A Mitochondria-Specific Orange/Near-Infrared-Emissive Fluorescent Probe for Dual-Imaging of Viscosity and H2O2 in Inflammation and Tumor Models

Article abstract of DOI:10.1002/cjoc.202000725

Elucidating the intrinsic relationship between viscosity/H₂O₂ and mitochondria-associated diseases remains a great challenge owing to the lack of research on multiple diseases models, such as inflammation and malignant tumor models.

Full text of DOI:10.1002/cjoc.202000725

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For published articles: https://onlinelibrary.wiley.com/journal/16147065
中
国 化 学
Cite this paper: Chin. J. Chem. 2021, 39, 1303—1309. DOI: 10.1002/cjoc.202000725
A Mitochondria-Specific Orange/Near-Infrared-Emissive
Fluorescent Probe for Dual-Imaging of Viscosity and
H2O2 in Inflammation and Tumor Models
Li Fan,*,a Qi Zan,a Xiaodong Wang,a Shuohang Wang,b Yuewei Zhang,*,b Wenjuan Dong,a Shaomin Shuang,a and
Chuan Dong*,a
a Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, Shanxi 030006, China
b School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin, , Jilin 132022, China
Keywords
Viscosity | H2O2 | Mitochondria-specific | Fluorescent probes | Fluorescence spectroscopy
Main observation and conclusion
Elucidating the intrinsic relationship between viscosity/H2O2 and mitochondria-associated diseases remains a great challenge owing
to the lack of research on multiple diseases models, such as inflammation and malignant tumor models. In this work, we have devel-
oped a mitochondria-specific orange/near-infrared-emissive fluorescent probe TTPB, for dual-imaging of viscosity and H2O2 levels in
two different channels. The probe exhibited a remarkable response to viscosity with NIR emission round 666 nm, and was highly sen-
sitive to H2O2 in orange channel with emission peak at 586 nm. Moreover, TTPB has good mitochondria-specific ability and permits
individual detecting of viscosity in NIR channels and H2O2 levels in orange channel in living cells. More notably, TTPB was successfully
applied to simultaneously image the viscosity and H2O2 levels in inflammation and cancer models.
Comprehensive Graphic Content
*E-mail: fanli128@sxu.edu.cn; zhangyueweichem@163.com;
dc@sxu.edu.cn
View HTML Article
Supporting Information
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reactive unit in dual-sensing H2O2/viscosity for the first time,
while the reported single probes employed phenylborate ester as
H2O2-sensitive group.[20,28,31] Meanwhile, the triphenylamine
combined with thiophene acted as the viscosity-sensitive unit due
to the more rotations than that in the reported single probes for
dual-sensing H2O2/viscosity,[20,28,31] which was predicted to display
a high sensitive response to viscosity. The detailed synthetic route
for probing TTPB and TTP was shown in Scheme 2, and the struc-
Background and Originality Content
Cellular viscosity, as an essential micro-environmental pa-
rameter, plays a critical role in many diffusion-mediated biological
processes, such as signal transport, electron transport and so on.[1]
Mitochondria, serving as the cellular power workshop, are closely
linked to cellular viability and physiological activity.[2] Abnormal
viscosity changes can cause mutations in mitochondrial network
organization, and subsequently affect metabolite diffusion, which
have been found to associate with inflammation, diabetes, neu-
rodegenerative disease and even cancer.[3]
As a prominent member of reactive oxygen species (ROS), hy-
drogen peroxide (H2O2) is mainly propagated within mitochondria,
and plays regulatory roles in cell growth, immune response, host
defense as well as signaling pathways.[4-5] However, the aberrant
production or accumulation of H2O2 within mitochondria can in-
duce mitochondrial swelling, fracture and fine structure mutation,
thereby leading to mitochondria malfunction and some diseases,
including cancer, inflammatory, obesity as well as neurodegener-
ative diseases.[6-8] To better understand the internal relationship
between the levels of mitochondrial viscosity/H2O2 and mito-
chondria-associated diseases, it is urgently need to exploit a sim-
ple but reliable and precise strategy for dual-detection of mito-
chondrial viscosity and H2O2 changes in vivo.
1
tures were fully characterized by H NMR, 13C NMR and HR-MS in
the Supporting Information. As expected, in a non-viscous or
low-viscous environment, the molecule (TTPB) is in a twisted in-
tramolecular charge transfer (TICT) state due to the free rotations,
resulting in the non-radiative decay of the excited state and the
non/weak fluorescence emission. However, as the high-viscosity
solvent presents, the rotations are restricted and the TICT state is
inhibited, leading to efficient conjugation of the molecule and
further the enhancement of near-infrared (NIR) fluorescence
emission. On the other hand, after the addition of H2O2, the aryl-
boronic acid in TTPB is deprotected to phenol, followed by an
elimination reaction to give TTP, which exhibits a remarked or-
ange fluorescent emission due to the inhibition of the TICT pro-
cess. We then demonstrated in detail the ability of TTPB for im-
aging mitochondrial viscosity and H2O2 changes individually in
living cells. Besides, TTPB was successfully applied to simultane-
ously monitor the variation of mitochondrial viscosity and H2O2 in
inflammation or malignant tumor models.
Fluorescent probes in combination with fluorescence imaging
technology have become the powerful tools for detecting bio-
molecules and biological parameters in live samples, because of
their simple operation, high sensitivity, excellent spatiotemporal
resolution and non-disruptive nature. So far, a lot of outstanding
Scheme 1 The structure and mechanism of TTPB for dual-responding to
mitochondrial viscosity and H2O2
fluorescent probes for single-imaging mitochondrial viscosity or
[9-29]
H2O2 have been reported,
yet they could not simultaneously
detect the two markers. Compared with the combination of sev-
eral fluorescent probes in one system, a single probe with dual-
sensing ability can avoid the spectral cross-talk, different localiza-
tions and metabolism of these probes.[30] In 2017, Lin’s group
designed a single fluorescent probe (Mito-VH) based on styrylpyri-
dine fluorophore, for dual-detection of viscosity and H2O2 in mi-
tochondria with different imaging channels in living cells for the
first time.[20] However, this probe possessed a short emission
wavelength and small Stokes shifts, which might suffer from ex-
cessive autofluorescence in living systems and shallow penetra-
tion depth. Subsequently, Huang et al. synthesized a two-photo
fluorescent probe (Mito-LX) with a dual-response towards chang-
es in the viscosity within near-infrared (NIR) emission and H2O2 in
visible channel in mitochondria, and extended the application into
Parkinson’s disease models.[28] Very recently, Liu and coworkers
reported a single NIR fluorescent probe with a large Stokes shift
for simultaneous imaging of mitochondrial viscosity and H2O2 in
Alzheimer’s disease models.[31] To the best of our knowledge, only
these three dual-imaging fluorescent probes have been devel-
oped for monitoring both mitochondrial viscosity and H2O2, and
the limited diseases models prevent us from delving into the in-
trinsic relationship between viscosity/H2O2 and mitochondria-
associated diseases due to the lack of suitable fluorescent probes
and research on multiple diseases models, such as inflammation
and malignant tumor models.
Scheme 2 The synthetic route of TTPB and TTP
In this contribution, we designed a single orange/near-infra-
red-emissive fluorescent probe (TTPB, Scheme 1) for dual-imaging
of viscosity and H2O2 within mitochondria. TTPB is comprised by a
pyridinium cation (electron acceptor moiety, A), a phenylboronic
acid unit, an ethylene (π-bridge), a thiophene fragment (π-bridge
and electron donor group, D), and a triphenylamine segment (D),
indicating a typical D-π-A molecular configuration, therefore
leading to NIR fluorescence emission. The pyridinium cation was
well-known as a mitochondrial-specific motif owing to the elec-
trostatic interaction with the negative membranes potential in
mitochondria. The phenylboronic acid was chosen as the H2O2
Results and Discussion
Spectroscopic response of TTPB to viscosity
The optical response of TTPB to viscosity was performed by
measuring their absorbance and emission spectra in methanol
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and glycerol, respectively. As shown in Figure S1 and Table S1,
TTPB exhibited a maximum absorption at 488 nm (ε488 = 3.472 ×
104 L·mol–1·cm–1) in pure methanol, while red-shifted to 502 nm
(ε502 = 2.910 × 104 L·mol–1·cm–1) in 99% glycerol. To precisely iden-
tify the viscosity-sensitive behavior of TTPB, the fluorescence
spectra of TTPB in methanol/glycerol mixtures were recorded
(Figure 1a), where the viscosity levels (cP) were tuned by adjust-
ing the glycerol fraction (fG). Upon increasing the viscosity from
0.59 cP (0% glycerol) to 950 cP (99% glycerol), a significant emis-
sion enhancement was observed at 666 nm, suggesting TTPB
could be potentially used as a NIR-emissive viscosimeter for bio-
imaging with minimum autofluorescence and photo damage.
Meanwhile, the fluorescent color changed from light-pink to red
(Inset of Figure 1a). The viscosity-dependent property of TTPB
might be attributed to the fact that the rotations of the molecule
were restricted and the TICT state was inhibited in high viscous
environment (Scheme 1). Interestingly, according to the Förster-
Hoffmann equation, a plot of log I666 vs log η was linear over the
viscosity range from 0.59 to 950 cP (R2 = 0.9909, slope = 0.3725)
(Figure 1b), indicating the high sensitivity of TTPB to micro-vis-
cosity changes. Meanwhile, TTPB had a remarkably large Stokes
shift of 178 nm that could effectively reduce the excitation inter-
ference. Subsequently, the fluorescence quantum yield (QY) of
TTPB in different methanol/glycerol mixtures (glycerol from 0% to
99%) was investigated. As shown in Table S1, the QY value of
TTPB was only 0.55% in pure methanol (0.59 cP), whereas it
reached up to 8.06% in 99% glycerol (950 cP) solution. Additional-
ly, the fluorescence lifetimes of TTPB in different methanol/glyc-
erol mixtures also exhibited a similar variation tendency toward
viscosity (Figure S2), further comfirming the viscosity-sensitive
feature of TTPB.
μmol/L, the fluorescence emission at 586 nm exhibited a dramatic
increase and the fluorescent color of the solutions turned to
bright orange. Notably, there was about 80 nm emission shift
between the emission peak of the response to viscosity and that
response to H2O2, which was beneficial for TTPB to fluorescence
image the viscosity and H2O2 in different channels. Additionally,
an excellent linear relationship between the fluorescence intensi-
ty (I586) and the H2O2 concentrations was established over the
range of 0—30 μmol/L (R2 = 0.9891, Figure 2b), and the detection
limit was calculated as 0.141 μmol/L (3σ/slope method), which is
much lower than physiological H2O2 level of the organization in
inflammation state (10—100 μmol/L).[32]
Figure 2 (a) Fluorescence response of TTPB (5 μmol/L) to various con-
centrations of H2O2 (0-100 μmol/L) in DMSO/PBS (1/1, V/V, pH 8.4), excit-
ed at 405 nm. (b) The linear relationship between the fluorescence inten-
sity at 588 nm and H2O2 concentration in the range of 0—30 μmol/L.
We then demonstrated that TTPB was highly selective for
H2O2 change in complex biological matrixes (Figure S6), and exhib-
ited significant fluorescence enhancement around pH 8.4 within
the alkaline mitochondrial matrix environment (pH ~8.0) (Figure
S7). We further confirmed the response mechanism of TTPB to
H2O2 (proposed in Scheme 1), on the base of high resolution mass
spectrometry (HR-MS) analysis (Figure S8). After reaction of TTPB
with H2O2, the mass peak at m/z = 565.2110 ([TTPB]+, calcd:
565.2116) for free TTPB disappeared, while a new peak at m/z =
431.1587 corresponding to the formation of adduct ([TTPB-H2O2]+)
was observed, which was very similar to TTP ([TTP]+, calcd:
431.1576).
Mitochondria-specific ability of TTPB
Figure 1 (a) Fluorescence spectral changes of TTPB (5 μmol/L) in
different methanol/glycerol mixtures (glycerol from 0% to 99%), excited at
488 nm. Inset: the fluorescent colour of the solution changed from
light-pink to red. (b) The linear relationship between log I and log η in the
range of η = 0.59 cP to η = 950 cP.
To confirm the mitochondria-specific ability of TTPB, a co-lo-
calization experiment between TTPB and MitoTracker Deep Red
(MTDR, a mitochondria-specific dye) was performed. As expected,
TTPB exhibited clear filament morphologies (Figure 3a) and
matched well with MTDR (Figures 3b, 3c), with an average Pear-
son's co-localization coefficient (PC) as high as 0.94 (Figure 3d).
On the contrary, TTPB co-stained with LysoBrite NIR (LB-NIR, a
commercial lysosome-specific dye) displayed poor co-localization
imaging, with a low PC of 0.13 (Figures 3e—3h). These results
suggested that TTPB predominantly localized in the mitochondria.
Moreover, a CCK-8 assay demonstrated the non-toxicity of TTPB
to live cells under the imaging conditions (Figure S9), and the IC50
value was calculated to be 47.44 μmol/L.
Furthermore, we confirmed that TTPB was highly selective
toward viscosity change in the presence of various polarities
solvents, including glycerol, ethanol, water, methanol, DMSO and
acetonitrile (Figure S3), and was insensitive to changes in pH
ranging from 3.0—10.0, in methanol/glycerol with glycerol 0% or
90% (Figure S4), indicating the high selectivity of TTPB toward
viscosity changes.
Spectroscopic response of TTPB to H2O2
Fluorescence imaging of TTPB to viscosity in live cells
To verify whether TTPB was responsive to H2O2 as designed,
the optical properties of probe TTPB and TTP were measured in
the absence and presence of H2O2. TTPB itself displayed a maxi-
mum absorption around 485 nm (ε485 = 2.845 × 104 L·mol–1·cm–1),
while reacting with H2O2, the maximum absorption blue-shifted to
408 nm (ε408 = 2.434 × 104 L·mol–1·cm–1), which was similar to that
of TTP (ε408 = 2.549 × 104 L·mol–1·cm–1) (Figure S5). Figure 2 de-
picted the fluorescence emission spectra of TTPB to various H2O2
levels. When adding the concentrations of H2O2 from 0 to 100
With its highly viscosity-sensitive in vitro and excellent mito-
chondria-specific ability confirmed, TTPB was investigated for its
viscosity-dependent fluorescence response in live cells. As re-
ported, the ionophores (nystatin and monensin) can cause struc-
tural changes and swelling of mitochondria, further leading to
mitochondrial malfunction accompanied by mitochondrial viscos-
ity changes.[28] After incubating with TTPB for 30 min, HeLa cells
were further treated by nystatin or monensin for another 30 min,
respectively. As depicted in Figure 4, the cells treated with
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We then examine whether TTPB could visualize endogenous H2O2,
by adding phorbol myristate acetate (PMA) or rotenone (ROT),
which can promote cellular ROS levels.[28,31] As predicted, a dra-
matic fluorescence enhancement was observed upon PMA or ROT
induction. Remarkably, the following addition of N-acetyl-L-cyste-
ine (NAC, an efficient ROS scavenger), almost quenched the fluo-
rescence signals in the orange channel. Thus, the results suggest-
ed that TTPB could detect both exogenous and endogenous H2O2
in live cells.
Figure 3 Fluorescence images of HeLa cells co-stained with (a, e) TTPB (5
μmol/L, 30 min) and (b) MTDR (0.3 μmol/L, 30 min) or (f) LB-NIR (0.3
μmol/L, 30 min) for 30 min, respectively. (c, g) Merged images. (d) The
Pearson's co-localization correlation of TTPB with MTDR intensities (PC =
0.94), and (h) the Pearson's co-localization correlation of TTPB with LB-NIR
intensities (PC = 0.13). TTPB: λex = 488 nm, λem = 560—670 nm; MTDR and
LB-NIR: λex = 633 nm, λem = 680—770 nm. Scale bar: 20 μm.
Figure 4 (a) Fluorescence images of HeLa cells incubated with TTPB (5
μmol/L, 30 min), and then treated without or with nystatin (10 μmol/L, 30
min) or monensin (10 μmol/L, 30 min). (b) The mean fluorescence intensi-
ties of images shown in (a). Error bars represent mean deviation (± S.D.),
n = 3. NIR channel for TTPB: λex = 488 nm, λem = 650—740 nm. Scale bar:
20 μm.
Figure 5 (a) Fluorescence images of HeLa cells incubated with TTPB (5
μmol/L, 30 min), and then treated without and with H2O2 (100 μmol/L, 40
min), rot (5 μmol/L, 40 min), rot (5 μmol/L, 40 min) with NAC (50 μmol/L,
40 min), PMA (5 μg/mL, 40 min) or PMA (5 μg/mL, 40 min) with NAC (50
μmol/L, 40 min). The mean fluorescence intensities of images shown in (a).
Error bars represent mean deviation (± S.D.), n = 3. Orange channel for
TTPB: λex = 405 nm, λem = 540—640 nm. Scale bar: 20 μm.
TTPB only displayed dim fluorescence in the NIR channel (650-740
nm), while the fluorescence intensity in nystatin or monensin
treated cells obviously enhanced, indicating a viscosity rise in
mitochondria, and the ability of TTPB for monitor mitochondrial
viscosity fluctuations.
Fluorescence imaging of TTPB to H2O2 in living cells
Dual-imaging of viscosity and H2O2 in inflammation model
The potential ability of TTPB to visualize H2O2 in live cells was
further investigated upon varying stimulation. As illustrated in
Figure 5, compared with only TTPB-stained cells, the fluorescence
emission within H2O2-treated cells showed a remarkable fluores-
cence enhancement in the orange channel (540—640 nm), indi-
cating the ability of TTPB for imaging exogenous H2O2 in live cells.
Having verified the ability of TTPB to separately image mito-
chondrial viscosity and H2O2 in two different channels, we then
evaluate its capability of simultaneously tracking mitochondrial
viscosity and H2O2 in an inflammation model induced by lipopoly-
saccharide (LPS), which has been verified to trigger cells to pro-
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duce inflammation.[33-34]
Dual-imaging of viscosity and H2O2 in malignant tumor model
As illustrated in Figure 6, the TTPB-stained cells showed faint
fluorescence in both orange channel (for H2O2) and NIR channel
(for viscosity), while obvious enhanced fluorescence were
observed in both of the two channels, implying the increase in pH
viscosity and H2O2 in the LPS-mediated inflammatory cells. The
results suggested that TTPB holds great potential for dual-imaging
viscosity and H2O2 levels in pathogenic cells.
Building on the prominent results from live cells imaging,
TTPB was finally applied to simultaneously monitor H2O2 level and
viscosity in live tissues slices. Specifically, the normal tissues (in-
cluding heart, liver, spleen, lung, kidney and thymus) and tumor
tissue were treated with TTPB for 30 min, respectively. Obviously,
in comparison with the very dim fluorescence signals from normal
tissues (Figure 7 and Figure S10), the tumor tissue exhibited
Figure 6 (a) Fluorescence dual-images of HeLa cells incubated with TTPB (5 μmol/L, 30 min), and then treated without or with LPS (100 μg/mL, 2h). (b)
The mean fluorescence intensities of images shown in (a). Error bars represent mean deviation (± S.D.), n = 3. Orange channel (H2O2 channel): λex = 405
nm, λem = 540—640 nm. NIR channel (viscosity channel): λex = 488 nm, λem = 650—740 nm. Scale bar: 20 μm.
Figure 7 (a) Fluorescence dual-images of TTPB (10 μmol/L, 30 min) in living normal tissues and tumor. (b) The mean fluorescence intensities of images
shown in (a). Error bars represent mean deviation (± S.D.), n = 3. Orange channel (H2O2 channel): λex = 405 nm, λem = 540—640 nm. NIR channel (viscosity
channel): λex = 488 nm, λem = 650—740 nm. Scale bar: 50 μm.
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significantly enhanced fluorescence in both orange channel (for
H2O2) and NIR channel (for viscosity), implying the elevated H2O2
level and increased viscosity of tumor tissue. Taken together, the
results vividly illustrated that TTPB could penetrate into the tis-
sues, and dual-image the contents of viscosity and H2O2 in malig-
nant tumor models.
ed with TTPB (5 μmol/L) for 30 min at 37 °C, and then treated
with ionophores (nystatin or monensin) (10 μmol/L) for another
30 min, respectively. The fluorescence images were acquired
through a confocal laser scanning microscope (Zeiss, LSM880)
with NIR channel (λex = 488 nm, λem = 650—740 nm).
For imaging of H2O2 in live cells. HeLa cells were incubated
with TTPB (5 μmol/L) for 30 min at 37 °C, and then treated with
H2O2 (100 μmol/L), rot (5 μmol/L), rot (5 μmol/L) + NAC (50
μmol/L), PMA (5 μg/mL), or PMA (5 μg/mL) + NAC (50 μmol/L) for
another 40 min, respectively. The fluorescence images were ac-
quired through a confocal laser scanning microscope (Zeiss,
LSM880) with orange channel (λex = 405nm, λem = 540—640 nm).
For dual-imaging of H2O2 and viscosity in inflammation mod-
el. For dual-detection of H2O2 and viscosity, the HeLa cells were
incubated with TTPB (5 μmol/L) for 30 min, and then treated with
LPS (100 μg/mL) for 2 h. The fluorescence images were acquired
through a confocal laser scanning microscope (Zeiss, LSM880)
with orange channel (λex = 405 nm, λem = 540—640 nm) and NIR
channel (λex = 488 nm, λem = 650—740 nm).
Conclusions
In summary, a mitochondria-specific orange/near-infrared-
emissive fluorescent probe TTPB was developed for simultane-
ously imaging the viscosity and H2O2 levels in inflammation and
cancer models. The probe emitted NIR emission at about 666 nm,
and exhibited a good linear relationship over the viscosity range
from 0.59 to 950 cP. Meanwhile, TTPB displayed high selective
and sensitive response to H2O2 levels with orange emission
around 586 nm. Moreover, we revealed that TTPB has satisfactory
mitochondria-specific ability and permits the visual monitoring of
viscosity and H2O2 changes in different channels in live cells. Most
importantly, using the single probe TTPB, we demonstrated the
simultaneous rise of viscosity and H2O2 levels in inflammation and
tumors models for the first time. Therefore, we believe that the
fluorescence probe will be a potential imaging tool for investigat-
ing mitochondria-associated diseases.
Tissue slices imaging experiment
KM mice (18—20 g) were purchased from Laboratory Animal
Center of Shanxi Cancer Hospital (Taiyuan, China) for in tissue
slices experiment. Herein, H22 cells were subcutaneously injected
into the right axillae of KM mice, and the tumor was obtained
after 12 d. For tissue slices imaging, the normal organs (heart,
spleen, liver, lung, kidney and thymus) and tumor were isolated
from the mice, then sectioned as 4 μm thicknesses. These slices
were incubated with TTPB (10 μmol/L) for 30 min, respectively,
and finally subjected for fluorescence imaging through a confocal
laser scanning microscope (Zeiss, LSM880) with orange channel
(λex = 405 nm, λem = 540—640 nm) and NIR channel (λex = 488 nm,
λem = 650—740 nm).
Experimental
Materials and apparatus
The reagents, apparatus and methods in the work were listed
in Supporting Information. The synthetic route of TTPB and TTP
was illustrated in Scheme 2, and the detailed synthesis process
and characterizations were shown in the Supporting Information.
Spectroscopic response of TTPB to viscosity
Supporting Information
TTPB (1.0 mmol/L) was prepared in DMSO to obtain the stock
solution. The solvents were obtained by mixing a methanol/glyc-
erol system in different proportions. The solutions of TTPB of
different viscosity were prepared by adding the stock solution
(1.0 mmol/L) 10 µL to 2 mL of methanol/glycerol system to obtain
the final concentration of Mito-TPB (5.0 µmol/L). These solutions
were sonicated for 10 min to eliminate air bubbles. Excitation and
emission bandwidths were both set at 2.0 nm, and the excitation
wavelength was fixed at 405 nm.
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.202000725.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Nos. 81901814 and 21874087) and Natural
Science Foundation of Shanxi Province (No. 201801D121040). The
animal experiments were approved by the Animal Care and Use
Committee of Shanxi University, and performed in compliance
with the Animal Management Rules of the Ministry of Health of
the People's Republic of China (Document no. 55, 2001).
Spectroscopic response of TTPB to H2O2
5 μmol/L TTPB was used in spectroscopic determination by
addition of 10 μL stock solution (1.0 mmol/L) to 2.0 mL PBS con-
taining 50% DMSO (pH 8.4). Absorption and emission spectra
were recorded after the test solution was incubated at 37 °C for 2
h with H2O2. Excitation and emission bandwidths were both set at References
2.0 nm, and the excitation wavelength was fixed at 488 nm.
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For co-localization experiments. HeLa cells were stained with
5 μmol/L TTPB for 30 min at 37 °C, and then co-incubated with
0.3 μmol/L MitoTracker Deep Red or 0.3 μmol/L LysoBrite NIR for
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with green channel (λex = 488 nm, λem = 560—670 nm) for TTPB,
red channel (λex = 633 nm, λem = 680—770 nm) for MitoTracker
Deep Red or LysoBrite NIR, respectively.
For imaging of viscosity in live cells. HeLa cells were incubat-
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A Dual‐response fluorescent probe for the detection of viscosity and
Manuscript received: December 29, 2020
Manuscript revised: February 9, 2021
Manuscript accepted: March 4, 2021
Chin. J. Chem. 2021, 39, 1303－1309
© 2021 SIOC, CAS, Shanghai, & WILEY‐VCH GmbH
www.cjc.wiley‐vch.de
1309