A photostable AIE luminogen for specific mitochondrial imaging and tracking

Article abstract of DOI:10.1021/ja310324q

Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in deman

Full text of DOI:10.1021/ja310324q

Communication
pubs.acs.org/JACS
A Photostable AIE Luminogen for Specific Mitochondrial Imaging
and Tracking
Chris Wai Tung Leung,†,# Yuning Hong,†,# Sijie Chen,† Engui Zhao,† Jacky Wing Yip Lam,†
and Ben Zhong Tang*,†,‡
†Department of Chemistry, Institute for Advanced Study, Division of Biomedical Engineering, State Key Laboratory of Molecular
Neuroscience and Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology, Clear Water
Bay, Kowloon, Hong Kong, China
‡Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials
and Devices, South China University of Technology, Guangzhou 510640, China
S
* Supporting Information
dynamic changes in a certain period of time, the probe must be
ABSTRACT: Tracking the dynamics of mitochondrial
morphology has attracted much research interest because
of its involvement in early stage apoptosis and
degenerative conditions. To follow this process, highly
specific and photostable fluorescent probes are in demand.
Commercially available mitochondria trackers, however,
suffer from poor photostability. To overcome this
limitation, we have designed and synthesized a fluorescent
agent, tetraphenylethene-triphenylphosphonium (TPE-
TPP), for mitochondrial imaging. Inherent from the
mitochondrial-targeting ability of TPP groups and the
aggregation-induced emission (AIE) characteristics of the
TPE core, TPE-TPP possesses high specificity to
mitochondria, superior photostability, and appreciable
tolerance to environmental change, allowing imaging and
tracking of the mitochondrial morphological changes in a
long period of time.
photostable under the continual irradiation of light from
fluorescent microscopes. Conventional fluorescent dyes for
mitochondria staining have been developed.9,10 Their photo-
stability, however, leaves much to be desired. Very diluted
solutions of these dyes are used in the imaging process, and
such small numbers of dye molecules can be quickly
photobleached when a harsh laser beam is used as the
excitation light source. The photostability cannot be improved
by using higher fluorophore concentration due to the
accompanying concentration-quenching effect.
Luminogens with aggregation-induced emission (AIE)
characteristics exhibit opposite phenomena: They are almost
nonfluorescent when molecularly dissolved but become highly
emissive in the aggregate state with fluorescence increasing
along with the increase of fluorophore concentration.11
Restriction of intramolecular motions (RIM) is proposed as
the main cause of the AIE effect.12 Lipophilic AIE molecules
form nanoaggregates in aqueous solution spontaneously
because of their hydrophobic nature.13 These nanoaggregates
have been successfully applied for long-term cell tracking in our
previous work.14 It is envisaged that the nanoaggregates of the
AIE molecules would possess better photostability than single
fluorescent molecule in dilute solutions.
To achieve the specificity to mitochondria, AIE luminogens
are decorated with mitochondria targeting moieties. In this
study, tetraphenylethene (TPE), an archetypal AIE luminogen,
was synthesized and functionalized with triphenylphosphonium
(TPP) groups (TPE-TPP, Figure 1). TPP is a well-known
functional group that facilitates the entrance of molecular
probes into mitochondria by its lipophilicity and electro-
phoretic force.2 We here demonstrate that TPE-TPP can light
up mitochondria specifically in live cells with superior
photostability, enabling the observation of mitochondrial
morphological changes.
itochondria, the organelle found in almost all eukaryotic
M
cells, play a vital role in the life and death of cells.1 The
most prominent function of mitochondria is to produce ATP,
the energy currency of the cell. The production of ATP
involves a series of electron-transport systems in the oxidation−
phosphorylation pathway, which is also found to be associated
with the generation of reactive oxygen species (ROS).1,2 The
production of ROS in mitochondria leads to the propagation of
free radicals and damaging cells and contributes to cell death,
which is known as mitochondria-mediated apoptosis.3,4 The
morphology of mitochondria, though varies upon cell type, cell-
cycle stage, and intracellular metabolic state, is affected by and
thus reflects cell functioning.5 The morphology is controlled by
a set of proteins, mutations of which will cause several human
diseases including degenerative diseases, such as Parkinson’s
and Alzheimer’s diseases.6 Recent reports also show that
proteins participating in apoptosis can affect the morphology of
mitochondria.7,8 Tracking the mitochondrial morphological
change may give insight for studying apoptosis and
degenerative conditions.
TPE-TPP was prepared by the synthetic route, as shown in
Scheme S1. The TPE core (Figure 1, TPE-TPP, blue) was
synthesized simply from a McMurry coupling reaction. TPP
(Figure 1, TPE-TPP, red) was attached through bromination
Fluorescent probes that can selectively illuminate cellular
mitochondria are powerful tools for monitoring the morpho-
logical changes and studying these processes. For observing the
Received: October 24, 2012
Published: December 17, 2012
© 2012 American Chemical Society
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experiment with MitoTracker red FM (MT), a commercially
available mitochondria imaging agent,15,16 suggests that the
observed fluorescence from TPE-TPP is localized to the
mitochondria of the living HeLa cells (Figure 2). Pearson’s
correlation coefficient (Rr; from +1 to −1), indicating the
degree of linear dependence between two variables, is used to
quantify the staining region overlap between TPE-TPP and
MT. Fluorescence signals of the two dyes collected from two
different channels are perfectly overlapped with Rr = 0.96,
demonstrating the specific targeting of TPE-TPP on
mitochondria. In Figure 2A, cells were incubated with TPE-
TPP for 1 h. However, staining time as short as 15 min is
sufficient for TPE-TPP to enter and light up mitochondria in
cells (data not shown). Incubation time between 30 min to 2 h
gives similar high signal-to-noise ratio (Figure S6).
The working concentrations of TPE-TPP and MT in our
experiemnts were 5 μM and 50 nM, respectively. At first glance,
one may think that MT is more sensitive than TPE-TPP. In a
sense, it is true. But MT has a disadvantage at such low
concentration because the dye molecules can be easily
photobleached by the strong excitation light especially in
confocal microscopes. At higher concentrations, these probes
tend to lose the specificity and stain other cellular structures.17
Photostability is one of the most important criteria for
developing fluorescent imaging agents. Continuous scanning by
confocal microscope (Zeiss laser scanning confocal microscope
LSM7 DUO) was used to quantitatively investigate the
photostability of TPE-TPP and MT. Two dishes of HeLa
cells subcultured from the same source were stained with 5 μM
TPE-TPP for 1 h and 50 nM MT for 30 min, respectively. With
the help of a power meter, excitation power from 405 and 560
nm channels of the microscope were unified (65 μW) and used
to irradiate the TPE-TPP and MT stained cells. The initial
intensity referred to the first scan of TPE-TPP and MT stained
cells was normalized, and the percentage of fluorescence signal
loss was calculated. As shown in Figure 3, during 50 scans with
total irradiation time of ∼7 min, the signal loss of TPE-TPP is
less than 20%, and no significant difference was observed
between the first and the 50th scan (video 1). Since live cells
are dynamic, the movements of cells may attribute to the slight
Figure 1. Chemical structure of TPE-TPP; PL spectra of TPE-TPP in
solid and solution (soln) states. Inset: Photographs of DMF solution
(left) and solid powder (right) of TPE-TPP taken under UV
irradiation. Concentration of TPE-TPP: 10 μM; excitation wavelength:
321 nm.
and the subsequent reaction with triphenylphosphine. The
product was characterized by NMR and MS, and both gave
satisfactory analysis data corresponding to their molecular
structure (Figures S1−S3). Although carrying positive charges,
TPE-TPP has relatively poor solubility in aqueous solution but
is completely soluble in polar solvents, such as DMF and
DMSO.
TPE-TPP shows typical AIE features, as shown in Figure 1.
The DMF solution of TPE-TPP was almost nonemissive, while
the solid state is strongly luminescent at 466 nm. Particle size
analysis reveals the existence of particles with average size of
144 nm in aqueous solution containing 0.1% DMSO, the
identical condition for cell staining, confirming that the TPE-
TPP molecules have indeed formed aggregates in nanoscale
(Figure S4).
Before the application for cell imaging, the cytotoxicity of
TPE-TPP was evaluated using a 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyltetrazolium bromide (MTT) assay (Figure S5).
The result shows that the cell viability is not significantly
altered when up to 7.5 μM TPE-TPP is added to the culture
medium. TPE-TPP was then assessed for its ability to localize
and stain mitochondria in living cells by fluorescence
microscope. Cervical cancer HeLa cells were incubated with 5
μM TPE-TPP for 1 h, and excess dyes were washed away by
buffer solution. As shown in Figure 2A, TPE-TPP stains
specifically the mitochondrial region in HeLa cells. The
reticulum structures of mitochondria are clearly visible with
the aid of the blue fluorescence of TPE-TPP. The costaining
Figure 3. Signal loss (%) of fluorescent emission of TPE-TPP (solid
circle) and MT (open circle) with increasing number of scans. Inset:
fluorescent images of living HeLa cells stained with TPE-TPP (5 μM)
with increasing number of scans (1−50 scans; the number of scans
shown in upper right corner). Excitation wavelength: 405 nm (for
TPE-TPP) and 560 nm (for MT); emission filter: 449−520 nm (for
TPE-TPP) and 581−688 nm (for MT); irradiation time: 7.75 s/scan.
Figure 2. Fluorescent images of HeLa cells stained with (A) TPE-TPP
(5 μM) for 1 h and (B) MitoTracker red FM (MT, 50 nM) for 15
min. (C) Panels A and B merged. Excitation wavelength: 330−385 nm
(for TPE-TPP) and 540−580 nm (for MT).
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fluctuation of the signals. In contrast, the fluorescence signals of
MT almost disappear after only 6 scans with <25% signal
intensity remaining (video 2; Figure S7).
targeting upon the change of ΔΨm. The lipophilicity may
also play an important role in retaining the specificity and
sensitivity of TPE-TPP in CCCP-treated HeLa cells. The
lipophilicity of TPE-TPP is greatly enhanced by the TPE core
with four phenyl rings rationalizing this phenomenon. On the
other hand, MT carries only one positive charge (structure
shown in Figure S8), and its selectivity is too susceptible to
subtle change of ΔΨm in mitochondria.
The high tolerance of TPE-TPP to the decrease of ΔΨm
enables the observation of change in mitochondrial morphol-
ogy induced by CCCP (video 3).22 Upon exposure to CCCP,
the reticulum-like mitochondria are gradually transformed to
small and dispersed fragments (Figure 5). The early stage of
The formation of nanoaggregates of TPE-TPP when
dispersed in aqueous media may facilitate the dye to diffuse
across the cell membrane to accumulate in the mitochondrial
region. Due to the AIE feature, the nanoaggregates of TPE-TPP
are much brighter emitters than its single molecular form
because the condensed packing in the aggregate state constrains
the intramolecular motions and blocks the nonradiative decay
channels.12 When exposed to excitation light, the outermost
layer of the nanoaggregates may be photobleached. However,
the condensed particles can prevent further photobleaching and
photo-oxidation by avoiding oxygen diffusion into the particles.
For MT, unfortunately the working concentration is so low that
even when accumulated in the mitochondrial matrix, it presents
as an individual molecule which will be destroyed with ease by
the strong excitation light.
Mitochondria are the organelles where celluar respiration
occurs. Mitochondria continuously oxidize substrates and
maintain a proton gradient across the lipid bilayer with very
large membrane potential (ΔΨm) of around −180 mV.18 This
value is double the plasma membrane of excitable cells and
about six times larger than nonexcitable cell plasma membranes.
Due to this large membrane potential gradient, mitochondria
drive cationic species, such as TPP, into the matrix.19 As a
result, their accumulation in mitochondria is 100−500-fold
higher than in other parts of the cell. To test the tolerance of
TPE-TPP and MT to the change of mitochondrial ΔΨm,
carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used
to treat the cells prior to the staining procedure. CCCP is an
uncoupler that causes rapid acidification of the mitochondria
and dysfunction of ATP synthase resulting in the decrease of
the mitochondrial ΔΨm.20 Upon treatment with 20 μM CCCP,
the pH of mitochondria will decrease about 0.7,21 and ΔΨm will
thus decrease around 40 mV according to Nernst equation.
When the cells were treated with 10 μM CCCP, MT had no
more specificity to mitochondria, and the sensitivity became
worse (Figure 4A). Since the targeting of MT to mitochondria
Figure 5. Fluorescent images of CCCP (20 μM) treated living HeLa
cells stained with TPE-TPP (5 μM) with increasing scan time (the
scan time shown in the upper left corner of the panel). Excitation
wavelength: 405 nm; emission filter: 449−520 nm; irradiation time:
15.49 s/scan.
apoptosis involves remodeling of mitochondrial cristae and the
consequent occurrence of morphological change of mitochon-
dria, which is considered an irreversible process associated with
the collapse of ΔΨm.7 TPE-TPP has excellent specificity to
mitochondria with outstanding photostability and tolerance to
microenvironment change, representing a potential candidate
of tracking agent for apoptosis studies.
In conclusion, an AIE-active TPE derivative, TPE-TPP, has
been synthesized and utilized as a fluorescent agent for
mitochondrial imaging. Possessing high specificity to mitochon-
dria, superior photostability, and appreciable tolerance to
microenvironment change, TPE-TPP is a well-suited imaging
agent for mitochondrial targeting and morphological change
tracking. Because of its synthetic accessibility, TPE-TPP can be
further modified as a dual-functional probe for an array of
applications, such as sensing of ROS, metal ions, or pH change
in mitochondria. Further studies on the development of AIE
materials with different emission colors for organelles imaging
and their biomedical applications are ongoing in our
laboratories.
Figure 4. Fluorescent images of CCCP (10 μM) treated HeLa cells
stained with (A) MT (50 nM) for 15 min and (B) TPE-TPP (5 μM)
for 30 min. Excitation wavelength: 540−580 nm (for MT) and 330−
385 nm (for TPE-TPP).
ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. The material is
available free of charge via the Internet at http://pubs.acs.org
■
S
is driven by the huge ΔΨm, the decrease of ΔΨm will affect the
direction and accumulation of cationic MT to mitochondria. It
was expected that similar phenomena would be observed for
TPE-TPP as it shares a similar working principle. Surprisingly,
under the same condition, the specificity and sensitivity of TPE-
TPP to mitochondria are perfectly retained in CCCP-treated
cells (Figure 4B).
AUTHOR INFORMATION
Corresponding Author
tangbenz@ust.hk
■
Author Contributions
#These authors contributed equally.
Compared with MT, TPE-TPP carries two positive charges,
which allows a wider dynamic range for mitochrondrial
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was partially supported by the Research Grants
Council of Hong Kong (604711, 602212, HKUST2/CRF/10,
and N_HKUST620/11), the University Grants Committee of
Hong Kong (AoE/P-03/08), HKUST (RPC10SC13,
RPC11SC09, and SRFI11SC03PG), the Innovation and
Technology Commission (ITCPD/17-9), and the National
Science Foundation of China (20974028). B.Z.T. thanks the
support from Guangdong Innovative Research Team Program
of China (201101C0105067115).
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