Super-Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore

Article abstract of DOI:10.1002/smll.201802166

Combining luminescent transition metal complex with super-resolution microscopy is an excellent strategy for the long-term visualization of the dynamics of subcellular structures in living cells. However, it remains unclear whether iridium(III) complexes are applicable for a particular type of super-resolution technique, structured illumination microscopy (SIM), to image subcellular structures. Herein, an iridium(III) dye, to track mitochondrial dynamics in living cells under SIM is described. The dye demonstrates excellent specificity and photostability and satisfactory cell permeability. While using SIM to image mitochondria, an ≈80 nm resolution is achieved that allows the clear observation of the structure of mitochondrial cristae. The dye is used to monitor and quantify mitochondrial dynamics relative to lysosomes, including fusion involved in mitophagy, and newly discovered mitochondria–lysosome contact (MLC) under different conditions. The MLC remains intact and fusion vanishes when five receptors, p62, NDP52, OPTN, NBR1, and TAX1BP1, are knocked out, suggesting that these two processes are independent.

Full text of DOI:10.1002/smll.201802166

FULL PAPER
Iridium(III) Luminophores
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Super-Resolution Tracking of Mitochondrial Dynamics with
An Iridium(III) Luminophore
Qixin Chen, Chengzhi Jin, Xintian Shao, Ruilin Guan, Zhiqi Tian, Chenran Wang, Fei Liu,
Peixue Ling, Jun-Lin Guan, Liangnian Ji, Fengshan Wang,* Hui Chao,* and Jiajie Diao*
tools for investigating the dynamics of
Combining luminescent transition metal complex with super-resolution
microscopy is an excellent strategy for the long-term visualization of the
dynamics of subcellular structures in living cells. However, it remains unclear
whether iridium(III) complexes are applicable for a particular type of super-
resolution technique, structured illumination microscopy (SIM), to image
subcellular structures. Herein, an iridium(III) dye, to track mitochondrial
dynamics in living cells under SIM is described. The dye demonstrates
excellent specificity and photostability and satisfactory cell permeability.
While using SIM to image mitochondria, an ≈80 nm resolution is achieved
that allows the clear observation of the structure of mitochondrial cristae.
The dye is used to monitor and quantify mitochondrial dynamics relative to
lysosomes, including fusion involved in mitophagy, and newly discovered
mitochondria–lysosome contact (MLC) under different conditions. The MLC
remains intact and fusion vanishes when five receptors, p62, NDP52, OPTN,
NBR1, and TAX1BP1, are knocked out, suggesting that these two processes
are independent.
subcellular structures, including mito-
chondria. At the same time, they have
also added to the requirements of fluores-
cent dyes, which need especially low cyto-
toxicity and high photostability to make
imaging living cells possible. Currently,
imaging subcellular structures relies upon
fluorescent proteins,[18,19] organic dyes,[20]
and quantum dots;[21] however, none of
those dyes are suitable for tracking the
dynamics of subcellular structures due to
their poor photostability and vulnerability
to photobleaching.[22] Studies have shown
that using luminescent transition metal
complexes (LTMCs),[23–26] including Ru,
Re, Pt, Au, and Zn, is an excellent alter-
native strategy that can overcome those
drawbacks. For example, to image mito-
chondria, Tang et al. reported a Zn(II)
complex dye whose fluorescence inten-
sity decayed to 10% after a short period
of continuous scan under STORM.[27] More recently, the lab of
Jim A. Thomas developed a Ru(II) complex dye with extreme
photostability, large Stokes shift and subcellular targeting to
image nuclear chromatin and mitochondria at a resolution of
less than 50 nm under STED.[28] However, it remains unclear
whether third-row LTMC dyes are applicable with SIM to image
the dynamics of subcellular structures.
Among LTMC dyes, iridium(III) complexes are the most
attractive candidate for bioimaging applications due to their
high phosphorescent quantum yield at room temperature, high
penetration and emission spectrum that can be extended to
1. Introduction
Highly mobile and dynamic in living cells, mitochondria are
the energy-generating organelles of cells,[1–3] the disorder
of which is associated with various diseases, including Alz-
heimer’s, Parkinson’s, and cancer.[4,5] Recently developed
super-resolution fluorescence microscopies such as stimu-
lated emission deletion (STED),[6–8] structured illumination
microscopy (SIM),[9–11] and stochastic optical reconstruction
microscopy (STORM),[12–14] as well as other single-molecule
super-resolution imaging techniques,[15–17] are enhanced new
Dr. Q. Chen, Dr. X. Shao, Z. Tian, Prof. C. Wang, Prof. J.-L. Guan,
Prof. J. Diao
Department of Cancer Biology
Dr. Q. Chen, Dr. X. Shao, Dr. F. Liu, Prof. P. Ling
Shandong Academy of Pharmaceutical Science
Key Laboratory of Biopharmaceuticals
University of Cincinnati College of Medicine
Cincinnati, OH 45267, USA
E-mail: jiajie.diao@uc.edu
Engineering Laboratory of Polysaccharide Drugs
National-Local Joint Engineering Laboratory of Polysaccharide Drugs
Jinan 250101, China
Dr. Q. Chen, Dr. X. Shao, Dr. F. Liu, Prof. P. Ling, Prof. F. Wang
School of Pharmaceutical Sciences
Shandong University
Dr. C. Jin, Dr. R. Guan, Prof. L. Ji, Prof. H. Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry
Jinan 250101, China
Sun Yat-sen University
E-mail: fswang@sdu.edu.cn
Guangzhou 510275, China
E-mail: ceschh@mail.sysu.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201802166.
DOI: 10.1002/smll.201802166
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near-infrared areas, excellent photochemical and physicochem-
ical stability that allows for long-term imaging, high biological
safety, and low cytotoxicity.[4,29] Those unique structures and
photophysical properties make it possible to develop innovative
bioimaging applications based on phosphorescence.[30,31]
As described herein, we developed a small molecule dye
based on iridium(III) complex for tracking mitochondrial
dynamics under an SIM. The dye not only has exceptional
cell permeability, light stability and mitochondrial specificity
but can also allow the observation of mitochondria at up to
80 nm resolution in living cells under SIM. Moreover, the dye
permits the clear observation of the structure of mitochon-
drial cristae, as well as the recording of dynamic approach
and separation processes of mitochondria. We applied the
dye to monitor fusion involved in mitophagy and mitochon-
dria–lysosome contact (MLC) in living cells and found that
five receptors, p62, NDP52, OPTN, NBR1, and TAX1BP1,
for mitophagy played no role in regulating MLC upon stim-
ulation. Our findings thus illustrate a novel perspective for
using LTMC dyes such as iridium(III) complexes to image
the dynamic processes of subcellular structures in living cells
under SIM.
2.2. Characterization of the Iridium(III) Complex Dye in Living Cells
To investigate the cell permeability of the dye in living cells, con-
centrations of dye ranging from 0.1 × 10−6 to 50 × 10−6 m were
incubated with HeLa cells for 30 min prior to observation under
a fluorescence microscope. We observed high phosphorescence
accumulation, and even nuclei were stained when cells were
exposed to dye at concentrations of (5–50) × 10−6 m, whereas
at concentrations of 0.1 × 10−6, 0.25 × 10−6, and 0.5 × 10−6 m,
phosphorescence intensity was too low to be visible (Figure S5,
Supporting Information). Taking both into consideration,
we determined that the dye at concentrations of 1 × 10−6 and
2.5 × 10−6 m could be used to image mitochondria well.
Next, we used flow cytometry to quantitatively analyze the
cellular uptake of the dye (Figure S6, Supporting Informa-
tion). Results indicated that when the cells were exposed to dye
at concentrations greater than 5.0 × 10−6 m, the cells took up
excessive dye, whereas at a concentration less than 2.5 × 10−6 m,
the cellular uptake of the dye gradually decreased in a dose-
dependent manner. Thereafter, we evaluated the cytotoxicity of
the dye at concentrations of (0.1–50) × 10−6 m during different
durations of incubation using a CCK-8 assay (Figure S7, Sup-
porting Information). Under continuous 12 h coculture, results
revealed no significant differences in cell viability between the
control group and the groups treated with the dye at concentra-
tions of less than 10 × 10−6 m.
2. Results and Discussion
2.1. Synthesis and Optical Characterization
Although it is feasible to observe and record dynamic pro-
gresses at the cellular level with confocal optical fluorescence
microscopy, it is difficult to distinguish subcellular structures
at resolutions less than 200 nm due to the Abbe diffraction
limit.[4,33,34] In response to that problem, after clarifying our
dye’s cell permeability, high specificity, and low cytotoxicity,
we investigated differences between the dye imaging of mito-
chondria under confocal microscopy and SIM (Figure S8 and
Movies S1 and S2, Supporting Information). To avoid the poten-
tial cytotoxicity of high-concentration dyes, we selected 0.5 ×
10−6, 1, 2.5× 10−6, and 5 × 10−6 m as experimental concentra-
tions. Results revealed that the dye had a low phosphorescence
background in confocal microscopy and SIM, as well as that the
resolution of the dye at a 1 × 10−6 m concentration was superior
to those at 0.5 × 10−6, 2.5 × 10−6, and 0.5 × 10−6 m concentrations
(Figure S8c,f,i,l, Supporting Information). When concentrations
exceeded 1 × 10−6 m, a large amount of the dye was taken up by
the cells, which caused strong phosphorescence (Figure S8a–f,
Supporting Information) that did not meet the requirement
for imaging mitochondria at the nanoscale level. By contrast,
when the concentration was less than 1 × 10−6 m, less dye was
taken up by the cells, which resulted in weak phosphorescence
due to which not even mitochondrial morphology was visible
(Figure S8i,j, Supporting Information). Therefore, we chose
1 × 10−6 m as an optimal concentration for further study.
First, we designed and synthesized an iridium(III) complex
dye with a molecular weight of 970 Da to specifically image
mitochondria in living cells under SIM (Figure 1a). The
main synthesis reaction route consisted of three major steps
(Figure 1b), and the characterizations with electrospray mass
spectrometry and nuclear magnetic resonance spectroscopy
appear in Figures S1–S3 in the Supporting Information. The
dye had higher phosphorescence at a wavelength of ≈700 nm
and different optical phosphorescence characteristics in phos-
phate-buffered saline at pH 4–10 (Figure 1c,d), which indicates
that it can be used in living cells in different pH environments.
The emission spectra show that the luminescent intensity in
pH 4–6 is relatively low compared to that in pH 6–10. This phe-
nomenon may contribute to the presence of pyrazine ring in
the auxiliary ligands of iridium(III) complexes. Since the phos-
phorescence intensity of this iridium(III) complex is relatively
stable in the pH range of 6.0–10.0, it is acceptable for mito-
chondrial imaging (physiological pH: 6.50 to 8.20).[32] To fur-
ther investigate the optical properties of the dye in other solvent
media, we performed fluorescence detection in 100% fetal calf
serum (FBS) and Dulbecco’s modified eagle medium (DMEM;
Figure S4a,b, Supporting Information). The dye exhibited high
fluorescence intensity in FBS and low fluorescence in DMEM,
which indicates that it can obtain a lower background in DMEM
under SIM. Upon testing the phosphorescence properties of
the dye at different temperatures, we found that it had the same
optical properties at all temperatures tested (Figure S4c,d, Sup-
porting Information), which suggests using the dye can afford
consistent data acquisition at physiological temperature (37 °C)
and ambient room temperature (25 °C) for SIM detection in
living cells.
To investigate the photostability and penetration depth of
the dye at 1 × 10−6 m for imaging mitochondria in living cells
under SIM, we recorded a single mitochondrion image at every
0.2 µm of depth (Figure 1e). Results indicated that the dye had
uniform tissue permeability at different depths in living cells.
The photobleaching properties of the dye directly affected the
monitoring of mitochondrial dynamic processes.[35,36] Next,
we performed a long-term continuous laser (165 s) to stimulate
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Figure 1. Synthesis and optical characterization of the iridium(III) complex dye. a) Schematic representation of the iridium(III) complex dye while
imaging mitochondria under SIM. b) Synthesis route of the iridium(III) complex dye. c) Fluorescence image of the iridium(III) complex dye in
phosphate-buffered saline at different pH levels. d) Photoluminescence mapping of the iridium(III) complex dye. e) Mitochondrial images at different
depths using the iridium(III) complex dye. White dotted lines show fluorescence intensity of a single mitochondrion image at every depth of 0.2 µm.
f) Photobleaching properties of the iridium(III) complex dye under laser stimulation for 165 s continuously recorded in living cells. SIM frames were
deliberately spaced at 15 s intervals. Scale bars: (e) 10 µm, (f) 10 µm.
cells in order to monitor the photostability of the dye in the
same section of the cell (Figure 1f). We found that the dye had
high phosphorescence within 60 s of continuous laser stimu-
lation and could image mitochondria within 135 s without
photobleaching. Those properties allowed us to record more
dynamic information while monitoring mitochondria in living
cells under SIM with the dye.
2.3. Whole-Cell 3D SIM Images of Mitochondrial Ultrastructures
Using the Iridium(III) Complex Dye
Visualizing mitochondrial ultrastructures affords new under-
standings of the pathology and diagnosis of mitochondria-
related diseases.[37,38] To investigate the capability of obtaining
more information about mitochondrial ultrastructures, we used
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Figure 2. Iridium(III) complex dye images of mitochondrial ultrastructure. a) Iridium(III) complex dye images of mitochondria in 1 × 10−6 m concen-
tration under 3D SIM. White solid line 1 shows fluorescence intensity, and red rectangle shows partial amplification. b) Single mitochondrial local
enlargement of (a). Red solid line 2 shows the length of a single mitochondrion, and blue solid line 3 shows its width. c) Individual mitochondria
fluorescence intensity with length as red solid line 2 and width as blue solid line 3. d) iridium(III) complex dye 3D map distributed in mitochondria
under SIM. e) Fluorescent intensity distribution of white solid line 1. f) Local magnification of (e), with resolution of iridium(III) complex dye image
of mitochondria up to 80 nm. Scale bars: (a) 5 µm, (b) 1 µm.
the dye to image mitochondrial ultrastructures in living cells
under 3D SIM. Shown in Figure 2a, results revealed that after
cell incubation with the dye for 30 min, intracellular mitochon-
dria showed spherical, rod-shaped, or filamentous particles
≈2.0 µm long (Figure 2c-2) and ≈0.7 µm wide (Figure 2c-3),
which is consistent with normal mitochondrial volume in HeLa
cells (width: 0.5–1.0 µm, length: 1.5–3.0 µm). The lamellar
cristae in mitochondrial were also visible using the dye-cristae
thickness was ≈105 nm (Figure 2b; Figure S9, Supporting
Information) and the dye could be evenly distributed on mito-
chondria (Figure 2d; Movie S3, Supporting Information), which
suggests that it can be located on the mitochondrial membrane
and have high specificity. To further clarify the resolution of the
dye for imaging mitochondria, we obtained a full-width at the
half-maximum (FWHM) up to 80 nm under SIM (Figure 2e,f),
which is similar to the Atto 647N mitochondrial dye reported
by Han et al. (FWHM: 91 nm).[36] Such results suggest that our
dye offers higher resolution and precision for tracking mito-
chondria under SIM.
2.4. Iridium(III) Complex Dye for Tracking Mitochondrial Dynamics
Mitochondria rank among the most dynamic organelles
in cells,[36] and understanding their dynamic processes is
important for analyzing the causes of many diseases.[39]
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Figure 3. Iridium(III) complex dye tracking of mitochondrial dynamics under SIM. a) Iridium(III) complex dye images of mitochondria under SIM. Red
rectangles indicate dynamic processes of approach, and blue rectangles indicate dynamic processes of separation. b) Trend of fluorescence intensity in
the dynamic process of approach. c) Trend of fluorescence intensity in the dynamic process of separation. d–f) Frames 1–3 of the dynamic approach
process in the red rectangle of (a). g–i) Frames 4–6 of the dynamic separation process in the blue rectangle of (a). The time interval between each
frame is ≈2 s. Both red and blue solid lines show fluorescence intensity. Scale bars: (a) 5 µm, (d) 1 µm.
Therefore, a strategy for visualizing the dynamics of mito-
chondria at nanoscale has important implications for under-
standing mitochondria-related diseases. With that goal in
mind, we recorded the process of mitochondrial dynamics
using our dye after incubating it in live cells for 30 min
(Figure 3; Movie S4, Supporting Information). We observed
that two mitochondria (Figure 3d–f, red arrow) maintained a
distance of ≈0.6 µm in Frame 1 (Figure 3b-1) and gradually
approached each other in Frames 1 and 2 (Figure 3b,e,f). We
also observed the separation process of mitochondria, particu-
larly the gradual disintegration from an intact mitochondria
(Figure 3c,g–i, blue arrows), which suggests that our dye can
be used to study the dynamic changes such as fusion and fis-
sion of mitochondria.
form MLC, similar to what has been previously reported. We
observed that MLC events were normal in wild-type (WT) cells
(Figure 4a–f; Movies S5–S7, Supporting Information), but did
not observe fusion. Moreover, we found two types of MLCs
(Figure 4c), point contact with limited overlap (Figure 4d)
and extended contact showing an elongated contact surface
(Figure 4e).
For the application of our dye to image damaged mitochon-
dria related to pathological conditions, we performed another
experiment with inducer treatment. We used 10.0 × 10−6
m
carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a common
mitochondria damage inducer, to treat cells 12 h prior to
staining with our dye and LysoTracker Green. Compared to
untreated cells (Figure 4g–j; Movie S8, Supporting Informa-
tion), we observed a significant increase of large overlaps
(yellow spots) of mitochondria and lysosomes corresponding to
the fusion after CCCP treatment.
2.5. Application of the Iridium(III) Complex Dye to Track MLC
and Fusion
The interactions of mitochondria and lysosomes, including
their fusion involved in mitophagy, are essential for repairing
damaged mitochondria. Recently, a direct contact between
mitochondria and lysosome was demonstrated in normal living
cells.[40] To investigate the crosstalk of mitochondria and lys-
osomes, we used our dye together with commercial LysoTracker
Green to label lysosomes in mammalian cells. We confirmed
that mitochondria and lysosomes were close to each other to
2.6. Iridium(III) Complex Dye to Track MLC in Penta Knockout
HeLa Cells
MLCs were observed in both normal and stimulated condi-
tions, while fusion involved in mitophagy was only found after
CCCP treatment. Pathologically, PINK1 is recruited to the mito-
chondrial membrane to phosphorylate Ser65 of ubiquitin ligase
and trigger mitophagy.[41] During that process, several receptors
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Figure 4. Iridium(III) complex dye tracking of mitochondria–lysosome contact (MLC) and fusion events in living cells. a) MLC events in WT cells. White
rectangles represent two types of MLCs, point (1–4) and extended (5–8) contacts. b) A representative MLC event. White rectangle represents the amplifica-
tion. c) Point and extended MLCs in (a). d,e) White solid lines indicate the fluorescence intensity. f) Partial enlargement of (d)-3. g) Fusion events in WT
cells after treatment CCCP. White rectangle shows the representative fusion event, and the white solid line shows fluorescence intensity. h) A representative
fusion event detected by using the iridium(III) complex dye (red, mitochondria) and LysoTracker Green (green, lysosome). i) Fluorescence intensity of the
solid line of (g)-9 inset. j) 3D SIM surface plot of merged image in (h). Scale bars: (a) 10.0 µm, (b) 0.5 µm, (e) 0.2 µm, (g) 5.0 µm, (g) 9 0.5 µm, (j) 0.5 µm.
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(i.e., p62, NDP52, OPTN, NBR1, and TAX1BP1) play an impor-
tant role.[41] Therefore, it is reasonable to check whether these
mitophagy proteins are involved in MLC events other than the
recently reported RAB7GTP hydrolysis.[40] To investigate the
correlation between MLC and fusion, we performed the SIM
experiment in Penta knockout (KO) HeLa cells which are defi-
cient of mitophagy. After using 10 × 10−6 m CCCP to treat cells
and staining with our dye and LysoTracker Green, we found
MLC events including 2–3 faint yellow spots for point MLC
throughout the cells (Figure 5a–c; Movie S9, Supporting Infor-
mation). In addition, we recorded the evolution of an MLC event
(Figure 5d–e; Movie S10, Supporting Information), in which a
mitochondrion and a lysosome underwent approach, contact,
and separation. Meanwhile, fusion event involved in mitophagy
Figure 5. Iridium(III) complex dye for tracking MLC in Penta knockout HeLa cells. a) MLC events in Penta KO cells treated with 10 × 10−6 m CCCP for
12 h. White rectangle represents the MLC shown in (d). b) 3D SIM surface plot of (a) after 30° rotation. White arrows indicate the MLC events. c) A repre-
sentative MLC event. d) Time evolution of one MLC in living cell. e) White solid lines indicate fluorescence intensity. Scale bars: (a) 10.0 µm, (b) 1.0 µm.
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in Penta KO HeLa cells (Figure 5a) was dis-
appeared compared to what was found in the
WT HeLa cells (Figure 4g). To that end, we
performed a controlled experiment in which
Penta KO HeLa cells did not receive CCCP
treatment. We detected no significant change
of MLC events in Penta KO cells (Figure 5g;
Movie S11, Supporting Information), which
indicates that fusion between mitochondria
and lysosomes are independent to MLC.
Again, our dye can be used to monitor MLC
events in living cells.
2.7. Quantitative Analysis of the Interaction
between Mitochondria and Lysosomes
To quantitatively assess the distance
changes between mitochondria and lyso-
some under different conditions, we propose
a Di-value driven from FWHM by the calcu-
lation formula shown in Figure 6a. FWHM
refers to the full width of the image at half-
maximum value and is a direct indicator of
the resolution. We then apply the Di-value to
quantify the distance change between mito-
chondria and lysosomes under different
conditions. We first calculated the Di-value
Figure 6. Quantitative analysis of the interaction between mitochondria and lysosomes. a) The
schematic illustration of a Di-value for quantitative analysis of the distance between mitochon-
dria and lysosomes. RFWHM indicates the FWHM of red color (mitochondria); GFWHM indicates
the FWHM of green color (lysosomes); XR indicates the X-axis value of the RFWHM center; and
(0.884
0.116, n = 10) in untreated WT
cells for MLC events. We then analyzed the
Di-value of WT cells treated with CCCP. We
found a much lower Di-value (0.146 0.118,
n = 10) for fusion events, indicating a signifi-
cant difference upon stimulation (Figure 6b).
For Penta KO cells with or without CCCP, no
XG indicates the X-axis value of the GFWHM center. b) Di-values of mitochondria and lysosome in
WT cells with or without CCCP treatment (n = 10). c) Di-values of mitochondria and lysosome
in Penta KO cells with or without CCCP treatment (n = 10).
significant difference in Di values was observed (1.123 0.176
vs 1.128 0.140, n = 10) (Figure 6c).
iridium(III) complex dye, we have demonstrated the capability
of imaging these dynamics with a high special resolution.
Moreover, this new iridium(III) complex dye can also be used
to quantitatively study the functional crosstalk between mito-
chondria and lysosomes, such as the fusion and MLC. The
fusion between mitochondria and lysosomes is an important
step of mitophagy for recycling damaged mitochondria.[43] We
observed that the fusion of mitochondria and lysosomes can
be significantly enhanced upon mitochondrial damage. How-
ever, the recently reported formation of MLCs differed from
these fusion events of mitophagy in which mitochondria tar-
geted to lysosomes for destruction.[40] Five receptors (i.e., p62,
NDP52, OPTN, NBR1, and TAX1BP1) that play essential roles
in mitophagy were not involved in the formation of MLCs,[40]
which suggests MLCs and mitophagy are independent
processes.
3. Conclusion
We discovered that a third-row transition metal complex fluo-
rescent dye based on iridium(III) can be used to track mito-
chondrial dynamics under SIM. Using our dye, we obtained
a mitochondrial image with ≈80 nm resolution. We observed
mitochondrial cristae and recorded the dynamic approach and
separation of mitochondria. Our results provide a novel perspec-
tive on using LTMC dyes such as an iridium(III) complex to
image and quantify the dynamic process of subcellular structures
in living cells under SIM. Given the synthesis of the iridium(III)
complex, future work could focus on developing a variety of
novel iridium(III) dyes for the specific imaging of lysosomes,
mitochondria, cell membranes, nuclei, and other organelles
under SIM toward the eventual goal of mapping the organelle
interaction network and thereby clarifying the network’s estab-
lishment, maintenance, dynamic changes, and regulatory mech-
anisms and to reveal its physiological and pathological functions.
Mitochondrial dynamic processes including fusion and
fission are closely related to many diseases.[42] With our
4. Experimental Section
Materials: IrCl3·xH2O were purchased from Alfa Aesar, 4,4′-dimethyl-
2,2′-bipyridine, diatomite, selenium dioxide 1-phenyl-1,2-propane-dione,
2-aminobenzenethiol, and benzene-1,2-diamine were purchased from
J&K Scientific. LysoTracker Green were purchased from Invitrogen
(Invitrogen, Eugene, OR, USA); cell counting kit-8 (CCK-8) was obtained
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Dojindo Laboratories (Dojindo Laboratories, Kumamoto, Japan); FBS,
DMEM, and other cell culture reagents were obtained from Gibco BRL
(Grand Island, NY, USA).
confocal laser scanning microscopy or OMX 3D-SIM super-resolution
microscope.
Confocal Laser Scanning Microscopy: The images were obtained using
an LSM-710 confocal laser scanning microscope (Carl Zeiss, Inc.)
equipped with a 63×/1.49 numerical aperture oil-immersion objective
lens and were analyzed with ZEN 2012 (Carl Zeiss, Inc.) and ImageJ
software (National Institutes of Health). All fluorescence images
were analyzed and the background subtracted with ImageJ software.
Pearson’s coefficient was quantified using the Colocalization Analysis
plugin for ImageJ.
OMX 3D-SIM Super-Resolution Microscope Imaging: Super-resolution
images were acquired on OMX 3D-SIM Microscope (Bioptechs, Inc)
equipped with a Olympus 100×/1.49 numerical aperture oil-immersion
objective lens and solid-state lasers. Images were captured with an electron-
multiplying charge coupled device camera (Photometrics Cascade II) with
a gain value of 3000 at 10 MHz. The exposure time was set to 50 ms for
each raw data capture. Picture was obtained at 512 × 512 using Z-stacks
with step size of 0.2 µm. SIM frames were deliberately spaced at 1, 2, 8 or
15 s intervals according to the purpose of each experiment. SIM images
were analyzed with Nikon Elements and ImageJ software.
Synthesis and Characterization of Iridium(III) Complex Dye: The dye
was synthesized by using a previously reported protocol. 4′-methyl-
[2,2′-bipyridine]-4-carbaldehyde[44] and the auxiliary ligand 2-methyl-3-
phenylquinoxaline (mpq)[45] were synthesized according to literature
[46]
methods. The iridium(III) dimmer [Ir(mpq)2Cl]2 was synthesized by
using the similar method of [Ir(ppy)2Cl]2. The synthetic process of the
main ligand and the iridium(III) complex were according to the previous
work. The main ligand (2-(4′-methyl-[2,2′-bipyridin]-4-yl)benzo[d]thiazole
(mbbt)) was synthesized by slowly dropping 2-aminobenzenethiol (196
mg, 1.55 mmol) into the EtOH solution of 4′-methyl-[2,2′-bipyridine]-
4-carbaldehyde (297 mg, 1.5 mmol). After stirring overnight in RT,
the product was condensed and then recrystallized by using CH2Cl2/
ethanol to get yellow flaky solid Yield, 80.5%, 367 mg. Anal. Calcd.
for C18H13N3S (%): C, 71.26; H, 4.32; N, 13.85. Found (%): C, 71.03;
H, 4.63; N, 13.61.1H NMR (500 MHz, CDCl3) δ 9.27 (s, 1H), 8.60
(d, J = 1.0 Hz, 2H), 8.42 (s, 1H), 8.11 (s, 1H), 8.00 (d, J = 4.5 Hz, 2H),
7.49 – 7.45 (m, 2H), 7.20 (s, 1H), 2.54 (s, 3H). ES-MS, (CH3OH): m/z =
304.15 [M+H]+). The goal iridium complex were synthesized by mixing
mbbt (30.4 mg, 0.1 mmol) and [Ir(mpq)2Cl]2 (66.3 mg, 0.0525 mmol)
in a degassing mixture of chloroform and MeOH (1:1, 40 mL). Then the
solution was refluxed overnight in argon atmosphere. After the reaction
was stopped, the solvent was removed and further purification was
conducted by using alumina column chromatography to get crimson
microcrystal. Yield, 51.3%, 49.7 mg. Anal. Calcd. for C48H35N7SClIr (%):
C, 59.46; H, 3.64; N, 10.11. Found (%): C, 59.19; H, 3.91; N, 10.01.
1H NMR (500 MHz, CD3OD): δ 8.78 (d, J = 1.5 Hz, 1H), 8.57 – 8.48
(m, 3H), 8.30 (s, 3H), 8.27 (dd, J = 6.0, 1.5 Hz, 1H), 8.23 (d, J = 6.0
Hz, 1H), 8.08 (t, J = 8.0 Hz, 2H), 7.87 (t, J = 8.0 Hz, 2H), 7.59 (s, 1H),
7.57 – 7.51 (m, 4H), 7.42 (t, J = 10.0 Hz, 2H), 7.32 – 7.26 (m, 2H),
7.11 (d, J = 12.0 Hz, 2H), 6.89 (d, J = 8.0 Hz, 2H), 6.72 (dd, J = 18.0,
7.5 Hz, 2H), 3.40 (d, J = 15.1 Hz, 6H), 2.49 (s, 3H). 13C NMR (125 MHz,
CD3OD) δ164.62, 164.55, 162.30, 156.57, 154.36, 153.18, 152.99, 152.87,
152.81, 152.68, 148.37, 146.82, 144.41, 143.47, 140.06, 139.90, 139.85,
130.62, 130.59, 129.97, 129.88, 129.80, 129.17, 128.66, 127.15, 127.01,
125.14, 125.01, 123.74, 122.98, 122.02, 120.63, 26.14, 26.09, 19.79.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
Q.C., C.J., X.S., and R.G. contributed equally to this work. This
research was supported by 973 Program (Nos. 2015CB856301 and
2015CB856304) from Ministry of Science and Technology of China,
the National Science Foundation of China (Nos. 21525105, 21471164,
21778079, and 21701196), the Fundamental Research Funds for the
Central Universities (7lgjc11), China Postdoctoral Science Foundation
(20173100041090767), Natural Science Foundation of Shandong
Province (ZR2017PH072), Key Research and Development Plan of
Shandong Province (2018GSF121033), and National Institutes of
Health (NIH, R35GM128837 to J.D., R01NS094144 and R01CA211066
to J.G.). The Light Microscopy Imaging Center (LMIC) is supported in
part with funds from Indiana University Office of the Vice Provost for
Research. The OMX 3D-SIM microscope was provided by NIH grant
S10 RR028697. The authors also thank Dr. Ehmer Birgit at University of
Cincinnati for assistance with laser scanning confocal microscope and
flow cytometry.
1
ES-MS (CH3OH): m/z = 933.95 [M-Cl−]+. H and 13C NMR spectra were
recorded using a Bruker 500 nuclear magnetic resonance spectrometer
using CDCl3 or CD3OD as the deuterated solvent. The electronic
absorption 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 and FLS 980 luminescence
spectrometer. Microanalysis (C, H, and N) was carried out using an
Elemental Vario EL elemental analyzer. Electro spray mass spectra were
recorded using an LCQ system (Finnigan MAT, USA).
Cell Culture: HeLa cells were gifted from Dr. Carolyn M. Price lab
(University of Cincinnati). Penta knockout HeLa cells were gifted from
Dr. Richard J. Youle lab (National Institutes of Health). Cells were
cultured in Dulbecco’s modified Eagle medium supplemented with 10%
FBS, penicillin (100 units mL−1), and streptomycin (100 µg mL−1) in a
5% CO2 humidified incubator at 37 °C.
Conflict of Interest
The authors declare no conflict of interest.
Cell Viability and Cytotoxicity Assay: Cells were treated in a 96-well
plate at density of 5 × 105 cell mL−1. The viability was determined by
using a cell counting kit-8 (CCK-8). 10 µL CCK-8 solution was added to
each well and the OD value for each well was read at wavelength 450 nm
on a microplate reader (Multiskan, Thermo, USA).
Keywords
iridium(III)
illumination microscopy
luminophore,
mitochondrial
dynamic,
structured
Flow Cytometry Analysis: Cells were seeded on 6-well plate at density of
Received: June 6, 2018
Revised: August 9, 2018
Published online:
1 × 105 cell mL−1 in 1 mL of complete medium for 24 h. After treatment
with iridium(III) complex dye (0.1 × 10−6, 0.25 × 10−6, 0.5 × 10−6
,
1 × 10−6, 2.5 × 10−6, 5 × 10−6, 10 × 10−6, 25 × 10−6, and 50 × 10−6 m)
for 30 min, cells were collected by trypsinization and washed two times
with cool PBS. Cells were resuspended by 500 µL binding buffer while
avoiding light prior to detection by flow cytometry.
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