Phospholipid-Biomimetic Fluorescent Mitochondrial Probe with Ultrahigh Selectivity Enables in Situ and High-Fidelity Tissue Imaging

Article abstract of DOI:10.1021/acs.analchem.7b00710

In situ and directly imaging mitochondria in tissues instead of isolated cells can offer more native and accurate information. Particularly, in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy, it is a routine examination item to directly observe mitochondrial morphology and number in muscle tissues from patients. However, it is still a challenging task because the selectivity of available probes is inadequate for exclusively tissue imaging. Inspired by the chemical structure of amphiphilic phospholipids in mitochondrial inner membrane, we synthesized a phospholipid-biomimetic amphiphilic fluorescent probe (Mito-MOI) by modifying a C₁₈-alkyl chain to the lipophilic side of carbazole-indolenine cation. Thus, the phospholipid-like Mito-MOI locates at mitochondrial inner membrane through electrostatic interaction between its cation and inner membrane negative charge. Simultaneously, the C₁₈-alkyl chain, as the second targeting group, is deeply embedded into the hydrophobic region of inner membrane through hydrophobic interaction. Therefore, the dual targeting groups (cation and C₁₈-alkyl chain) actually endow Mito-MOI with ultrahigh selectivity. As expected, high-resolution microscopic photos showed that Mito-MOI indeed stained mitochondrial inner membrane. Moreover, in situ and high-fidelity tissue imaging has been achieved, and particularly, four kinds of mitochondria and their crystal-like structure in muscle tissues were visualized clearly. Finally, the dynamic process of mitochondrial fission in living cells has been shown. The strategy employing dual targeting groups should have reference value for designing fluorescent probes with ultrahigh selectivity to various intracellular membranous components.

Full text of DOI:10.1021/acs.analchem.7b00710

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Article
A phospholipid-biomimetic fluorescent mitochondrial probe with
ultrahigh selectivity enables in-situ and high-fidelity tissue imaging
Ruoyao Zhang, Yuming Sun, Minggang Tian, Ge Zhang, Ruiqing Feng,
Xuechen Li, Lifang Guo, Xiaoqiang Yu, Jing Zhi Sun, and Xiuquan He
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017
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Analytical Chemistry
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A phospholipid-biomimetic fluorescent mitochondrial probe with ul-
trahigh selectivity enables in-situ and high-fidelity tissue imaging
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Ruoyao Zhang,† Yuming Sun,‡ Minggang Tian,† Ge Zhang,† Ruiqing Feng,† Xuechen Li,† Lifang Guo,†
Xiaoqiang Yu,*,† Jing Zhi Sun*,§ and Xiuquan He*,ǁ
†Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan
250100, P. R. China. Fax: +86 0531 88364263; Eꢀmail address: yuxq@sdu.edu.cn
‡School of Information Science and Engineering, Shandong University, Jinan 250100, P. R. China
§MoE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering,
Zhejiang University, Hangzhou 310027, P. R. China. Fax: +86 0571 87953734; Eꢀmail address: sunjz@zju.edu.cn
ǁDepartment of Anatomy, Shandong University School of Medicine, Jinan 250012, P. R. China. Fax: +86 0531 88364263; Eꢀ
mail address: hxq3000@sdu.edu.cn
ABSTRACT: Inꢀsitu and directly imaging mitochondria in tissues instead of isolated cells can offer more native and accurate inꢀ
formation. Particularly, in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy, it is a routine examinaꢀ
tion item to directly observe mitochondrial morphology and number in muscle tissues from patients. However, it’s still a challengꢀ
ing task because the selectivity of available probes is inadequate for exclusively tissue imaging. Inspired by the chemical structure
of amphiphilic phospholipids in mitochondrial inner membrane, we synthesized a phospholipidꢀbiomimetic amphiphilic fluorescent
probe (Mito-MOI) by modifying a C18ꢀalkyl chain to the lipophilic side of carbazoleꢀindolenine cation. Thus the phospholipidꢀlike
Mito-MOI locates at mitochondrial inner membrane through electrostatic interaction between its cation and inner membrane negaꢀ
tive charge. Simultaneously, the C18ꢀalkyl chain, as the second targeting group, is deeply embedded into the hydrophobic region of
inner membrane through hydrophobic interaction. Therefore, the dual targeting groups (cation and C18ꢀ alkyl chain) actually endow
Mito-MOI with ultrahigh selectivity. As expected, highꢀresolution microscopic photos showed that Mito-MOI indeed stained miꢀ
tochondrial inner membrane. Moreover, inꢀsitu and highꢀfidelity tissue imaging has been achieved, and particularly, four kinds of
mitochondria and their crystalꢀlike structure in muscle tissues were visualized clearly. Finally, the dynamic process of mitochondriꢀ
al fission in living cells has been shown. The strategy employing dual targeting groups should have reference value for designing
fluorescent probes with ultrahigh selectivity to various intracellular membranous components.
Different from the microscopic images obtained in cultured
cells, inꢀsitu and directly imaging mitochondria in intact tisꢀ
sues can offer more native and accurate information.1 On one
hand, cells in living tissues are surrounded by native extracelꢀ
lular matrix (ECM) with indispensable functions compared to
culture medium,2,3 such as mediating tissue morphogenesis
and providing clews for cell proliferation and differentiation.4,5
On the contrary, cells cultured are often immortalized for infiꢀ
nite proliferation, which will damage their original differentiꢀ
ated functions.6 More importantly, the change of mitochondriꢀ
al morphology and number in muscle tissues have clinical
diagnostic value in mitochondrial diseases, such as mitochonꢀ
drial myopathy, encephalopathy, lactic acidosis, and stroke‑
like episodes (MELAS).7ꢀ10 For example, in muscular tissues
with MELAS, the smooth muscle cells of the succinateꢀ
dehydrogenaseꢀreactive blood vessels show marked proliferaꢀ
tion of mitochondria.10 Thus it is a routine examination item to
directly observe mitochondria in muscle tissues from patients
and available observing method by scanning electron microꢀ
scope (SEM) is complex.7,9,10 However, it is still a challenging
task to inꢀsitu and directly image mitochondria in tissues by
fluorescent microscopy due to the low selectivity of fluoresꢀ
cent probes available.
So far, available mitochondrial probes generally bear an orꢀ
ganic cationic unit,11ꢀ18 which enables them to target mitoꢀ
chondria with a negative membrane potential of ꢀ180 mV.19ꢀ21
Generally, their selectivity to mitochondria is enough to image
cells, but inadequate for tissue imaging. In tissues, the subꢀ
stances are far more complex than that in cells, such as various
amounts of collagens, adhesion molecules, proteoglycans,
growth factors and cytokines in ECM.4,5 As a result, some
probes are nonspecifically bound to other components and
thus produce high background noise. When staining cells, the
background fluorescence can be eliminated by washing out
unbound probes,22ꢀ24 while washing process cannot be adopted
when staining living tissues. On the other hand, due to maꢀ
tured cell culture technology, the incubation time for staining
cells is allowed for 0.5 hours or longer.13, 25ꢀ27 However, the
viability of isolated living tissues will rapidly decay and the
mitochondrial membrane potential may also rapidly decrease.
Hence, the staining time must be as short as possible. Thus, to
image tissues, a probe with ultrahigh selectivity is urgent.
One possible and rational way is to design a probe with dual
targeting groups. A key fact is that the tight bilayer of mitoꢀ
chondrial inner membrane mainly composes of amphiphilic
phospholipids,28,29 such as phosphatidylcholine and phosphaꢀ
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tidyl ethanolamine, which contain a hydrophilic cation and Herein, a probe Mito-MOI was synthesized by carefully
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hydrophobic overlong alkyl chains (Scheme 1a). Inspired by
the chemical structure of phospholipids, we believe that the
ultrahigh selectivity can be realized by a phospholipidꢀ
biomimetic amphiphilic mitochondrial probe prepared by
modifying an overlong alkyl chain to the lipophilic side of a
common cationic mitochondrial probe (Scheme 1a). Accordꢀ
ing to Scheme 1b, the probes should be able to naturally and
stably arrange in inner leaflet of mitochondrial inner memꢀ
brane, like some phospholipids. In accordance with the orienꢀ
tation of amphiphilic phospholipids, the cations are located at
the hydrophilic side of inner leaflet due to the electrostatic
interaction with inner membrane negative charge. Simultaneꢀ
ously, the overlong alkyl chain, as the second targeting group,
is embedded into the hydrophobic region of the inner leaflet
through hydrophobic interaction. Therefore, compared with
common cationic mitochondrial probes, this phospholipidꢀ
biomimetic probe has an additional hydrophobic interaction
with mitochondria (Scheme 1b). As a result, the binding afꢀ
finity can be obviously enhanced, which offers ultrahigh selecꢀ
tivity required by tissue imaging. However, a serious concern
is that dyes with overlong alkyl chain most probably stay in
plasma membrane. Interestingly, not all probes with overlong
alkyl chains are hindered by plasma membrane. Another key
fact is that plasma membrane probes bearing long chains
sometimes show rapid internalization, such as Laurdan with a
C12ꢀchain.30 The two key facts tell us a crucial possibility that
a mitochondrial probe simultaneously possessing ultrahigh
selectivity and fast stainability can be synthesized by exquisꢀ
itely introducing an overlong alkyl chain to common cationic
mitochondrial probe.
modifying a C18ꢀalkyl chain to the lipophilic side of carbazoleꢀ
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indolenine cation (Scheme 2). The structure of Mito-MOI was
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characterized by H NMR, 13C NMR, IR and HRMS spectra,
as showed in Supporting Information. As expected, Mito-
MOI was able to light up mitochondria in living cells, and
high resolution microscopic photos have shown that Mito-
MOI exclusively stains mitochondrial inner membrane.
Moreover, mitochondria in living tissues have been inꢀsitu and
highꢀfidelity imaged. Particularly, four kinds of mitochondria
and their crystalꢀlike structure in muscle tissues were also
visualized clearly. Therefore, modifying a long alkyl chain to
the lipophilic side of a cation was a reasonable choice to create
a mitochondrial probe with ultrahigh selectivity.
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Scheme 2. The synthetic routines of Mito-MOI.
EXPERIMENTAL SECTION
Apparatus and general methods. The UVꢀvisibleꢀnearꢀIR
absorption spectra of dilute solutions were recorded on a
HITACH Uꢀ2910 spectrophotometer using a quartz cuvette
having 1 cm path length. Oneꢀphoton spectra were obtained on
a HITACH Fꢀ2700 spectrofluorimeter equipped with a 450ꢀW
Xe lamp. The mitochondrial probes MitoTracker Green (MTG)
and MitoTracker Red (MTR), and doubleꢀstranded DNAꢀ
specific dye Hoechst 33342 were purchased from Molecular
Probes. PBS buffer solution: 10 mM NaCl, Na2HPO4•12H2O,
NaH2PO4•2H2O, pH = 7.40.
Measurement of Φ. Quantum yield (Φ) can be calculated
by means of Eq. (1):31
ꢉ
ꢄ ꢆ ꢇ ꢊ
ꢅ
ꢅ
ꢈ
ꢈ
ꢀ ꢂ ꢀꢃ
ꢁ
(1)
ꢉ
ꢄ ꢆ ꢇ ꢊ
ꢈ
ꢈ
ꢅ
ꢅ
s and r refer to the sample and the reference materials, reꢀ
spectively. Φ is the quantum yield, F is the integrated emission
intensity, A stands for the absorbance, and n is the refractive
index. In this work, the quantum yields were calculated by
using fluorescein (Φ = 0.95, pH = 13) as the standard.
Cell culture and staining. All cells were grown in a 5%
o
CO2 incubator at 37 C. Human cervical cancer cell lines
(SiHa and HeLa) were grown in Dulbecco’s Modified Eagle
Medium supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin and streptomycin. Rat astrocytes and neuꢀ
rons were cultured in Dulbecco's Modified Eagle Media: Nuꢀ
trient Mixture Fꢀ12 supplemented with 10% bovine calf serum
in a 5% CO2 incubator at 37 °C. Mito-MOI was dissolved in
DMSO at a concentration of 5 mM. Hoechst 33342 was preꢀ
pared as 1 mM aqueous solution. MTG and MTR were disꢀ
solved in DMSO at a concentration of 0.1 mM. HeLa and
SiHa cells were placed on glass coverslips and allowed to adꢀ
here for 24 h. Rat astrocytes were placed on glass coverslips
and allowed to adhere for 6 days. For living cells imaging
experiment of probes (Mito-MOI, MTG and MTR), cells
were incubated with probes (Mito-MOI: 5 µM; MTG: 0.2 µM;
MTR: 0.2 µM) in DMEM for 30 min (Mito-MOI for 5 min) at
37 °C. Every time (expect for the experiment of realꢀtime obꢀ
servation), the cells were washed to remove the unbound
Scheme 1. Chemical structures of phosphatidyl ethanolamine
and Mito-MOI (a), and binding model of Mito-MOI within
mitochondrial inner membrane (b).
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Analytical Chemistry
probe before stained with another probe. After rinsing with
PBS twice, cells were imaged immediately.
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Cellꢀviability assay: the study of the effect of Mito-MOI on
viability of cells was carried out using the methylthiazolyldiꢀ
phenylꢀtetrazolium bromide (MTT) assay. HeLa and SiHa
cells growing in log phase were seeded into 96ꢀwell plates (ca.
1 × 104 cells/well) and allowed to adhere for 24 h, respectively.
Mito-MOI (200 µL/well) at concentrations of 10 µM, 5 µM,
and 2.5µM was added into the wells of the treatment group,
and 200 µL/well DMSO diluted in DMEM at final concentraꢀ
tion of 0.2% to the negative control group, respectively. The
cells were incubated for 24 h at 37 °C under 5% CO2, respecꢀ
tively. Then MTT (5 mg/mL in DMEM) was added into each
well. After 4 h incubation at 37 °C, 200 µL DMSO was added
to dissolve the purple crystals. After 20 min incubation, the
optical density readings at 492 nm were taken using a plate
reader. Cytotoxic experiment was repeated for four times.
Tissue staining: the rat skeletal muscle, cardiac muscle and
hepatic tissues were directly removed from just killed adult
wistar rat (purchased from Laboratory Animal Center, Shanꢀ
dong University). Then they were stained with Mito-MOI (5
µM, 5 min) at room temperature in HꢀDMEM supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin and
streptomycin.
Fluorescent imaging. Wideꢀfield fluorescent images were
acquired with an Olympus IX71 inverted microscope coupling
with a CCD and display controller software. The fluorescence
of Mito-MOI was excited and collected through the red chanꢀ
nel. Confocal fluorescent images were obtained with a LSM
780 confocal laser scanning microscope or an Olympus FV
300 laser Confocal Microscope. The overlap coefficients and
fluorescence intensity of the images were determined by the
software with the LSM 780 confocal microscope. For the
Olympus Microscope, emission was collected with a beam
splitter DM570 and an IF565 nm pass filter combination.
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Figure 1. Absorption and fluorescence spectra of Mito-MOI
in DMSO, EtOH, glycerol and water. Excited wavelengths:
the corresponding maximum absorption wavelengths. Comꢀ
pound concentration: 10 µM.
can find that Φ value of Mito-MOI is very low in aqueous
solutions, and turns larger in some organic solvents. It should
be noted that the Φ value is obviously enhanced in glycerol, an
organic solvent with rather high viscosity, which should be
attributed to the restrain of its intramolecular motions. Since
Mito-MOI can target mitochondrial inner membrane which
can also provide highꢀviscosity conditions,15 this physical
property of the probe greatly favors its imaging of mitochonꢀ
dria with highꢀfidelity.
RESULTS AND DISCUSSION
Photophysical properties. The absorption and fluorescence
spectra of Mito-MOI have been plotted in Figure 1. At the
same time, the relevant photophysical parameters of Mito-
MOI have been summarized in Table S1. From Table S1, one
Figure 2. Confocal fluorescent images of SiHa (a) and astrocytes (b) stained with Mito-MOI (5 µM, 5 min) and MTG (0.2 µM, 30
min). Coꢀlocalization coefficients of Mito-MOI and MTG (c) in SiHa and astrocytes. λex = 488 nm, λem = 600ꢀ700 nm to Mito-
MOI, λem = 490ꢀ520 nm to MTG. Bar = 20 µm (the mean ± SD from three independent experiments).
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Selectivity to mitochondria. The absorption and fluoresꢀ
High-resolution imaging mitochondrial inner membrane.
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cence spectra of Mito-MOI, MTG and MTR were studied
(Figure S1). When excited by 488 nm, both Mito-MOI and
MTG emitted fluorescence. If collecting 600ꢀ700 nm for Mi-
to-MOI and 490ꢀ520 nm for MTG, there would not be optical
interferences between the two dyes. Thus, coꢀstaining experiꢀ
ment with MTG was carried out to investigate the intracellular
localization of Mito-MOI.32 The confocal microscopic photos
of SiHa and rat astrocytes incubated with Mito-MOI and
MTG were shown in Figure 2. The average coꢀlocalization
coefficients of Mito-MOI and MTG in SiHa and astrocytes
were 0.93 and 0.95, respectively. These results indicated that
Mito-MOI was capable of imaging intracellular mitochondria
exclusively.
In SiHa cells stained with Mito-MOI, the deconvolved threeꢀ
dimensional (3D) reconstruction image (Figure 3) was obꢀ
tained by Airyscanning. Each individual mitochondrion with
oval morphological and hollow structure was imaged without
background noise. In the magnified pictures, mitochondria
were stained as hollow spheres, indicating that Mito-MOI
were rich in mitochondrial membrane. Considering that catiꢀ
onic mitochondrial probes were collected by mitochondria due
to the negative membrane potential across mitochondrial inner
membrane,19ꢀ21 Mito-MOI should target the inner membrane.
Moreover, the cristae, a characteristic structure of mitochonꢀ
drial inner membrane (as shown in Figure S2, SEM picture),
can be also observed though vague (as marked by yellow arꢀ
rows in Figure 3b and c). These results further proved that
Mito-MOI indeed stained mitochondrial inner membrane.
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Figure 3. a) Deconvolved highꢀresolution 3D reconstruction
image by Airyscanning in SiHa cells stained with Mito-MOI
(5 µM, 5 min); b) and c), The enlargement images of the yelꢀ
low box in (a) and the yellow arrows are pointed to the cristae.
λex = 488 nm, λem = 490ꢀ700 nm. Bar = 2 µm.
Figure 5. Morphology of mitochondria in skeletal muscle
tissue stained with Mito-MOI (5 µM, 5 min) and Hoechst
33342 (5 µM, 30 min). a) Confocal fluorescence images of
longitudinal plane at 20× magnification; b) Crossꢀsectional
plane at 40× magnification; c) Longitudinal plane at 60× magꢀ
nification; d) Enlargement of the yellow box in c). Mito-MOI,
λex = 488 nm, λem = 490ꢀ700 nm; Hoechst 33342, λex = 405 nm,
λem = 420ꢀ470 nm. Bar = 20 µm.
Fast and high-fidelity imaging mitochondria in living
cells. SiHa cells were stained with Mito-MOI, MTG and
MTR for 30 min, respectively. In Figure 4I and Video S1a
from Mito-MOI, nearly no background signal was observed
in cytoplasm and culture medium. Particularly, mitochondria
were visualized in less than 5 min. While in Figure 4II and
Video S1b, MTG showed intense background noise. Moreover,
MTG firstly stained plasma membrane in 5 min and mitoꢀ
chondria were visualized after 20 min. In Figure 4III and Vidꢀ
eo S1c from MTR, there was still some detectable background
noise in culture medium. Similar to MTG, MTR also firstly
stained plasma membrane and then stained mitochondria in
more than 10 min. These results showed that Mito-MOI had
fast stainability and could highꢀfidelity image mitochondria
with washꢀfree procedure, while MTG and MTR cannot. From
Figure 4. Realꢀtime images of living SiHa cells stained with
Mito-MOI (I, 5 µM), MTG (II, 0.2 µM), MTR (III, 0.2 µM).
The observation time: 3 min (a), 5 min (b), 10 min (c), 20 min
(d), 30 min (e). Mito-MOI and MTG, λex = 488 nm, λem
=
490ꢀ700 nm; MTR, λex = 561 nm, λem = 570ꢀ700 nm. Bar = 20
µm.
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Analytical Chemistry
spectra of Mito-MOI, MTG and MTR in organic solvent and ages was obtained (Figure 6), which showed a clear intracelluꢀ
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water, the relative intensity ratios of Mito-MOI in organic
solvents and water were higher than that of MTG and MTR
(Figure S3), which proved that Mito-MOI had higher signal to
noise ratio (SNR).
lar mitochondrial networks and the spatial organization of
nucleus and different mitochondria. In addition, we also got
crossꢀsectional images every 1.7 µm along the z axis (Figure
S5). The results showed that mitochondria at a 50.9 µm depth
can be visualized by Mito-MOI.
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At the same time, hepatic and myocardial tissues were also
studied. According to Figure S6a, mitochondria filled cytoꢀ
plasm of polygonal hepatocytes. And the staining depth was
40 µm. In Figure S6b, the longitudinal section of the myocarꢀ
dial tissue was shown. Moreover, a staining depth of 36 µm
was exciting as myocardial tissue was also denser.
Tracking dynamic process of mitochondrial fission. The
variations of the location and orientation of mitochondria were
realꢀtime tracked in living rat astrocytes. (Figure 7I, Video S2).
In tracking process of 20 min, little intracellular and extracelꢀ
lular background fluorescence was observed, and only mitoꢀ
chondria emitted bright fluorescence. Thus the dynamic proꢀ
cess of mitochondrial fission from 480 s to 520 s was shown
clearly in situ. Delightfully, mitochondrial morphology and
distribution in a neuron was also visualized clearly (Figure
7II). In particular, mitochondrial morphology in neuronal soꢀ
ma was clearly shown in the enlargement image.
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Figure 6. Zꢀstack images were taken of every 1 µm section
from the top to bottom and 3D reconstruction of one muscle
tissue (135 µm × 135 µm × 37 µm,) showing spatial relationꢀ
ships between different mitochondria and nucleus. Mito-MOI,
λex = 488 nm, λem = 490ꢀ700 nm; Hoechst 33342, λex = 405 nm,
λem = 420ꢀ470 nm. Bar = 20 µm.
Fast and high-fidelity imaging mitochondria in living tis-
sues. A piece of skeletal muscle tissue from a sevenꢀweek old
rat without fixation was stained with Mito-MOI and Hoechst
33342 by washꢀfree procedure. Examination of mitochondrial
morphology in both longitudinal and crossꢀsectional (transꢀ
verse) planes was achieved by optical sectioning of confocal
microscopy. The longitudinal view (Figure 5a) revealed that
mitochondria were regularly arranged and formed reticulum in
muscle, whereas the transverse view (Figure 5b) revealed the
actual tubular morphology of intermyofibrillar (IMF) mitoꢀ
chondria. The results above were consistent with the mitoꢀ
chondrial structure in skeletal muscle obtained by SEM.33ꢀ35
Particularly, four kinds of mitochondria with different morꢀ
phologies have been inꢀsitu observed (Figure 5c and d): paraꢀ
cascular mitochondria (PVM), Iꢀband mitochondria (IBM),
fiber parallel mitochondria (FPM), and cross fiber connection
mitochondria (CFCM). Four kinds of mitochondria formed
crystalꢀlike networks and provided a conductive pathway for
skeletal muscle energy distribution.36,37
Figure 7. Realꢀtime tracking images of living rat astrocytes (I)
and wideꢀfield fluorescence image of living rat neuron (II)
stained with Mito-MOI (5 µM). Enlargement images from
480 s (Ia) to 520 s (Ic) showing mitochondrial fission. Enꢀ
largement image in (II) showing mitochondrial morphology in
neuronal soma. I) λex = 488 nm, λem = 490ꢀ700 nm; II) λex
510ꢀ550 nm, λem > 570 nm. Bar = 20 µm.
=
Photostability and other advantages. To quantitatively
compare the photostability of Mito-MOI, MTG and MTR,
one of the most crucial criteria on fluorescent imaging
38ꢀ40
agents,25,
HeLa cells stained by them were exposed to
constant laser beam. The fluorescence intensity of intracellular
dyes was normalized and plotted as a function of time. In Figꢀ
ure 8, Mito-MOI exhibited constant fluorescence emission
during continuous laser irradiation of 15 min. In contrast, unꢀ
der the same laser intensity, the signal intensity of MTG and
MTR decayed sharply in 2 min and 1 min, respectively, indiꢀ
By optical sectioning of the tissue, a series of fluorescent
images was captured every 1 µm along the z axis (Figure 6
and S4), and their computerꢀgenerated 3D reconstruction imꢀ
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cating the superior photostability of Mito-MOI. Moreover, the alkyl chain). As expected, high resolution microscopic photos
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cytotoxicity of Mito-MOI with different concentrations in
HeLa and SiHa cells was investigated and the cell viability
assay data were quantified. As shown in Figure 9a and b, the
viability of HeLa and SiHa cells incubated with 10 µM Mito-
MOI was more than 85% after incubation for 24 h. Thus the
micromolar concentrations of Mito-MOI should be nontoxic.
showed that it stained mitochondrial inner membrane without
background noise. Different from conventional mitochondrial
probes, Mito-MOI had faster stainability, higher SNR and
removed washing process. Therefore, highꢀfidelity mitochonꢀ
drial fluorescent images in intact living tissues have been sucꢀ
cessfully obtained. Particularly, four kinds of mitochondria
and their crystalꢀlike structure in muscle tissues were visualꢀ
ized clearly. Furthermore, Mito-MOI could track the dynamic
process of mitochondrial fission and has excellent photostabilꢀ
ity as well as low toxicity. The strategy of dual targeting
groups should have important value for designing probes with
ultrahigh selectivity to various intracellular membranous comꢀ
ponents.
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ASSOCIATED CONTENT
Supporting Information
Additional information as noted in text. This material is available
free of charge via the Internet at http://pubs.acs.org.
The synthetic details and characterization of Mito-MOI,
photophysical properties of Mito-MOI, SEM imaging of
muscle fiber, relative intensity ratio of Mito-MOI, MTG
and MTR, fluorescent images of optical sectioning of
skeletal muscle tissues, confocal fluorescence images of rat
hepatic tissue and myocardial tissue. (PDF)
Figure 8. Confocal fluorescence microscopy images (left) of
the living HeLa cells incubated with Mito-MOI (5 µM, 5 min),
MTG (0.2 µM, 30 min), MTR (0.2 µM, 30 min) and photostaꢀ
bility comparison was shown in the right figure. Mito-MOI
and MTG, λex = 488 nm, λem = 490ꢀ700 nm; MTR, λex = 561
nm, λem = 570ꢀ700 nm. Bar = 20 µm.
Videos: Realꢀtime observation of living SiHa cells stained
with Mito-MOI, MTG and MTR, respectively. Realꢀtime
tracking of living rat astrocytes stained with Mito-MOI.
(AVI)
AUTHOR INFORMATION
Corresponding Author
Xiaoqiang Yu
Fax: +86 0531 88364263;
Eꢀmail address: yuxq@sdu.edu.cn
Jing Zhi Sun
Fax: +86 0571 87953734;
Eꢀmail address: sunjz@zju.edu.cn
Xiuquan He
Fax: +86 0531 88364263;
Eꢀmail address: hxq3000@sdu.edu.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Our Laser confocal scanning imaging of living cells was perꢀ
formed at The Microscopy Characterization Facility, Shandong
University. For financial support, we thank the National Natural
Science Foundation of China (51273107, 51273175 and
21672130), Natural Science Foundation of Shandong Province,
China (ZR2012EMZ001), Shandong University 2011JC006, Open
Project of State Key Laboratory for Supramolecular Structure and
Materials (SKLSSM201524).
Figure 9. MTT results of HeLa (a) and SiHa (b) cell viabilities
after incubation with Mito-MOI (10 µM, 5 µM, 2.5 µM) for
24 h.
REFERENCES
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To summarize, we have synthesized a phospholipidꢀ
biomimetic mitochondrial probe Mito-MOI with ultrahigh
selectivity due to its dual targeting groups (cation and C18ꢀ
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