensity, and n is the re-
fractive index of solvent. The subscript r indicates the reference
sample, and s represents the test sample.
ICP-MS analysis
HeLa cells were plated at a density of approximately 3”105 cells
per mL in 25 cm2 culture plates (Corning) with 6 mL of DMEM with
10% FBS. IrL1–IrL5 (200 nm) was added to the culture medium
and incubated for 5 min or for a time within the range of 0–30 min
at 378C. After digestion with trypsin (Gibco), the HeLa cells were
counted and divided into two samples. The first sample was sub-
jected to nuclear isolation using a nucleus extraction kit. The
second sample was subjected to mitochondrial extraction using
a cytoplasm and mitochondria extraction kit (Shanghai Sangon Bio-
logical Engineering Technology & Services Co. Ltd.). The samples
were digested in 65% HNO3 at room temperature for at least one
day. Each sample was diluted with double distilled water to obtain
2% HNO3 sample solutions. The iridium concentration in the two
samples was determined by inductively coupled plasma mass spec-
trometry (ICP-MS Thermo Elemental Co., Ltd.).
IsArns
r IrAsnr
ð1Þ
fs ¼ f
Flow cytometry analysis
Cell viability assay
HeLa cells at a density of 1”105 cells per mL were cultured in 6-
well plates for 24 h in an incubator and then treated with IrL2
(200 nm) in DMEM supplemented with 10% FBS for 5 min at 378C.
Then, the cells were trypsinized and washed with PBS three times.
These cell uptake samples were analyzed with a FACSCanto II in-
strument (BD Biosciences).
HeLa cells were maintained as monolayer cultures in DMEM sup-
plemented with 10% FBS and 1% antibiotic solution at 378C in
a humidified atmosphere with 5% CO2. The cytotoxicity of the iri-
dium(III) complexes against HeLa cells was evaluated using the
MTT assay. The exponentially grown HeLa cells were seeded in trip-
licate into 96-well plates at 1”104 cells per well. After incubation
for 24 h, the cells were treated with IrL1–IrL5 at the concentration
used for staining (200 nm) for various durations. To stain the viable
cells, 12 mL of MTT (5 mgmLÀ1, Sigma) was added to each well.
Then, the cells were incubated for another 4 h at 378C. The media
was carefully removed to avoid disturbing the formazan crystals
that had formed, and then 150 mL of DMSO was added to dissolve
the formazan crystals. The absorbance of the samples was mea-
sured at 595 nm using an ELISA reader (BioTek Instruments, Wi-
nooski, VT).
Acknowledgements
This work was supported by the 973 program (Nos.
2015CB856301), the National Science Foundation of China
(Nos. 21172273, 21171177, and 21471164), and the Program for
Changjiang Scholars and Innovative Research Team in the Uni-
versity of China (No. IRT1298).
Confocal luminescence imaging
Keywords: cyclometalation
· imaging agents · iridium ·
mitochondrial tracking · two-photon excitation
HeLa cells were incubated at a density of approximately 1”104
cells per mL in DMEM supplemented with 10% FBS as well as 1%
penicillin and streptomycin (Gibco) at 378C in a humidified atmos-
phere with 5% CO2. After incubation for 1 day, the cells were treat-
ed with 200 nm IrIII complexes in DMEM for 15 min. After removing
the DMEM and washing with PBS buffer three times to remove the
remaining dye, the HeLa cells were further stained with MTG for
30 min. The cells were washed three times carefully with PBS
buffer prior to luminescence imaging measurements. The cell
images were captured with a monochromatic CoolSNAP FX camera
(Roper Scientific, USA) and analyzed using the AxioVision 4.2 soft-
ware (Carl Zeiss).
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Received: May 13, 2015
Published online on July 21, 2015
Chem. Eur. J. 2015, 21, 12000 – 12010
www.chemeurj.org
12010
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim