A water-soluble two-photon fluorescence chemosensor for ratiometric imaging of mitochondrial viscosity in living cells

Article abstract of DOI:10.1039/c6tb01240j

A convenient and rationally designed water-soluble two-photon excited fluorescence (TPEF) sensor for monitoring viscosity with specific targeting at the subcellular level remains a challenge. Herein, we reported a novel water-soluble ratiometric TPEF chem

Full text of DOI:10.1039/c6tb01240j

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A water-soluble two-photon fluorescence chemosensor for
ratiometric imaging mitochondrial viscosity in living cells
Meng Zhao,a Yingzhong Zhu,a Jian Su,a Qian Geng,b Xiaohe Tian*,b Jun Zhang,c Hongping Zhou,a
Shengyi Zhang,a Jieying Wu,a Yupeng Tian*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Convenient and rational design water-soluble two-photon excited fluorescence (TPEF) sensor for monitoring viscosity with
specific targeting at subcellular level remain a challenge. Herein, we reported a novel water-soluble ratiometric TPEF
chemosensor EIN that is specifically responsive and singularly sensitive to mitochondria viscosity in living cells. Because its
fluorescence emission bands at two different wavelengths show obvious enhancement with the increased solvent
viscosity, we found that fluorescence intensity ratio (I569 / I384) versus the logarithm of the viscosity (log η) enable to
monitor and quantify the abnormal changes of mitochondria viscosity. More importantly, EIN was successfully utilized to
distinguish normal and nystatin treated HepG2 cells viscosity difference of mitochondria. In a word, the merits of NIR
property, high selectivity and signal ratio provide more convenience for studying biological processes and mitochondria
www.rsc.org/
viscosity
related
diseases.
micro-viscosity-targeted TPEF sensors have been introduced.9,10
Such ratiometric detection allows signal rationing and has been
widely applied in microviscosity imaging and quantification.
However, in view of the fact that many viscosity-related diseases
and malfunctions are organelle phenotypic, how to design
Introduction
Intracellular viscosity is a significant factor governing the mass or
signal transportation, biomacromolecules interactions and reactive
metabolites diffusion.1,2 Abnormal in vivo viscosity reflect diseases
and malfunctions, such as Alzheimer disease, diabetes,
atherosclerosis and cell malignancy.3-5 In addition, unusual viscosity
variation in mitochondrial matrix may induce changes in
mitochondrial network organization, and further influence
organelle-specific chemosensor still remains
a challenge for
biological applications. Since double-layer mitochondria offer high
viscosity micro-environments, viscosity sensors capable of
exhibiting strong fluorescence emission once combined to the
metabolite diffusion.6 Therefore, rational designing targeted mitochondria, while viscosity sensors exhibit weak or quenching
background emission in the uncombined state.11
molecules capable of quantifying the mitochondrial viscosity in
living cells are of great significance.
Considering above, a novel chemosensor EIN was designed and
synthesized by introducing flexible diethyl 2,2’-
It is known that molecular rotors possess the rotatable conjugated
moiety, that is, a certain part of a whole molecular can rotate to
another part. On the one hand, the intramolecular rotation relaxes
the excitation energy resulting in significant decreasing or
quenching of fluorescence in low-viscosity environments. On the
other hand, intramolecular rotation is inhibited in viscous
environments, which can reduce the probability of nonradiating
pathways and further restore fluorescence.7,8 Hence, molecular
rotors with appropriate biocompatibility are highly suitable as
viscosity-sensitive fluorescent chemosensors in living cells and
tissues.
(phenylazanediyl)diacetate moiety and cationic 1,2,3,3-tetramethyl-
3H-indol-1-ium moiety. Compared with reported viscosity
sensors,12-16 ratiometric TPEF chemosensor EIN has great water
solubility, low cytotoxicity, reasonable photostability, large stokes
shift as well as longer excitation wavelengths of two-fluorescence
microscopy (TPM). Importantly, by means of the fluorescence
intensity ratio of 569 and 384 nm, the precise mitochondria
viscosity in normal and nystatin treated HepG2 cells was also
measured.17-19
In the past decades, molecular rotors with dual emission as the
Results and discussion
Design and synthesis of EIN
The organic molecules based on the push-pull π-conjugated
system might possess the main and most promising
foundation for two-photon materials,20,21 especially in the field
of
chemosensor
applications.22-25
Diethyl
2,2’-
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(phenylazanediyl)diacetate moiety is not only an electron-
donating group, but also possess good biocompatibility as well,
which suggested that it is highly prospective for constructing
biocompatible TPEF sensor for bioimaging in living cells and
tissues. Cyanine dye, 1,2,3,3-tetramethyl-3H-indol-1-ium, is a
cationic fluorophore with high fluorescence quantum yields,
NIR property and great water stability, and it is well
established that cationic dyes can be easily taken up and
accumulate in the mitochondria of living cells.26,27 To design
water-soluble
mitochondrial
ratiometric
TPEF
chemosensor
diethyl
with
2,2’-
specificity,
(phenylazanediyl)diacetate was connected with 1,2,3,3-
tetramethyl-3H-indol-1-ium to produce novel cationic
chemosensor EIN by using extended π-conjugation double
a
bond, as shown in Scheme 1
.
Spectral properties
Fig. 1 (a) Changes of fluorescence spectra with the variation of solution viscosity in
water-glycerol system (excited at 320 nm). (b) Relationship of fluorescence intensity
ratio (I569 / I384) with the logarithm of the viscosity (log ). (c) Changes of fluorescence
spectra with the variation of solution viscosity in water-glycerol system (excited at 490
nm). (d) Relationship of fluorescence intensity ratio (I569 / I0) with the logarithm of the
viscosity (log ). Inset in (a), fluorescence changes of EIN in 0.01% or 99.9 % glycerol
system under UV light (365 nm excitation).
The linear absorption and emission spectra of EIN in H2O
buffered with HEPES are shown in Fig. S2. It can be seen that
EIN exhibits two absorption peaks (λabs = 320 nm and 483 nm)
and two emission peaks (λem = 378 nm and 562 nm) in pure
HEPES buffer. Furthermore, water and glycerol solubility
experiments were shown in Fig. S3, which suggested that EIN
possesses good water solubility (50
(20 M).
ꢀM) and glycerol solubility
571 nm) of EIN with increasing proportions of glycerol in the
mixture solvent. The emission of EIN was greatly enhanced
(24-fold) with the increased solvent viscosity when excited at
490 nm (Fig. 1c). And the fluorescence intensity ratio (I / I0) at
ꢀ
One-photon fluorescence response to solvent viscosity
As shown in Fig. S4, generally solvents with different polarity
influence the absorption and emission maxima to some extent.
EIN gives very small responses upon polarity, and such limited
polarity influence is vital to the molecular rotor to sense
environmental viscosity.28-31
To investigate the relationship between the viscosity and the
fluorescent intensity, fluorescence emission spectra of EIN was
measured upon 320 nm at room temperature as shown in Fig.
1a. It is obvious that EIN showed two emission bands at 384
nm and 569 nm, where both emission intensities were found
to be enhanced by increasing proportions of glycerol in the
mixture solvent. The fluorescence intensity of EIN at 569 nm
increased 29-fold while the blue emission at 384 nm gave
weaker responses with the 11-fold fluorescence enhancement.
As a result, we found a better linear relationship (R2 = 0.994,
571 nm showed
logarithm of the viscosity (log
a
reasonable linear relationship with
) of the solution (R2 = 0.970,
η
the slope x = 8.299), which was identical to that measured for
the dual emission (Fig. 1d).
The study of quantum yield (QY) and fluorescence lifetime of
EIN were also carried out as shown in Fig. 2. Quantum yields
were very low, approximately 0.01-0.05, in low-viscosity
solvents such as water, methyl alcohol and dimethyl sulfoxide,
and was not apparently affected by solvent polarity. However,
in water and glycerol mixed solvents (viscosity range from
1.005 cP to 1385 cP), the quantum yields of EIN obviously
increased with increasing glycerol volume fraction. In 99.9 %
glycerol, EIN exhibited the strongest and naked eye visible red
emission with the highest quantum yield 0.33. Namely, EIN can
exhibit strong fluorescence intensity only in viscous medium
restricting intramolecular free rotation and reducing the
probability of nonradiating pathways, and other factors such
the slope x = 0.464) between fluorescence intensity ratio (I569
η
/
I384) and the logarithm of the viscosity (log
).32,10 The results
exhibited that EIN can be employed to ratiometrically detect
viscosity in various environments including biological systems
(Fig. 1b and Fig. S5).
In addition, we investigated the red emission spectra (λem
=
Fig. 2 (a) The fluorescence quantum yields of EIN (excited at 490 nm) in different
solvents: (1) dichloromethane, (2) dimethyl sulphoxide, (3) ethanol, (4) methanol,
(5) water, (6) water/glycerol (8:2, v/v), (7) water/glycerol (6:4, v/v), (8)
water/glycerol (4:6, v/v), (9) water/glycerol (2:8, v/v), (10) glycerol (99.9 %). (b)
Changes of fluorescence decay of EIN (10 μM) with the variation of solution
viscosity, excited at 490 nm.
Scheme 1. Schematic representation of the synthesis procedures of EIN
.
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Fig. 4 Confocal laser fluorescence microscopic images of HepG2 cells treated with 10
μM EIN. (a) Fluorescent image of EIN, collected at 520-580 nm and excited at 840 nm;
(b) bright fields of HepG2 cells; (c) Merged image of (a) and (b).
evaluated the precise subcellular localization of EIN
,
colocolization experiments using commercial dyes were
evaluated (Mito Tracker Red: panels (a) – (c), Lyso-Tracker blue:
panels (d) – (f)) as showed in Fig. 5. Apparently, EIN exhibited
superior mitochondria-targeting ability than lysosomes with
colocalization coefficient of 0.90 and 0.05, respectively (Fig. 6).
The above experimental results implied that EIN is indeed a
mitochondria-targeted two-photon fluorescence sensor.
Fig. 3 Two-photon (TP) action cross-section spectra of EIN with the variation of solution
viscosity in the wavelength region 700-840 nm.
In addition, we inspected whether mitochondrial membrane
potential could influence the fluorescence signals of EIN since
it accumulates in mitochondria (Fig. 7). Then HepG2 cells
treated with a membrane-potential uncoupler CCCP that
as pH did not affect bond rotation and alter its fluorescence
intensity (Fig. S6).
Two-photon absorption response to solvent viscosity
Since the push-pull π-conjugated system has been reported to
possess two-photon properties, the two-photon cross sections
of EIN were recorded using two photon excited fluorescence
measurements (Fig. 3). The two-photon process was also
confirmed by a power dependence experiment (Fig. S7). In the
range of 700-840 nm, two-photon absorption spectra of EIN
enhanced remarkably (6.9-fold at 700nm) with increasing
proportions of glycerol in the mixture solvent. And the two-
photon action cross-section (Φδ) at 700 nm increased from
18.5 (20 % glycerol, 1.76 cP) to 124.1 GM (99.9 % glycerol,
1385 cP) (1 GM = 10-50 cm4 s photon-1). Such remarkable
enhancement of two-photon action cross-sections (Φδ) is
crucial to permit a molecular rotor to reflect environmental
viscosity in biological system.
disrupt
mitochondrial
membrane
potential
were
.
simultaneously incubated Mito Tracker Red or EIN 33-35
Staining with Mito Tracker Red whose uptake is dependent of
mitochondria membrane potential was inconsistent in the
absence or presence of CCCP. Similarly, fluorescence
brightness of EIN changed distinctly upon treatment with CCCP.
The above results implied that EIN locate in mitochondria in
living are membrane potential dependent.
Ratiometric two-photon fluorescence imaging
Since one of major advantages of mitochondria-targeted EIN is
that it possesses dual absorption and emission, that is
independent of the dye concentration or intracellular
distribution, and avoid most of the interferences from
In addition, interference experiments exhibited the
fluorescence intensity of EIN rarely responded in the presence
of DNA, RNA, BSA and various aminophenol within one hour
(Fig. S8).
Cell cytotoxicity (MTT)
The study of the effect of EIN on viability of HepG2 cells was
carried out using the methylthiazolyldiphenyl-tetrazolium
bromide (MTT) assay as shown in Fig. S9. With the
concentration ranging from 10 M to 50 M, EIN has little
ꢀ
ꢀ
cytotoxicity for 24 hours incubation. Subsequently, photon
resistance abilities were evaluated via standard photon-
bleaching experiments, indicating that EIN exhibited
reasonable photo-stability and showed no significant
fluorescence signals decrease under continuous irradiation for
518 s (Fig. S10). Above results promised further two-photon
biological imaging in living cells.
Cell localization of EIN
Fig. 5 Confocal laser fluorescence microscopic images of HepG2 cells treated
with 10 μM EIN and Mito-tracker Red (1.0 μM) or Lyso-Tracker Blue (1.0 μM). (a)
and (d) Fluorescence imaging of EIN in HepG2 cells, collected at 520-580 nm and
excited at 840 nm; (b) Fluorescent image of Mito-Tracker Red (1.0 μM), collected
by a 585-625 nm band path filter with excitation at 579 nm; (c) Merged image of
(a) and (b); (e) Fluorescent image of Lyso-Tracker Blue (1.0 μM), collected by a
filter of 410-450 nm upon excitation at 408 nm; (f) Merged image of (d) and (e).
To investigate intracellular distribution of EIN, cell imaging was
first performed with HepG2 cells under confocal fluorescence
microscopy. As illustrated in Fig. 4 and Fig. S11, micrographs
demonstrated EIN (10 µM, 30 min) stained tubular-like
structure with superb signal-to-noise ratio. Furthermore to
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Fig. 8 Confocal laser fluorescence microscopic images of 10 μM EIN before and
after treatment of HepG2 cells by 10 μM nystatin for 30 minutes, respectively; (a)
and (e) Bright fields of HepG2 cells; (b) and (f) Red channels fluorescence images
detected at 520-580 nm nm upon excited at 840 nm; (c) and (g) Green channels
fluorescence images detected at 375-450 upon excited at 840 nm; (d) and (h)
Ratiometric image of EIN obtained by the ImageJ software.
Fig. 6 (a) Correlation plot of Mito-Tracker Red and EIN intensities. (b) Intensity
profile of ROIs across HepG2 cells.
microenvironments.36-38 Therefore we consider EIN is highly
suitable to quantify mitochondrial viscosity and monitor its
abnormal change in living cells. As shown in Fig. 8, the
fluorescence images were obtained by collecting the blue
emission region (375 – 450 nm) and the red emission region
(520 – 580 nm) when excited at 840 nm. The ratio image
(panels (d) and (h)) based on blue emission and red emission
clearly displayed the distribution of viscosity in the cells. And
the average fluorescence intensity ratio in HepG2 cells is 1.27
accuracy and reliability.
Conclusion
To summarize,
a
viscosity-sensitive water-soluble TPEF
± 0.05 (panels (d)), which is equivalent to 56.7 cP according to chemosensor
with a flexible diethyl 2,2’-
linear relationship between fluorescence intensity ratio and
the logarithm of the viscosity. Moreover, upon treatment with
nystatin (panels (e) – (h)), which can induce mitochondrial
malfunction caused by structure changes or swelling of
mitochondria,39,40 the average viscosity of mitochondria in
HepG2 cells increased to 199.3 cP (average fluorescence
intensity ratio is 1.52 ± 0.05, panels (h)). Obviously, the green
regions in panels (d) and (h) represent higher viscosity than
blue regions. Above experimental results agrees with previous
reports and indicates that nystain can induce abnormal
increasing of mitochondria viscosity. Accordingly, EIN as a
mitochondrial viscosity chemosensor can monitor and quantify
abnormal metabolic changes in mitochondria with high
(phenylazanediyl)diacetate moiety and cationic 1,2,3,3-
tetramethyl-3H-indol-1-ium moiety have been developed. As a
water-soluble cationic sensor, it has dual absorption and
emission, as well as large two-photon action cross-sections
(Φδ). Other factor such as the polarity of solvents has limited
influence on sensor EIN. Also, it exhibits reasonable photo-
stability and low cytotoxicity and can effectively stain
mitochondria by mitochondrial membrane potential.
Fluorescence intensity ratio (I569 / I384) of EIN is in better linear
relationship with the logarithm of the viscosity (log
0.994, the slope x 0.464). Thus the EIN is able to
η =
) (R2
=
quantificationally track and image the intracellular
mitochondria viscosity by fluorescence ratiometry with
excellent self-calibrating ability. Furthermore, EIN was
successfully applied for monitoring the viscosity distinction in
normal and nystatin treated HepG2 cells. It is anticipated that
EIN provide
a promising strategy for the pathology
investigation and diagnoses of mitochondria viscosity related
diseases.
Experimental
Materials and physical measurements
All chemicals and solvents used in the synthesis were of
reagent grade and used without further purification while
handling them. The 1H-NMR spectra were recorded at 25 °C on
a Bruker Advance 400 spectrometer, and the chemical shifts
are reported as parts per million from TMS (δ). The 13C-NMR
spectra were recorded on 150 MHz spectrometers. Chemical
shifts were reported in parts per million relative to
tetramethylsilane (δ = 0). Mass spectra were determined with
a Micro-mass GCT-MS (EI source). Elemental analyses were
measured on a Perkin Elmer 240C elemental analyzer. FT-IR
spectra (KBr pellets) were recorded with a NEXUS-870 (Nicolet)
spectrometer in the 4000-400 cm-1 region. The linear
absorption spectra were measured on a SPECORD S600
spectrophotometer. The single-photon emission fluorescence
Fig. 7 Confocal laser fluorescence microscopic images of HepG2 cells treated
with 10 μM EIN or 1.0 μM Mito-tracker Red, then the cells were incubated in the
absence or presence of 10 μM CCCP for 30 min. (a) and (b) Fluorescent image of
EIN , collected by a 520-580 nm band path filter with excitation at 840 nm; (c)
and (d) Fluorescent image of Mito-tracker Red, collected by a 585-625 nm band
path filter with excitation at 579 nm.
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(SPEF) spectra measurements were performed using a Hitachi 8.7 Hz, 2H), 7.78 (dd, J = 15.8, 7.7 Hz, 2H), 7.55 (dd, J = 14.0,
F-7000 fluorescence spectrophotometer. The two-photon 7.4 Hz, 2H), 7.37 (d, J = 16.0 Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H),
emission fluorescence (TPEF) spectra were measured at 4.43 (s, 4H), 4.15 (q, J = 7.0 Hz, 4H), 4.02 (s, 3H), 1.75 (s, 6H),
femtosecond laser pulse and Ti: sapphire system (680-1080 nm, 1.22 (t, J = 7.1 Hz, 6H). 13C-NMR (d6-DMSO, 101 MHz, ppm) δ =
80 MHz, 140 fs) as the light source. The viscosities of the 180.5, 169.4, 153.8, 152.8, 142.9, 141.9, 133.3, 128.7, 128.1,
solvents were determined using capillary Ubbelohde dilution 123.8, 122.6, 114.1, 112.5, 107.2, 60.8, 52.5, 51.3, 33.4. FT-IR
type viscometers, and higher viscosities of the aqueous (KBr, ν, cm-1): 2985(vw), 2363(vw), 2345(vw), 1744(m), 1577(s),
solution of glycerol were taken from published tables.19 The 1529(vs), 1479(m), 1420(w), 1396(m), 1374(w), 1332(w),
fluorescent quantum yields were measured with integrating 1301(m), 1189(vs), 1173(s), 1164(s), 1117(m), 1021(w), 972(w),
sphere.
932(vw), 840(vs), 792(w), 763(w), 708(vw), 558(w). HRMS-ESI:
HepG2 cells were luminescently imaged on a Zeiss LSM 710 m/z, cal: 449.2440, found: 449.2451 [M+]. M.p. = 188-189 °C.
META upright confocal laser scanning microscope. Image data
acquisition and processing was performed using Zeiss LSM
Acknowledgments
Image Browser, Zeiss LSM Image Expert and Image J.
This work was supported by the National Natural Science
Foundation of China (51372003, 21271004, 51432001,
21271003, 51472002, 21275006), Focus on returned overseas
scholar of Ministry of Education of China, the Higher Education
Revitalization Plan Talent Project (2013).
Synthesis of compound 1
2,3,3-Trimethylindolenine (1.59 g, 10.0 mmol) was dissolved in
iodomethane (3.98 g, 28.0 mmol) and with constant stirring,
the solution was refluxed for 24 hours. The precipitate
produced was filtered under suction, washed with n-hexane
and dried in vacuo to yield the product as a pink solid (1.7 g,
yield 56 %).41
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brown solution. Then the mixture was cooled, the filtrate was
concentrated to yield brown oil as crude product (4.4 g,
86 %).42
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Synthesis of EIN
Compound
(20.0 mL), followed by the addition of compound
3
(0.98 g, 3.40 mmol) was dissolved in acetonitrile
(1.00 g,
1
12. N. Gupta, S. I. Reja, V. Bhalla, M. Gupta, G. Kaur and M.
3.20 mmol). With constant stirring, five drops of piperidine
were added to the mixture, and the solution was refluxed for
12 hours to produce a red solution. After cooling, AgPF6 (0.81 g,
3.20 mmol) was added dropwise into the solution with
constant stirring at 60 °C for 1 hour. The solution was
concentrated under reduced pressure to yield red oil, which
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was
purified
by
column
chromatography
using
dichloromethane/methanol (10:1, v/v) as the eluent, and EIN
was collected as red solid (1.20 g, 61%).
Elemental analysis for EIN, C27H33F6N2O2P, Calcd (%): C, 57.65;
H, 5.91; N, 4.98. Found: C, 55.30; H, 5.57; N, 4.75. 1H-NMR (d6-
DMSO, 400 MHz, ppm) δ = 8.32 (d, J = 15.9 Hz, 1H), 8.07 (d, J =
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Page 7 of 7
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C6TB01240J
Convenient and rational design water-soluble two-photon excited fluorescence
(TPEF) sensors for monitoring viscosity with specific targeting at subcellular level
remain a challenge. Herein, we reported a novel water-soluble ratiometric TPEF
chemosensor EIN that is specifically responsive and singularly sensitive to
mitochondria viscosity in living cells. Because its fluorescence emission peaks at two
different wavelengths show obvious enhancement with the increased solvent viscosity,
we found that fluorescence intensity ratio (I569 / I384) versus the logarithm of the
viscosity (log η) enable to monitor and quantify the abnormal changes of
mitochondria viscosity. More importantly, EIN was successfully utilized to
distinguish normal and nystatin treated HepG2 cells viscosity difference of
mitochondria. In a word, the merits of NIR property, high selectivity and signal ratio
provide more convenience for studying biological processes and mitochondria
viscosity related diseases.