Tracking mitochondrial viscosity in living systems based on a two-photon and near red probe

Article abstract of DOI:10.1039/c9nj03744f

A two-photon fluorescent probe, Mito-V, was designed and prepared for sensing the viscosity changes of mitochondria. The systematic investigations demonstrated that the fluorescence emission at 634 nm increased about 22-fold from methanol to 99% glycerol, indicating high viscosity dependence. Notably, using the probe, the mitochondrial viscosity fluctuations have been successfully monitored not only in living cells but also in zebrafish and living mice.

Full text of DOI:10.1039/c9nj03744f

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Tracking mitochondrial viscosity in living systems
based on a two-photon and near red probe†
Cite this: New J. Chem., 2019,
43, 16945
Min Peng, Junling Yin
and Weiying Lin
*
A two-photon fluorescent probe, Mito-V, was designed and prepared for sensing the viscosity changes
of mitochondria. The systematic investigations demonstrated that the fluorescence emission at 634 nm
increased about 22-fold from methanol to 99% glycerol, indicating high viscosity dependence. Notably,
using the probe, the mitochondrial viscosity fluctuations have been successfully monitored not only in
living cells but also in zebrafish and living mice.
Received 18th July 2019,
Accepted 16th September 2019
DOI: 10.1039/c9nj03744f
rsc.li/njc
Viscosity, as one of the crucial parameters of cells, directly with the merits of deeper penetration, higher spatial resolution as
19–22
affects the interaction between the transmission of chemical well as longer excitation wavelength and lower phototoxicity.
signals and intracellular biomolecules as well as facilitating the Thus, it is of great urgency to design a TP fluorescent probe for
1,2
diffusion of metabolites. Mitochondria, as a vital intracellular detecting viscosity variations in the mitochondrial region.
organelle, are present in the cytoplasm of almost all eukaryotic Fluorescent probes typically contain a molecular rotor and a
cells, and they play an important role in maintaining the balance fluorophore. For such a fluorescent probe, molecular rotors can
3
–5
of numerous physiological functions in human physiology.
be rotated relative to the fluorophore in non-viscous media
Additionally, mitochondria are known as the cellular energy providing relaxation of an electronically excited dye by a non-
2
2–27
factory due to the fact that their principal function is energy radiative process.
However, the fluorescence was enhanced
production. Deviation from normal mitochondrial viscosity levels in high viscous-media, which was ascribed to the fact that the
will result in inactivation of mitochondria, which will trigger rotation of the molecular rotor was inhibited and the likelihood
2
8,29
various diseases including diabetes, atherosclerosis, Parkinson’s of non-radiative pathways was reduced.
Furthermore,
6–13
disease (PD) and Alzheimer’s disease (AD). To this end, detection of changes in viscosity of a particular organelle can
developing a targeted molecule capable of real-time monitoring also be achieved by introducing a targeting group.
3
0,31
Inspired
the mitochondrial viscosity is crucial to better understand the by this, we aimed to design a mitochondrial targeting fluorescent
mechanism of mitochondria during pathological and biological probe that possesses the fluorophore and the rotor with the aim
processes.
of tracking and sensing mitochondrial viscosity changes in
Recently, various viscometers have been developed to detect living systems.
viscosity. Unfortunately, these traditional methods fail to mea-
Herein, we have rationally designed and synthesized a near
1
4–16
sure the viscosity at the cellular level.
Compared with red probe, Mito-V (Scheme 1), for tracking mitochondrial viscosity
traditional techniques, fluorescence technology has become a
powerful tool for detecting viscosity in living systems owing to
its excellent properties including high sensitivity, non-invasive
detection, high selectivity as well as real-time monitoring and
16–18
temporal resolution.
In particular, two-photon (TP) fluorescent
probes have been developed as robust tools for sensing viscosity
variations in biological systems. Compared with one-photon (OP)
fluorescent probes, TP fluorescent probes have received more favor
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of
Jinan, Jinan, Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
†
Electronic supplementary information (ESI) available: Characterization data,
and additional spectra. See DOI: 10.1039/c9nj03744f
Scheme 1 The design concept of Mito-V for monitoring viscosity.
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with two-photon excitation using hemicyanine and coumarin as an cross sections of Mito-V were obtained at 730 nm with 63 GM
acceptor and a donor, respectively. Coumarin, as an excellent (Fig. S2, ESI†) indicating the two-photon nature of the probe
donor, is widely used in the synthesis of dyes and two-photon Mito-V. As anticipated, the probe Mito-V also exhibits an obvious
fluorescent probes. Furthermore, hemicyanine, as a mitochondrial viscosity dependence in TP mode (Fig. S3, ESI†). Additionally,
targeting group, possesses a positive charge that facilitates considering that the cytoplasm is a complex heterogeneous fluid,
penetration into the mitochondria of cells through the plasma polarity would be a potential factor affecting probe behaviour. To
membrane. We envision that Mito-V would emit no fluorescence confirm whether Mito-V is affected by the polarity of the solvent,
in non-viscous media owing to free rotation of the coumarin the fluorescence spectra of Mito-V in different polarity solvents
moiety and hemicyanine moiety. However, the rotation would were measured. As shown in Fig. 1d, Mito-V exhibited almost no
be restricted and the fluorescence intensity would become fluorescence in different polar solvents compared to strong
dramatically enhanced in highly viscous media. Therefore, fluorescence in glycerol, indicating that the probe Mito-V was
Mito-V may be suitable for monitoring mitochondrial viscosity. almost completely unaffected by polarity. These results demon-
With the successful preparation of the probe, the optical strate that Mito-V may be applied for monitoring viscosity
properties of Mito-V in a mixture of glycerol–methanol were changes in complex biological environments.
investigated. Firstly, we measured the UV-vis absorption spectrum
It is important to evaluate probe selectivity because the cell
of Mito-V in glycerol and methanol solutions, respectively (Fig. S1, is a complex multicomponent system, which might generate
ESI†); Mito-V exhibited slightly heightened fluorescence from significant effects on cellular viscosity. Based on this, the fluores-
methanol to glycerol solution. As expected, Mito-V exhibited cence spectra of Mito-V were recorded in the presence of various
negligible fluorescence in methanol (Fig. 1a). However, the fluores- possible competitive biomolecules. As displayed in Fig. 2a, active
cence emission intensity at lex = 634 nm of Mito-V exhibited a nitrogen, anions, cations, active oxygen and amino acids triggered
remarkable increase by ca. 22-fold upon viscosity changing from almost no fluorescence fluctuation, indicating the high selectivity
1.7 cP to 953.0 cP. In addition, a good linear relationship exists of Mito-V. Moreover, other spectral experiments have indicated
between log I634 and log Z with a correlation coefficient of 0.99 that Mito-V exhibits excellent photostability (Fig. S4, ESI†) and
according to the F o¨ rster–Hoffmann equation (Fig. 1b), indicating negligible fluorescence responses to pH changes (Fig. S5, ESI†).
that Mito-V is able to detect viscosity. Moreover, the fluorescence Prior to this, the cytotoxicity of Mito-V toward HeLa cells was
quantum yield of Mito-V was calculated in different methanol/ evaluated by the MTT standard assay. As shown in Fig. 2b, the
glycerol fractions and the result is depicted in Fig. 1c and Table S1 survival rate of HeLa cells was more than 85%, indicating that
(
ESI†). A quantum yield of Mito-V of 0.0372 was obtained in Mito-V has low toxicity to HeLa cells. These results suggest that
methanol, an approximately 12-fold enhancement of intensity on Mito-V would be suitable for imaging living cells.
going from methanol to glycerol. Moreover, the two-photon action Considering the overall positive charge of Mito-V, the
spectra of Mito-V were recorded. The maximum TP absorption co-localization experiments of Mito-V were performed with
Mito-Tracker Green (MTG) by confocal laser microscopy. As
shown in Fig. 3, the imaging results indicate that Mito-V could
easily enter HeLa cells and a clear fluorescence image was obtained
in the TRITC channel (570–620 nm) (Fig. 3a). Simultaneously, MTG
imaging was collected in the FITC channel (500–550 nm) (Fig. 3b).
The merged image indicated that the red TRITC channel of Mito-V
and the green FITC channel of MTG overlapped pretty well (Fig. 3c).
Moreover, the Pearson correlation coefficients were calculated to be
as high as 0.92 (Fig. 3d) and the intensity profile variations in the
Fig. 1 (a) The fluorescence emission spectra of 10 mM Mito-V in the
varied viscosity media of methanol/glycerol (v/v) mixtures. (b) Linear
Fig. 2 (a). Fluorescence intensity of Mito-V (10 mM) at lem = 634 nm in
2
À
À
À
2À
À
relationship of log I634 and log Z, R = 0.99. (c) Fluorescence quantum
PBS buffer (pH = 7.4, 0.01 mM) 1: Ac ; 2: Br ; 3: Cl ; 4: CO
3
; 5: F ;
3À
À
2À
2À
2À
3+
3+
2+
yield (F
f
) of Mito-V in the methanol/glycerol (v/v) mixture. (d) Fluorescence
6: HCO ; 7: HPO
4
; 8: SO
3
; 9:SO
2+
4
; 10: NO ; 11: Al ; 12: Ba
;
;
2+
2+
2+
2+
2+
2+
emission of Mito-V in various solvents with different polarity. The spectra
were recorded at 25 1C, the fluorescence intensity was measured at
13: Ca ; 14: Co ; 15: Cu ; 16: Sn ; 17: Fe ; 18: Fe ; 19: Ni ; 20: Mg
21: Ala; 22: Arg; 23: Ser; 24: D-alanine; 25: GSH; 26: L-glutamic; 27: Gln;
28: Asp; 29: Val; 30: Thr; 31: Gly; 32: Ile; 33: Leu; 33: Hcy; 34: Glycerol;
(b). The cytotoxicity of Mito-V in HeLa cells.
l
7
ex = 561 nm with both excitation and emission slit widths of 5 nm, and
00 V PMT voltage.
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30 min, an obvious strong red fluorescence was observed in the
TRITC channel (Fig. 4A(c1–c3) and (d1–d3)). Besides, cuvette
experiments were carried out with the addition of nystatin or
monensin in glycerol or methanol, and the fluorescence exhibited
negligible perturbation (Fig. S6, ESI†), indicating that the cellular
fluorescence intensity increase can be attributed to viscosity
variations in mitochondria rather than the external stimulus. In
addition, the analysis results of the cells stimulated by exogen-
ous sources displayed that the average fluorescence intensity
was 4.3-fold higher than the normal values in the OP (Fig. 4B) or
TP mode (Fig. 4C). More importantly, the quality of the fluores-
cence image in TP mode is obviously superior to that in OP
mode (Fig. S7, ESI†). More importantly, we have performed the
real-time fluorescence imaging of dynamic changes in mitochondrial
viscosity at different time intervals, indicating that the fluorescence
intensity gradually increases with time and remains stable (Fig. S9,
Fig. 3 Co-localization fluorescence images of the probe Mito-V in HeLa
cells. (a) Mito-V (10 mM) stain, lex = 561 nm, collected at 570–620 nm.
(
(
b) Mitochondrial green stain, lex = 488 nm, collected at 500–550 nm.
c) The merged image of (a) and (b). (d) Intensity scatter plots of the probe ESI†). These phenomena suggest that Mito-V can sensitively monitor
Mito-V across the cells in the red and green channels. (e) Intensity profile viscosity of mitochondria in living cells.
of the two channels. Scale bar: 25 mm.
Furthermore, based on the above research, we evaluated the
feasibility of Mito-V for sensing the viscosity changes in zebrafish.
The zebrafish were initially non-fluorescent (Fig. 5A(a1–a4)) and
weak red fluorescence was observed after treating with Mito-V
two channels show a synchronous trend (Fig. 3e). As expected,
the experimental results indicate that Mito-V possesses favour-
able membrane permeability and specifically accumulates in
mitochondria.
(Fig. 5A(b1–b4)). However, in the presence of the external
stimulus (monensin and nystatin), zebrafish emitted stronger
red fluorescence (Fig. 5A(c1–c4) and (d1–d4)). Moreover, the
analysis results of the zebrafish demonstrated that the mean
fluorescence intensity triggered by the external stimulus was
Subsequently, the capacity of the probe Mito-V for the real-
time monitoring of the dynamic changes in mitochondrial
viscosity under an external stimulus was validated. External
stimuli (monensin and nystatin) were utilized to stimulate
3.1-fold higher than the normal ones in the OP (Fig. 5B) or TP
3
2,33 channel (Fig. 5C). Similarly, the quality of the fluorescence
image in TP mode is obviously superior to that in OP mode
a viscosity increase by inducing mitochondrial disorders.
The cells stained without Mito-V exhibited no fluorescence
(Fig. S8, ESI†). The above phenomena corresponded well with
(Fig. 4A(a1–a3)). When the HeLa cells were stained only with
the results observed in living cell imaging experiments, which
further demonstrated that the probe Mito-V could be applied for
making a distinction between viscous zebrafish and normal
zebra-fish by fluorescence imaging of mitochondrial viscosity.
Mito-V, the probe emitted weak red fluorescence (Fig. 4A(b1–b3)).
Excitingly, when the cells were pre-incubated with nystatin or
monensin for 30 min and then stained with Mito-V for another
Fig. 4 (A) (a1)–(a3) Confocal fluorescence images of the cells incubated
without Mito-V. (b1)–(b3) Confocal fluorescence images of the cells
incubated with 10 mM Mito-V. (c1)–(c3) Confocal fluorescence images of
the cells incubated with 10 mM Monensin for 30 min + 10 mM Mito-V for
another 30 min. (d1)–(d3) Confocal fluorescence images of the cells
incubated with 10 mM Nnystatin for 30 min + 10 mM Mito-V for another
Fig. 5 (a1)–(a4) Confocal fluorescence images of the zebrafish incubated
without Mito-V. (b1)–(b4) Confocal fluorescence images of the zebrafish
incubated with 10 mM Mito-V. (c1)–(c4) Confocal fluorescence images of
the zebrafish incubated with 10 mM monensin for 30 min + 10 mM Mito-V
for another 30 min. (d1)–(d4) Confocal fluorescence images of the zebra-
fish incubated with 10 mM nystatin for 30 min + 10 mM Mito-V for another
30 min. OP: lex = 561 nm; TP: lex = 730 nm; scale bar =250 mm.
3
0 min. (B) Normalized fluorescence intensity of HeLa cells in OP mode.
C) Normalized fluorescence intensity of HeLa cells in TP Mode. lem = 570–
20 nm; lex = 561 nm in OP mode and 730 nm in TP mode. Scale bar: 25 mm.
(
6
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Acknowledgements
This work was financially supported by NSFC (21472067,
1672083, 21877048), Taishan Scholar Foundation (TS201511041),
2
and the startup fund of University of Jinan (309-10004). The animals
were purchased from School of Pharmaceutical Sciences, Shandong
University, and the studies were approved by the Animal Ethical
Experimentation Committee of Shandong University. All animals
were kindly kept during experiment according to the requirements
of the National Act on the use of experimental animals (China).
Notes and references
1
T. Aron, K. M. Ramos-Tottes, J. A. Cotruvo Jr. and C. J.
Chang, Acc. Chem. Res., 2015, 48, 2434–2442.
2
P. Ning, J. C. Jiang, L. C. Li, S. X. Wang, H. Z. Yu, Y. Feng,
M. Z. Zhu, B. C. Zhang, H. Yin, Q. X. Guo and X. M. Meng,
Biosens. Bioelectron., 2016, 77, 921–927.
Fig. 6 (A1) Normal living mice + probe Mito-V (left) and mice triggered
with LPS + probe Mito-V (right); (A2) normal living mice + probe Mito-V
3
Z. G. Yang, Y. X. He, J. H. Lee, N. Y. Park, M. Suh, W. S. Chae,
J. F. Cao, X. J. Peng, H. Jung, C. Kang and J. S. Kim, J. Am.
Chem. Soc., 2013, 135, 9181–9185.
(left) and mice triggered with monensin + probe Mito-V (right); B1 refers to
the normalized fluorescence intensity of mice in A1 and B2 refers to the
normalized fluorescence intensity of mice in A2. Error bars represent
standard deviation (ÆS.D.), n = 3, the statistical analysis was performed
on three separate biological replicates. lex = 520 nm, lem = 670 nm.
4 P. Ning, P. Y. Dong, Q. Geng, L. Bai, Y. Q. Ding, X. H. Tian,
R. Shao, L. Li and X. M. Meng, J. Mater. Chem. B, 2017,
5, 2743.
5
6
7
8
A. Jim ´e nez-S ´a nchez, E. K. Lei and S. O. Kelley, Angew. Chem.,
Int. Ed., 2018, 57, 8891–8895.
Encouraged by the above results, tracking viscosity in mice was
also performed. As reported in the previous literature, inflammation
can stimulate an increase in the viscosity of a biological system with
the trigger of Lipopolysaccharide (LPS). To further study the
feasibility of Mito-V to monitor the viscosity changes in vivo, LPS
was injected into nude mice to induce inflammation The normal
mice and inflamed mice stimulated with LPS or monensin emitted
inconspicuous fluorescence (Fig. S7(a1 and a2), ESI†). However,
after injection of Mito-V, the inflamed mice incubated with LPS
exhibited obvious stronger fluorescence than the normal mice
N. Gupta, S. I. Reja, V. Bhalla, M. Gupta, G. Kaur and
M. Kumar, J. Mater. Chem. B, 2016, 11, 1968–1977.
J. P. Li, Y. Y. Zhang, H. Zhang, X. P. Xuan, M. S. Xie, S. Xia,
G. R. Qu and H. M. Guo, Anal. Chem., 2016, 88, 5554–5560.
S. J. Chen, Y. N. Hong, Y. Zeng, Q. Q. Sun, Y. Liu, E. G. Zhao,
G. X. Bai, J. N. Hao and Z. Tang, Chem. – Eur. J., 2015, 21,
34
4315–4320.
9
O. Morawski, B. Kozankiewicz, A. Miniewica and A. L. Sololewski,
Chem. Phys. Chem., 2015, 16, 3500–3510.
0 Roopa, N. Kumar, V. Bhalla and M. Kumar, Chem. Commun.,
(Fig. 6(A1)). In addition, monensin was also utilized to induce
1
1
1
1
increased viscosity in the nude mice, and the results corre-
sponded well with LPS observations (Fig. 6A2). Furthermore, the
analysis results of the inflamed mice demonstrated that
the fluorescence intensity was about 2.1-fold higher than for
the normal mice (Fig. 6(B1 and B2)). These results collectively
demonstrate that Mito-V could be applied for monitoring viscosity
in vivo.
In summary, a two-photon and mitochondria-targeting
fluorescent probe, Mito-V, was designed for sensing the viscosity
changes in cells and in vivo. The probe exhibited a 22-fold
enhancement with the increase of viscosity from 1.4 cP to
2015, 51, 15614–15628.
1 L. H. Feng, Y. Wang, F. Liang, X. J. Wang and L. W. Zhang,
Sens. Actuators, B, 2011, 156, 499–503.
2 H. L. Liu, Q. Peng, Y. D. Wu, D. Chen, X. L. Hou, M. Sabat
and L. Pu, Angew. Chem., Int. Ed., 2010, 49, 602–606.
3 Y. Ooyama, S. Inoue, T. Nagano, K. Kushimoto, J. Ohshita,
I. Imae, K. Komaguchi and Y. Harima, Angew. Chem., Int.
Ed., 2011, 50, 7429–7433.
1
4 X. B. Song, N. Li, C. Wang and Y. Xiao, J. Mater. Chem. B,
2017, 5, 360–368.
1
5 S. C. Lee, J. Y. Heo, J. W. Ryu, C. L. Lee, S. Kim, J. S. Tae,
B. O. Rhee, S. W. Kim and O. P. Kwon, Chem. Commun.,
956 cP. More interestingly, with the powerful sensor Mito-V,
the detection of mitochondrial viscosity has been successfully
achieved not only in living cells but also in vivo. Thus, Mito-V is
a promising tool for sensing viscosity in complex biological
systems.
2016, 52, 13695–13698.
1
6 D. D. Li, X. H. Tian, A. D. Wang, L. J. Guan, J. Zheng, F. Li,
S. L. Li, H. P. Zhou, J. Y. Wu and Y. P. Tian, Chem. Sci., 2016,
7, 2257–2263.
1
1
7 L. Yuan and W. Lin, Biomaterials, 2013, 34, 9566–9571.
8 H. Chen, Y. Tang and W. Lin, TrAC, Trends Anal. Chem.,
Conflicts of interest
2016, 16, 1397–1406.
There are no conflicts to declare.
19 M. Xue and K. L. Wang, Senors, 2012, 12, 11612–11637.
16948 | New J. Chem., 2019, 43, 16945--16949 This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
20 M. K. Kuimova, S. W. Botchway, A. W. Parker, M. Balza, 27 M. G. Ren, K. Zhou, L. Wang, K. Y. Liu and W. Y. Lin, Sens.
H. A. Collins, H. L. Anderson, K. Suhling and P. R. Ogilby,
Nat. Chem., 2009, 1, 69–73.
1 M. Ren, B. Deng, K. Zhou, X. Kong, J. Wang, G. Xu and
W. Lin, J. Mater. Chem., 2016, 4, 4739–4745.
2 M. Ren, B. Deng, K. Zhou, X. Kong, J. Wang and W. Lin,
Anal. Chem., 2017, 89, 552–555.
3 X. J. Peng, Z. G. Yang, J. Y. Wang, J. L. Fan, Y. X. He,
Actuators, B, 2018, 262, 452–459.
28 N. Jiang, J. L. Fan, S. Zhang, T. Wu, J. Y. Wang, P. Gao, J. L. Qu,
F. Zhou and X. J. Peng, Sens. Actuators, B, 2014, 190, 685–693.
29 W. Sun, Y. D. Shi, A. X. Ding, Z. L. Tan, H. Chen, R. Liu,
R. B. Wang and Z. L. Lu, Sens. Actuators, B, 2018, 276, 238–246.
30 F. Liu, Y. S. Luo and M. Y. Xu, Tetrahedron Lett., 2018, 59,
4540–4544.
2
2
2
F. L. Song, B. S. Wang, S. G. Sun, J. L. Qu, J. Qi and M. Yan, 31 L. Wang, Y. Xiao, W. M. Tian and L. Z. Deng, J. Am. Chem.
J. Am. Chem. Soc., 2011, 133, 6626–6635. Soc., 2013, 135, 2903–2906.
4 M. A. Haidekker, T. P. Brady, D. Lichlyter and E. A. 32 S. P. Soltoff and L. J. Mandel, J. Membr. Biol., 1986, 94, 153–161.
2
2
2
Theodorakis, J. Am. Chem. Soc., 2005, 128, 398–399.
5 G. S. Kottas, L. I. Clarke, D. Horinek and J. Michl, Chem.
Rev., 2005, 105, 1281–1376.
33 J. G. Molina, X. L. Zhang, M. Borghesan, J. M. D. Silva,
M. Awan, B. Fuller, N. Gavara and C. Selden, Biomaterials,
2018, 177, 113–124.
6 Y. Baek, S. J. Park, X. Zhou, G. Kim, H. M. Kim and J. Yoon, 34 E. Yeom, H. M. Kim, J. H. Park, W. Choi, J. Doh and S. J. Lee,
Biosens. Bioelectron., 2016, 86, 885–891. Sci. Rep., 2017, 7, 1801.
This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem., 2019, 43, 16945--16949 | 16949