A photostable fluorescent probe for rapid monitoring and tracking of a trans-membrane process and mitochondrial fission and fusion dynamics

Article abstract of DOI:10.1039/c5nj02821c

Rapid monitoring and tracking of a trans membrane process and mitochondrial fission and fusion dynamics play critical roles in judging the occurrence and development of disease, and can give insights for studying apoptosis and cell degeneration. However, the existing probes are not capable of rapid monitoring and tracking the above dynamics process. To solve this problem, we develop a unique functional mitochondria probe containing long alkyl chains, 3,5-bis((E)-2-(pyridin-4-yl)vinyl)-1H-indole monoiodide (MT-PVIM), which is capable of rapid real-time imaging and tracking mitochondrial fission and fusion dynamics. In addition, compared with a commercially available mitochondrial probe MTR, the MT-PVIM probe has excellent specificity to mitochondria with outstanding tolerance of micro-environmental changes, thus representing a potential candidate as a tracking agent for apoptosis studies. The good performance of our proposed approach demonstrates that this strategy might open up new opportunities for the development of rapid image mitochondria-targetable molecular tools for bioanalytical and biomedical applications.

Full text of DOI:10.1039/c5nj02821c

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DOI: 10.1039/C5NJ02821C
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ARTICLE
A photostable fluorescent probe for rapid monitoring and tracking
trans membrane process and mitochondrial fission and fusion
dynamics*
Received 00th January 20xx,
Accepted 00th January 20xx
Yong Liua, Fangfang Menga, Yonghe Tanga, Xiaoqiang Yub*, Weiying Lina*
DOI: 10.1039/x0xx00000x
Rapid monitoring and tracking trans membrane process and mitochondrial fission and fusion dynamics play critical roles
for judging occurrence and development of the disease, and can give insights for studying apoptosis and cell degeneration.
However, the existing probes are not capable of rapid monitoring and tracking the above dynamics process. To solve this
problem, we develop a unique functional mitochondria probe containing long alkyl chains, 3, 5-bis((E)-2-(pyridin-4-
yl)vinyl)-1H-indole monoiodide (MT-PVIM), which is capable of rapid real-time imaging and tracking mitochondrial fission
and fusion dynamics. In addition, compared with commercially available mitochrondrial probe MTR, the probe MT-PVIM
has excellent specificity to mitochondria with outstanding tolerance of micro-environmental changes, thus representing a
potential candidate as a tracking agent for apoptosis studies. The good performance of our proposed approach
demonstrates that this strategy might open up new opportunities for the development of rapid image mitochondria-
targetable molecular tools for bioanalytical and biomedical applications.
www.rsc.org/
mitochondrial fission and fusion dynamics processes can give
insight for studying occurrence and development of the
disease and apoptosis and cell degeneration.
Introduction
Mitochondria play a key role in various biological processes
including energy production through the respiratory chains,1-3
cell signaling via reactive oxygen species production,4,
regulation of Ca2+ homeostasis,6, 7 and the triggering of cell
death.8-11 Mitochondria are dynamic organelles that can
quickly change in number and morphology within a cell during
the development, the cell cycle, or when challenged by various
toxic conditions.12 They move from one location to another
and undergo continuous fusion and fission events quickly.
These behaviors are altogether classified as “mitochondrial
dynamics”.13 Mitochondrial dysfunction are associated with
In the past decade, fluorescence probes have been an
important detection means to image mitochondria and
intracellular target.17-20 To date, two classes of probes are
mainly used. As shown in table S1, the first class of probes can
real-time image mitochondria.14, 21 But, most probes enter the
cells and detect mitochondria are approximately 15-60 min.
Thus, the probes cannot achieve rapid detect and real-time
image mitochondria in a short period of time. Moreover, these
5
probes unable to track mitochondrial fission and fusion
dynamics processes. The second type of probes can track
23
mitochondrial dynamics changes.22,
For example, green
intrinsic apoptotic pathways and cause
a variety of
fluorescent proteins (GFP),24 CMT-red,14 NPA-TPP,23
Rhodamine 12325 and JC-1,26 which have been used to
specifically target mitochondria to reveal that the
mitochondria in vivo are highly dynamic. However, these
fluorescent probes showed poor photostability so that could
not rapid monitor and track trans membrane process and
mitochondrial fission and fusion dynamics. For example, JC-1 is
the most widely used fluorescent sensor for detecting
mitochondrial membrane potential (ΔΨm). However, though
the probe JC-1 is highly sensitive to dye loading concentration
and time, many literature studies reported false results of
using JC-1 for studying mitochondria.27 And it is not suitable
for rapid monitoring and tracking rapid trans membrane and
mitochondrial dynamics changes. All in all, the existing probes
showed poor photostability and poor permeability so that
could not monitor and track rapid trans membrane and
mitochondrial fission and fusion dynamics process. Therefore,
neurodegenerative diseases such as Alzheimer's disease,
cancer and diabetes.14 Mitochondrial fission and fusion are
closely connected with mitochondrial dynamic changes. It
plays role in maintaining protection of mitochondrial DNA
(mtDNA).15 And this process regulates mitochondrial
morphology, number, location, and function.16 Therefore,
Rapid acrossing cell membrane and real-time tracking
a Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Biological Science and Technology, University of
Jinan, Jinan, Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
b Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal
Materials, Shandong University, Jinan, 250100, PR China.
Electronic Supplementary Information (ESI) available: Experimental procedures,
Video S1-S4 and characterization data See DOI: 10.1039/x0xx00000x.
This journal is © The Royal Society of Chemistry 20xx
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the search for a probe that can monitoring and tracking rapid Cancer cells SiHa were cultured in Dulbecco’s modified Eagle’s
View Article Online
DOI: 10.1039/C5NJ02821C
trans membrane process and mitochondrial fission and fusion medium supplemented with penicillin/streptomycin and 10%
dynamics has always been an attractive and challenging goal.
bovine calf serum in a 5% CO2 incubator at 37°C. SiHa cells were
In this work, as shown in Fig 1, we develop a unique grown overnight on a 35 mm petri dish with a cover slip or a
mitochondria probe, 3,5-bis((E)-2-(pyridin-4-yl)vinyl)-1H-indole plasma-treated 25 mm round cover slip mounted to the bottom of a
monoiodide (MT-PVIM). We predict that this probe is capable 35 mm petri dish with an observation window. The living cells were
of monitoring and tracking trans membrane process and stained with 3 μM MT-PVIM for 3 min (by adding 2 μL of a 1 mM
mitochondrial fission and fusion dynamics compared with MTR. stock solution of MT-PVIM in DMSO to 1 mL culture medium) or
250 nM MTR for 20 min (by adding 3 μL of a 200 μM stock solution
Experimental
of MTR in DMSO to 1 mL culture medium).
Measurements equipments and materials
Tracking probe enter cells processes test
TLC analysis was performed on silica gel plates and column
chromatography was conducted over silica gel (mesh 200–300),
both of which were obtained from the Qingdao Ocean Chemicals.
All chemicals mainly were analytical grade, and 4-picoline, 1-
iodododecane and 5-bromo-1H-indole-3-carbaldehyde were
purchased from Sinopharm Chemical Reagent Co., MitoTracker Red,
Ltd (Shanghai, China), Palladium (II) acetate and tri-o-
tolylphosphine were purchased from J&K Chemical (Shanghai,
China). Tris and PBS were purchased from Seikagaku Corporation of
Japan. All solvents maimly used in the spectral measurement are of
chromatographic grade.
We used a Bruker Avanace 300/400 spectrometer to obtain
Nuclear magnetic resonance spectra (1H and 13C) of all compounds.
The HRMS spectra were obtained by LC/MS ThermoFisher LCQ
FLEET or Agilent Technologies 6510 Q-TOF. The elemental analyses
were performed on a Vario EI III instrument. The UV-visible-near-IR
absorption spectra of dilute solutions were recorded on a Cary 50
spectrophotometer using a quartz cuvette having 1 cm path length.
The fluorescence fluorescence spectra were obtained by a HITACHI
F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. All
confocal microscopic photos were obtained with Carl Zeiss
For the dye entering into cells process test, the cells in the 35
mm petri dishes without stain were loaded below the
microscope; Next, using microsyringe join probe to petri use
over time (starting from time 0, then at 5, 10 and 15 min) using
confocal microscope (Zeiss Laser Scanning Confocal
Microscope; LSM7 DUO) and ZEN 2009 software (Carl Zeiss).
The probe MT-PVIM was excited at 488 nm and the
fluorescence was collected at 490-604 nm. MTR was excited at
561 nm and fluorescence was collected at 569-691 nm.
Cell imaging with carbonyl cyanide m-chlorophenylhydrazone
(CCCP) treatment
Living SiHa cells were grown overnight using a 35 mm petri
dish and a cover slip. The cells were incubated with 10 μM
CCCP (by adding 1 μL of a 200 mM stock solution of CCCP in
DMSO to 2 mL culture medium) for 30 min. The CCCP treated
cells were then stained by 3 μM MT-PVIM for 3 min or 250 nM
MTR (by adding 0.5 μL of a 200 μM stock solution of MTR in
DMSO to 2 mL culture medium) for 20 min.
Microscopy LSM780 equipped with
coupled device (CCD) (Pixel: 512x512) and 60×objective oil lens. The
a high-resolution charge
Results and discussion
confocal microscopic image and differential interference contrast Preparation of probes
(DIC) image were taken with a 488 nm laser. Confocal microscopic
As we all know, a selective localization of hydrophobic
images of MitoTracker Red (MTR) were collected between 569-691
nm upon excitation at 561 nm; Fluorescence images of MT-PVIM
were collected between 490 -604 nm upon excitation at 488 nm.
fluorophore at the plasma membrane can be achieved by
conjugating it with zwitterionic groups and long hydrophobic
chains.28 In our previous work, we found some structures
containing indoles cationic salt were capable of sensing
mitochondria.29 Thus, in this work, we constructed a unique
Measurement of the fluorescence quantum yields
probe MT-PVIM, and the anchor group of the compound MT-
In this work, the fluorescence quantum yields (Ф) of the probe MT-
PVIM were calculated by means of equation (1):
PVIM was composed of long alkyl chains indoles cationic group,
allowing strong interaction with the lipid membrane (Fig. 1a).
First, this push-pull electronic system based on indole possess
superior membrane permeability so that may rapidly enter
cells and light up mitochondria in a short period of time. In
addition, we anticipated that this system with linear alkyl
chains may rapid across the cell membrane and track
mitochondrial fission and fusion dynamics process (Fig. 1b).
The compound MT-PVIM was synthesized by Knoevenagel

2 


r (r ) ns Fs









(1)
s  r
2

s (s )
Fr
nr


where the subscripts s and r represent the sample and the
reference materials, respectively. Ф stands for the quantum yield, F
expresses the integrated emission intensity, A stands for the
absorbance, and
n is the refractive index. In this paper,
Fluorescence quantum yields for MT-PVIM was determined by
using fluorescein (Φ = 0.95 in water) as fluorescence standard.
condensation
reactions
between
N-(2-dodecane)-4
methylpyridinium iodide and a substituted indole-3-carboxaldehyde
using piperidine as catalyst. The intermediate 3 was obtained by
Cell culture and image
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Heck coupling
carbaldehyde with 4-vinylpyridine. The separated MT-PVIM were
characterized by various spectroscopic methods, from which
satisfactory analysis data were obtained (Scheme 1).
ARTICLE
reaction
between
5-bromo-1H-indole-3-
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DOI: 10.1039/C5NJ02821C
Fig. 2. (a) Absorption spectra and (b) fluorescence responses of
MT-PVIM in various solvents. Buffer (
■), DMSO (●), EtOH (▲),
CH3CN ( ), THF ( ). [MT-PVIM]: 2 μM.
▼
◆
Table 1. The photophysical properties of MT-PVIM
Probe
Solvents
λa
λb
Фc
MT-PVIM
Buffer
DMSO
EtOH
CH3CN
THF
405
450
450
436
450
588
538
534
534
537
0.012
0.40
0.27
0.19
0.33
λa and λb are linear absorption and fluorescent maximum peak in
nm, repectively; Φ is fluorescence quantum yield (Error limit: 8%)
c
determined by using fluorescein (Φ = 0.95) in aqueous NaOH (pH
13) as the standard.
Fig. 1. (a) Structures of the probe MT-PVIM and commercially
available mitochondrial probe MitoTracker Red (MTR); (b) Overall
strategy of our switchable probe, MT-PVIM (red), that is capable of
rapid monitoring and tracking trans membrane process and
mitochondrial fission and fusion dynamics.
Colocalization experiment
Living SiHa cells were incubated with MT-PVIM and the
commercially available mitochondrial indicator MTR to examine
whether the probe could specifically stain the mitochondria (Fig. 3).
The confocal image of MT-PVIM merged well with that of the MTR.
Moreover, the Pearson’s colocalization coefficient was determined
as 0.90, demonstrating that MT-PVIM predominantly located in the
mitochondria.30-33
Scheme. 1. Synthesis of the probe MT-PVIM
Photophysical properties of the probe
With the probe MT-PVIM in hand, we proceeded to examine its
photophysical properties. We find that the absorbance and
fluorescence intensities are high in organic solvents and very low in
tris-HCl buffer solution. Similarly, quantum yield (Ф) of the probe
MT-PVIM in buffer solution only reach 1.2% (Fig. 2a and b). In
organic solvent, maximum Ф of the probe MT-PVIM reached 19-
40% (Table 1). As we all know, mitochondria are membrane
organelles composing of different organic phase. Thus, we think
that the MT-PVIM probe may occur strong fluorescence in
mitochondria. At the same time, in pH 4.0–7.5, a wide physiological
pH range, the MT-PVIM was weakly fluorescent (see Fig. S1 in the
ESI†). This result showed that the probe MT-PVIM was not affect by
pH.
Fig. 3. (a) Fluorescence images of MT-PVIM (3 μM, 3 min) collected
between 490 and 604 nm upon excitation at 488 nm; (b) Confocal
fluorescence images of SiHa cells stained with MTR (250 nM, 10
min) collected between 569 and 691 nm upon excitation at 541 nm.
(c) Merged image of a and b. (d) Intensity profile of linear region of
interest (white arrow) across the SiHa cells costained with MTR and
MT-PVIM; (e) Correlation plot of MTR and MT-PVIM intensities.
Scale bar = 20 μm.
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Rapid monitoring and tracking trans membrane and imaging Tracking mitochondrial fission and fusion dynamics
Journal Name
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DOI: 10.1039/C5NJ02821C
mitochondria
We have proved the probe MT-PVIM could rapid monitor and track
We investigated whether the MT-PVIM probe can rapidly image trans membrane and image mitochondria. Next, we will track
and track mitochondrial morphological changes. First, we studied mitochondrial fission and fusion dynamics within 620-899 s. As
the process that the dye enters into the cells and illuminates the shown in Fig. 5a, mitochondria appear to exist as a dynamic
mitochondria. The MT-PVIM dye was added to a 35 mm Petri dish reticulum of long, thin, branched tubules that spread throughout
with an observation window containing 1mL culture medium the cytoplasm of the cell (Fig. 5d). In addition to high mobility and
consisting of SiHa cells and was imaged over time (initially, and then frequent shape changes, the fusion (arrows) and fission
at 5 min intervals up to 15 min) using Zeiss laser scanning confocal (arrowheads) of mitochondria can be detected (See video S2 in the
microscope LSM780. As shown in Fig. 4a, during 30 scans with a ESI†). Changes of mitochondrial morphology are associated with a
total irradiation time of ∼899 s, the fluorescence emission signal of distinct increase in mitochondrial number, suggesting the
MT-PVIM at 488 nm excitation first increased, then leveled off and participation of mitochondrial fission machinery. The number and
finally gradually decreased in the cell membrane (See video. S1 in morphology of mitochondria within a cell are controlled at precisely
the ESI†). MT-PVIM clustered into the cell membrane in as little as regulated rates of organelle fusion and fission. Under different
93 s. However, the intracellular fluorescence emission signals growth conditions, fission or fusion rates may change and lead to an
continue to increase until reached the saturation point under 488 increase or decrease in mitochondrial number.
nm continued laser excitation. The results showed that the probe
Until recently, little was known about the molecular mechanisms
possessed superior permeability to living cells and high sensitivity to regulating mitochondrial number and morphology. However,
intracellular mitochondria (Fig. 4b).
combined genetic, biochemical and microscopic approaches have
revealed the existence of a number of proteins that participate in
mitochondrial fusion and fission.34 The probe MT-PVIM enable us to
have a first look at the molecular events governing the regulation of
mitochondrial morphology in cells. By contrast, we cannot visualize
the process how MTR enters cells or visualize the mitochondrial
dynamic behavior in cultured mammalian cells. Because the
fluorescence signals of MTR almost disappear after the second scan
(Fig. 6).
The working concentration of MT-PVIM and MTR was 3 μM and
250 nM in our experiments, respectively. At first glance, one may
think that MTR is more sensitive than MT-PVIM. In a sense, MTR
can easily be photobleached by strong excitation fluorescence. At
high concentrations, the probe MTR will lose the specificity and
stain other cellular structures. 35
Fig. 5. Imaging of SiHa cells stained with MT-PVIM (a) fluorescent
picture (b) DIC picture (c) merged picture. (d) Dynamic network-like
behavior of mitochondria in cultured mammalian cells (red squares).
In addition to high mobility and frequent shape changes, fusion
(775 s) and fission (arrowheads 806 s) of mitochondria can be
detected. Irradiation time: 30.98 s/scan. Scale bar = 10 μm.
Fig. 4. (a) SiHa cells were stained with the probe MT-PVIM and
imaged over time (starting from time 0, then at 5, 10 and 15 min, as
shown in video S1) using confocal microscopy. (b) Intensity change
of cell membrane (white arrow) and mitochondria at times from 0-
899 s (red squares). Images and fluorescence spectra were acquired
using 488 nm, excitation and emission windows of 490-604 nm;
irradiation time: 30.98 s/scan. Scale bar = 10 μm.
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to CCCP, the mitochondria exhibited small and dispersed
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fragmentation and different morphologicaDl OcIh: a10n.g10e3s9/oCv5eNrJ0a28lo2n1Cg
period of time. That is to say, before and after the cells were
treated, MT-PVIM possessed outstanding photostability and
tolerance of micro-environmental changes (Fig. 7c), thus
representing a potential candidate as a tracking agent for apoptosis
studies.
Fig. 6. SiHa cells were stained with the mitochondria fluorescent
probe MTR and imaged over time (at time 0, followed by 279 s)
using confocal microscopy. Images were acquired using 561 nm,
excitation and emission windows of 569 and 691 nm; irradiation
time: 30.98 s/scan. Scale bar = 20 μm.
Fig. 7. Fluorescence images of living SiHa cells stained with MT-
PVIM (3 μM) before (a) and after (b) treatment with CCCP (20 μM)
as increasing scan time. (c) Fluorescent intensity changes of living
SiHa cells stained with MT-PVIM (3 μM) without (a) and with (b)
CCCP (20 μM) treatment as increasing scan time (the scan time
shown in the upper left corner of the panel). Excitation wavelength:
488 nm; emission filter: 499−560 nm; irradiation time: 30.98 s/scan.
Scale bar = 20 μm.
Photostability and tolerance
Photostability is one of the most important criteria for developing
fluorescence imaging agents.36, 37 Continuous scanning by confocal
microscope (Zeiss laser scanning confocal microscope LSM780) was
used to quantitatively investigate the photostability of MT-PVIM (2
μM) and MTR (2 μM). Within 300 s, the fluorescence signals of MT-
PVIM decreased slightly (~ 5%); In contrast, MTR exhibited
significant photobleaching with only
~ 16% signal intensity
remaining (see Fig. S2 in the ESI†).
Conclusions
Changes of mitochondrial membrane potential (ΔΨm) value will
cause change about tolerance of probe. This part, we will use
carbonyl cyanide CCCP to treat the cells prior to the staining
procedure. The compound CCCP is an uncoupler that can result in
decrease of ΔΨm value. It mainly caused by rapid acidification of
the mitochondria and dysfunction of ATP synthase in cells.36 Join of
CCCP caused decreasing of intracellular pH.38
We study the influence of ΔΨm by cell imaging. The probe MTR
and MT-PVIM incubated SiHa cells treated by 5 μM CCCP. The
image results showed that MTR had no more specificity to
mitochondria, and the sensitivity worsened (see Fig. S3 in the ESI†).
Compared with MTR, the sensitivity of MT-PVIM to mitochondria is
perfectly retained in the CCCP-treated cells (Fig. 7). Because the
mitochondria imaging is closely related to ΔΨm, and decrease of
ΔΨm will directly affect accumulation of cationic MTR in the
mitochondria. Thus, compared with MTR, MT-PVIM allows a wider
dynamic range for mitochrondrial targeting upon the change of
ΔΨm.
The fluorescence intensity of the stained mitochondria can be
used to estimate the mitochondrial ΔΨm. By monitoring the time-
dependence of the fluorescence of the inner mitochondrial
membrane, one can obtain qualitative information about the
dynamics of the ΔΨm. The high tolerance of MT-PVIM to a decrease
of ΔΨm enables the observation of changes in the mitochondrial
morphology induced by CCCP (video S4 in the ESI†). Due to the
photostability of MT-PVIM, the mitochondrial reticulum-like
behavior was clearly observed (Fig. 7a, video S4 in the ESI†).
However, compared to reticulum-like mitochondria, upon exposure
In summary, rapid acrossing cell membrane and tracking
mitochondrial fission and fusion dynamics processes can give
insight for studying development of the disease and cell
degeneration. In this work, we designed and synthesized a
unique mitochondria-targetable fluorescent probe MT-PVIM
for monitoring and tracking trans membrane process and
mitochondrial fission and fusion dynamics. Compared with
commercially available mitochrondrial probe MTR, the probe
MT-PVIM has excellent specificity to mitochondria and
tolerance of micro-environmental changes. The good
performance of our proposed approach demonstrates that this
strategy might open up new opportunities for the
development of efficient subcellular molecular tools for
bioanalytical and biomedical applications.
Acknowledgements
For financial support, this work was financially supported by
the NSFC (51503077, 21172063, 21472067), Taishan Scholar
Foundation (TS 201511041), NSFSP (ZR2015PE001) and the
start up fund of University of Jinan and doctor start up fund
(160082102) of University of Jinan. In addition, we thank
Shandong University 2011JC006, Open Project of State Key
Laboratory for Supramolecular Structure and Materials
(SKLSSM201207)
，and the NSFC (51303097).
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Am. Chem. Soc., 2015, 137, 3017.
32. D. Kim, G. Kim, S. –J. Nam, J. Yin and J. Yoon, Sci. Rep., 2015, DOI:
10.1038/srep08488.
33. Y. –Q. Sun, J. Liu, H. Zhang, Y. Huo, X. Lv, Y. Shi and W. Guo, J.
Am. Chem. Soc., 2014, 136, 12520.
34. R. E. Jensen, A. E. Aiken Hobbs K. L. Cerveny and H. Sesaki.,
Microsc. Res. Tech., 2000, 51, 573.
35. I. Johnson, D. Ph, M. T. Z. Spence, C. Walker, G. Buller, M. T.
Ascp and J. Croissant, The Molecular Probes Handbook editors
scientific support Graphics Production and layout, 11th ed.; Life
Technologies Corporation: Carlsbad, CA, 2010.
36. Y. Song, M. Yang, S. V. Wegner, J. Zhao, R. Zhu, Y. Wu, C. He and
P. R. Chen, ACS. Chem. Biol., 2015, 10, 1610.
37. Y. Wu, C. J. Yang, L. L. Moroz and W. Tan, Anal. Chem., 2008, 80,
3025.
38. A. O. de Graaf, L. P. van den Heuvel, H. B. P. M. Dijkman, R. de
Abreu, K. U. Birkenkamp, T. de Witte, B. van der Reijden, J. M.
Smeitink and J. H. Jansen, Exp. Cell Res., 2004, 299, 533.
Notes and references
1. G. Wolken, V. Kostal and E. A. Arriaga, Anal. Chem., 2011, 83, 612.
2. K. L.Stauch, P. R. Purnell and H. S. Fox., J. Proteome Res., 2014, 13,
2620.
3. S. Komatsu, A. Yamamoto, T. Nakamura, M. –Z. Nouri, Y. Nanjo, K.
Nishizawa and K. Furukawa, J. Proteome Res., 2011, 10, 3993.
4. P. M. Arce, O. Khdour, M. R. Goldschmidt, J. S. Armstrong and S.
M. Hecht, ACS. Med. Chem. Lett., 2011, 2, 608.
5. Y. Wang, C. –M. Che, J. –F. Chiu, and Q. –Y. He, 2007, J. Proteome
Res., 6, 4703.
6. B. Glancy and R. S. Balaban, Biochem., 2012, 51, 2959.
7. F. Noritaka., T. Matsuda and T. Nagai, ACS. Chem. Biol., 2014, 9,
1197.
View Article Online
DOI: 10.1039/C5NJ02821C
8. D. R. Green and G. Kroemer, Science, 2004, 305, 626.
9. S. R. Jean, M. P. Pereira and S. O. Kelley, Mol. Pharmaceutics.,
2014, 11, 2675.
10. C. C. Wong, Y. Wang, K. –W. Cheng, J. –F. Chiu, Q. –Y. He and F.
Chen, J. Proteome. Res., 2008, 7, 2050.
11. M. Zbidah, A. Lupescu, K. Jilani, A. Fajol, D. Michael, S. M. Qadri
and F. Lang, J. Agric. Food. Chem., 2012, 60, 533.
12. J. Bereiter-Hahn and M. Voth, Microsc. Res. Tech., 1994, 27, 198.
13. L. Stiles, P. Fellow and O. S. Shirihai, Best. Pract. Res. Cl. En.,
2012, 26, 725.
14. A. R. Sarkar, C. H. Heo, H. W. Lee, K. H. Park, Y. H. Suh and H. M.
Kim, Anal. Chem., 2014, 86, 5638.
15. J. Nunnari, W. F. Marshall, A. Straight, A. Murray, J. W. Sedat
and P. Walter, Mol. Cell. Biol., 1997, 8, 1233.
16. S. Jakobs, Biochim. Biophys. Acta., 2006, 1763, 561.
17. F. Miao, W. Zhang, Y. Sun, Y. Liu, F. Guo, W. Zhang, M. Tian and
X. Yu, Biosens. Bioelectron., 2014, 55, 423.
18. C. Song, W. Yang, N. Zhou, R. Qian, Y. Zhang, K. Lou, R. Wang,
and W. Wang, Chem. Commun., 2015, 51, 4443.
19. X. Zhang, T. Dong, Q. Li, X. Liu, L. Li, S. Chen and X. Lei, ACS.
Chem. Biol., 2015, 10, 1676.
20. C. Liu, W. Chen, W. Shi, B. Peng, Y. Zhao, H. Ma and M. Xian, J.
Am. Chem. Soc., 2014, 136, 7257.
21. L. Yang, N. Li, W. Pan, Z. Yu and B. Tang, Anal. Chem., 2015, 87,
3678.
22. C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam and B. Z
Tang, J. Am. Chem. Soc., 2013, 135, 62.
23. S. Huang, R. Han, Q. Zhuang, L. Du, H. Jia, Y. Liu and Y. Liu,
Biosens. Bioelectron., 2015, 71, 313.
24. M. Karbowski and R. J. Youle, Cell. Death. Differ., 2003, 10, 870.
25. L. V. Johnson, M. Walsh and L. B. Chen, PNAS, 1980, 77, 990.
26. R. Rizzuto, A. W. Simpson, M. Brini and T. Pozzan, Nature, 1992,
358, 325.
27. S. P. Perry, J. P. Norman, J. Barbieri, E. B. Brown and H. A.
Gelbard, Biotechniques, 2011, 50, 98.
28. J. A. Mondal, S. Nihonyanagi, S. Yamaguchi and T. Tahara, J. Am.
Chem. Soc., 2010, 132, 10656.
29. L. Yuan, W. Lin, Y. Yang and H. Chen, J. Am. Chem. Soc., 2012,
134, 1200.
30. Y. Liu, K. Li, M. –Y. Wu, Y. –H. Liu, Y. –M. Xie and X. –Q. Yu, Chem.
Commun., 2015, 51, 10236.
31. H. S. Jung, J. Han, J. –H. Lee, J. H. Lee, J. –M. Choi, H. –S. Kweon,
J. H. Han, J. –H. Kim, K. M. Byun, J. H. Jung, C. Kang and J. S. Kim, J.
6 | J. Name., 2012, 00, 1-3
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New Journal of Chemistry
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DOI: 10.1039/C5NJ02821C
The probe MT-PVIM was capable of monitoring and tracking trans membrane process and
mitochondrial fission and fusion dynamics.