A novel mitochondria-targeted rhodamine analogue for the detection of viscosity changes in living cells, zebra fish and living mice

Article abstract of DOI:10.1039/c8tb00298c

Monitoring the intracellular viscosity changes is crucial for better understanding diffusion-controlled cellular processes. Herein, we synthesized a novel phenyl-substituted imidazole-fused rhodamine analogue RV-1 with a long absorption wavelength at 573

Full text of DOI:10.1039/c8tb00298c

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DOI: 10.1039/C8TB00298C
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ARTICLE
A novel mitochondria-targeted rhodamine analogue for detection
of viscosity changes in living cells, zebra fishes and living mice†
Received 00th January 20xx,
Accepted 00th January 20xx
Rui Guo,a Junling Yin,b Yanyan Ma, Qiuan Wang *, and Weiying Lin
b
a,
a, b,
*
DOI: 10.1039/x0xx00000x
Monitoring the intracellular viscosity changes is crucial to better understand the diffusion-controlled
cellular processes. Herein,we synthesized a novel phenyl-substituted imidazole fused rhodamine analogue
RV-1 with long absorption at 573 nm and a large Stokes shift about 82 nm for monitoring the change of
cellular viscosity The new probe RV-1 showed very strong fluorescence emission at around 655 nm, with a
www.rsc.org/
4
8.5-fold enhancement of fluorescence intensity from methanol to 99% glycerol. Significantly, the innovative
probe RV-1 was successfully applied for detection of the viscosity change not only in living cells,but also in
zebra fishes and living mice.
applied in biological field due to its simple operation and high
sensitivity as well as the excellent spatial and temporal
resolution. A class of fluorescent probes termed fluorescent
Introduction
The cytoplasm of virtually all living cells is highly crowded with
intracellular organelles and macromolecules such as proteins,
lipids, nucleic acids, and sugars.1, 2 The consequences of this
crowding in cells may hinder the solute diffusion to influence
the key cellular functions, including protein folding, enzyme
catalysis, intracellular signalling, intracellular transport, and
localization of molecules and organelles. One of the primary
factors which determine the solute diffusion is viscosity, and
therefore, it is crucial to monitor the viscosity changes in
various cellular processes. Moreover, Mitochondria, is the
principal energy-producing compartments in most eukaryotes,
which play critical roles in a number of vital cellular processes,
molecular rotors were developed for detection of the cellular
viscosity variations. The molecular rotors are fluorescent
molecules with an intramolecular charge transfer (ICT)
mechanism undergoing the twisting motion in the excited
7
state. When the molecule absorbs a photon, it first gives a
planar excited state with charge transfer, then forms a twisted
3
8
excited state by intramolecular rotation. The rotation typically
4
9
leads to a dark non emissive excited state. In low viscous
solutions, the fluorescent molecular rotor exhibits very weak
fluorescence due to their freely rotation. Whereas, in the high
viscous solutions, the rotation of the fluorescent molecular
rotor is hindered to emit strong fluorescence emission. There
5
such as ATP production, central metabolism, and apoptosis.
Viscosity changes in mitochondria may lead to the dysfunction
1
0-
have been numerous fluorescent molecular rotors reported,
13
but most of them have short absorption/excitation
of the
mitochondria. However, measuring the viscosity on
wavelength in the range of 300-500 nm, which may lead to
damage to living biological systems. Therefore, it remains
highly desirable for construction of the fluorescent molecular
rotors with longer absorption/excitation wavelengths for
detection of viscosity in living cells.
6
the microscopic scale such as in tiny living cells still difficult.
The local micro-viscosity in the cells changes widely from one
region to another, which is very different to the viscosity
observed on a macroscopic scale. Therefore, the methods for
detection of the viscosity changes in cells or on subcellular
level are still in high demand.
3
The fluorescence technology becomes a powerful tool
a.State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan
4
10082, P. R. China
b.
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 P. R. China
*
E-mail: weiyinglin2013@163.com (W. Lin) ； WangQA@hnu.edu.cn (Q.Wang)
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See DOI:
1
0.1039/x0xx00000x
Scheme 1 The structure and proposed mechanism of RV-1 for response to viscosity.
J. Name., 2013, 00, 1-3 | 1
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Rhodamine is a dye widely used as fluorescent probes for ratios which had varied viscosity were chosen to as tVeieswtiAnrgticmle Oendliinae.
various analytes and laser source due to its excellent spectroscopic We first examined the absorption spectraDoOfI:t1h0e.10p3r9o/bCe8TRBV00-12.98ACs
properties, such as long absorption and emission wavelengths, high shown in Fig.1a, it had almost no absorption in methanol at above
fluorescence quantum yield, large extinction coefficient, and high 500 nm. In contrast, a new absorption peaks at 573 nm appeared in
14-16
stability against light.
However, most of the rhodamine 99 % glycerol. Then, the fluorescent emission spectra were also
derivatives have the absorption/emission wavelengths which are investigated. As shown in Fig.1b, the probe exhibited almost no
below 560/580 nm and show a small stokes shift at the range of 10- fluorescence above 600 nm when excitated at 573 nm in methanol.
3
0 nm, which may result in self-absorption and fluorescence This might be attributed to the free rotation between the imidazole
1
7,18
detection error due to excitation backscattering effects.
Herein, ring and the 2, 3-dihydro-1H-xanthene core, which lead to a non-
we rationally designed and synthesized a new phenyl-substituted radiative decay of the excited state, weakening the fluorescent
imidazole fused rhodamine dye RV-1 with long emission of the probe. Whereas, in the high viscous media, the free
absorption/fluorescence wavelengths, at about 573/655 nm, and a rotation of the imidazole moiety of the probe RV-1 is hindered,
large Stokes shift about 82 nm (Scheme 1).We successfully utilized which reduces the non-radiative pathway and restored the
the probe RV-1 for detection mitochondrial viscosity changes and fluorescence emission. In addition, with the viscosity of the
zebra fishes. And more importantly, we applied the probe RV-1 for methanol-glycerol mixture gradually increasing from 0.6 to 953 cP,
monitoring viscosity variations in the inflammatory mice.
the fluorescent emission exhibits a remarkable increase (48.5-fold)
when excited at 573 nm (Fig.1c). Moreover, as shown in Fig.1d, the
probe shows a good linear relationship between the fluorescence
Results and discussion
2
intensity (log I655) and viscosity (log η), (R = 0.99, x =0.58) by fitting
20
the Förster–Hoffmann equation. All of the above results indicate
that RV-1 has the potential to serve as an excellent probe for
detection of viscosity changes.
Design and synthesis
The novel probe RV-1 is consisted of two moieties, the 2, 3-
dihydro-1H-xanthene core and the imidazole moiety which has two
phenyl substituent groups. We envisioned that the imidazole
moiety could serve as a molecular rotor because the imidazole
moiety is connected to the 2, 3-dihydro-1H-xanthene core through
a single bond, which could freely rotate. The single bond which
connected the imidazole moiety and the 2, 3-dihydro-1H-xanthene
core could cause a dark non-emissive excited state in low viscous
7
media. In contrast, these rotations could be hindered in high
viscous media, leading to an enhancement of fluorescence emission.
Furthermore, the restriction of these rotations may also render the
whole molecule staying at the planar configuration in the charge
transfer state, and forming a strong ICT system, which could cause a
19
large Stokes shift. Moreover, compared with the classic
rhodamine B, the strong ICT system in RV-1 may render it has
longer absorption/emission wavelength than those of rhodamine B.
The probe RV-1 was readily synthesized by a two-step
reaction (Scheme
characterized by HNMR, C NMR, and high-resolution mass
spectroscopy (HRMS, see ESI† for details).
2). The structure of the probe was Fig.1 (a) Normalized absorption of the probe RV-1 in methanol and glycerol. (b)
1
13
Normalized fluorescence emission of the probe RV-1 in methanol and glycerol,
ex=570 nm. (c) Fluorescence spectra of RV-1 (10 µM) with the variation of
λ
solution viscosity (methanol-glycerol system).
relationship of log I655 and log η, R = 0.99, x =0.58.
ex
λ = 570 nm. (d) Linear
2
We further measured the quantum yield of the probe RV-1 in
different solvents. As shown in Table 1, the quantum yield of the
probe in other solvents was very low, in a range of 0.003-0.
0
18.Howerver, with the percentage of glycerol gradually increasing
in methanol, the quantum yield of the solvents also increased, and
reached to 0.116 in 99% glycerol. The above results indicated that
the probe RV-1 was sensitive to the viscous variations of the
solvents.
The solvent polarity is an important factor which might
influence the fluorescence of the probe. Because the cellular
environment is very complex, it is hardly possible to distinguish the
effect of viscosity on fluorescence or that from the polarity.
Therefore, we measured the fluorescence spectra of the probe RV-
Scheme 2 The synthesis of the probe RV-1.
Optical response of probe RV-1 to viscosity
To investigate the effects of viscosity to the new probe RV-1,
various mixtures of methanol and glycerol with different volume
1
in solvents with different polarities to confirm that the probe RV-1
2
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Table 1 The photo-physical data of probe RV-1 in different solvent systems
ARTICLE
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DOI: 10.103 [9b /] C8TB00298C
[a]
Solvents
Dielectric Constant
η (cP)
λ
ab(nm)
λ
em(nm)
Fluorescence Quantum Φ
H
2
O
78.4
9.1
1.01
0.43
0.37
1.54
0.53
0.32
0.54
2.24
0.80
0.59
13.4
953
571
570
573
568
574
571
568
576
575
573
571
573
651
652
653
650
653
652
650
655
656
655
653
655
0.003
0.013
0.016
0.015
0.014
0.018
0.013
0.017
0.018
0.015
0.034
0.116
Dichloromethane
MeCN
37.5
2.2
Dioxane
THF
7.6
Acetone
Chloroform
DMSO
20.7
5.1
48.9
36.7
32.6
-
DMF
MeOH
MeOH/glycerol[c]
Glycerol
45.8
[a] The viscosity of the solvent; [b] Rhodamine B was used as a standard reference with a quantum yield of 0.97 in ethanol; [c] MeOH/glycerol=5:5, (v: v).
was independent of solvent polarity. As shown in Fig.2, the probe proved that the probe RV-1 could be rendered as a probe for
RV-1 exhibits strong fluorescence emission in 99% glycerol. In measuring the viscosity changes of solvents.
contrast, in other solvents with different polarities, the probe RV-1
has almost no fluorescence. These results indicate that the probe is
not sensitive to the solvent polarity, and that it may be applied for
monitoring viscosity changes in complex biological environments.
2
Fig.3 Linear relationship of log τ and log η, R = 0.99, x =0.18
Theoretical Calculation and Mechanism
Fig.2 Fluorescence emission of the probe RV-1 in various solvents with different
The structure of RV-1 was optimized by DFT/TDDFT in B3LYP/6-
1G (d) of Gaussian 09. As shown in Fig.4a and FigS5，the 2, 3-
polarities.
3
dihydro-1H-xanthene core and the imidazole moiety of RV-1 stayed
almost on a planar conformation, which caused the molecular
structure of the probe RV-1 in the optimal state displayed a large
conjugated system. The frontier molecular orbitals in Fig.4b showed
that in methanol, when the dihedral angle at around C13-C12-C53-
N55 is ether 0° or 90°, the HOMO centered at the at the imidazole
moiety of RV-1, and the LUMO centered at the 2, 3-dihydro-1H-
xanthene core. These phenomena demonstrate that the probe RV-1
underwent an ICT process when excited from the ground state to
the excited state, which rendered a large Stokes shift. Moreover, as
shown in Fig. 4a, when the dihedral angle at around C13-C12-C53-
Fluorescence lifetime measurement
Because the fluorescence intensity may be influenced by the
concentrations of the probe, we measured the fluorescence lifetime
of the probe RV-1, as shown in Table S1, with the viscosity of the
solvent systems increasing, the fluorescence lifetime gradually
increased, which further indicated that the probe was sensitive to
the viscosity changes of the solvents. Moreover, according to the
20
Förster–Hoffmann equation, as shown in Fig. 3, the fluorescence
lifetime of the probe RV-1 (log τ) and viscosity (log η) exhibited a
2
good linear relationship (R =0.99, x=0.18). These results further
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N55 is 0°, the oscillator strength fem was 0.8259; when the dihedral addition DNA or BSA to the solution of the probe RVV-i1ewoAnrtliycleleOdnlitnoe
angle at around C13-C12-C53-N55 is 90°, the oscillator strength fem small enhancement of the fluorescence eDmOiIs: s1i0o.1n0.39T/hCe8sTeB0r0e2s9u8ltCs
was only 0.0028(Fig.4b). These results indicated that the probe showed that the fluorescence of the probe RV-1 is not influenced
molecule could form a twisted excited state by intramolecular by the biological macromolecules DNA, BSA, ROS, RSS, and ions.
rotation, which led to weak non-fluorescence emission. From these
calculation results, it was obviously that the probe RV-1 could be properties are two forms of the rhodamine molecules.
Ring-opening form and lactone with different fluorescence
23-25
At acidic
rendered as a probe for detection of the viscosity changes of the conditions, the rhodamine derivatives stand on the ring-open forms,
solvents.
which usually have very strong fluorescence emission. In contrast,
the rhodamine derivatives are on spirocyclic forms at basic
conditions, which often have no fluorescence. Therefore, the
fluorescence emission of rhodamine dyes is very sensitive to pH of
solutions. Thus, we investigated the pH effect on the probe RV-1. As
shown in Fig.5b, the probe RV-1 had stronger fluorescence
emission at pH 4-7 than the fluorescence emission at pH 7-10,
which could be attributed to the ring opening form in the acidic
solutions. However, notably, all of the fluorescence emissions in
PBS buffer solutions (Fig.5b) are much lower than that in high
viscous solutions (Fig.2a). These data suggest that the probe RV-1
could be applied for monitoring viscosity at physiological pH
conditions.
Fig.5 (a) 1. Fluorescence intensity of RV-1 (10 µM) at λem = 655 nm in PBS buffer
(
pH=7.4, 0.01 mM); 2. Fluorescence intensity of RV-1 (10 µM) at λem = 655 nm in 99%
glycerol and various species (200 µM). 1. blank; 2. 99% glycerol; 3. NaClO; 4. H ; 5.
HO∙; 6. Cysteine(Cys); 7. Homocysteine(Hcy); 8. Glutathione(GSH); 9. Cu2+; 10. Zn2+; 11.
2 2
O
Mg2+; 12. Fe3+; 13. Na ; 14. K ; 15. Ca2+; 16. DNA (200 µL, 100 mg/L); 17. BSA (200 µL,
+
+
100 mg/L). (b) pH effects on the probe RV-1.
Cell cytotoxicity assay and cell imaging
Cytotoxicity may influence the potential of a probe for
application in living cell imaging. To determine the cytotoxic
Fig.4 (a) The optimized geometries of RV-1 in the excited states; (b) frontier molecular effect of the probe RV-1, we carried out the MTT experiment.
orbital of RV-1 in the excited states with a dihedral angles of 0° at around C13-C12-
C53-N55; (c) frontier molecular orbital of RV-1 in the excited states with a dihedral
angles of 90° at around C13-C12-C53-N55. Calculations were by the DFT method (PCM
cells survived after 24 h. The results indicate that the probe
model) with a B3LYP/6-31G (d) basis set by using Gaussian 09.
The HeLa cells were incubated with RV-1 (0, 1, 2, 5, 10, 15, 20,
and 40 µM) for 24 h. As shown in Fig. S6 (ESI†), >90% HeLa
RV-1 had low cytotoxicity and may be suitable for living cell
Selectivity of probe RV-1 to viscosity and pH dependence of the imaging.
Monensin and nystatin could induce structural changes or
probe RV-1
swelling of mitochondria, which resulted in the mitochondria
The selectivity is a crucial requirement for fluorescent probes. Living
cells are very complex, multicomponent systems, which contain
26, 27
viscosity changes.
As shown in Fig.6, the cells treated with
only the probe RV-1 exhibited almost no fluorescence in the
TRITC channel (570-620 nm). By contrast, when added
Monensin or nystatin to the cells and stayed for 30 min, and
then were incubated with the probe RV-1 for another 30 min,
the cells showed strong red fluorescence in the TRITC channel.
The above results clearly suggest that the probe RV-1 could be
utilized to measure the viscosity variations in living cells.
21,
numerous biological macromolecules such as proteins and DNA.
22
These biological macromolecules might have crucial effect on the
cellular viscosity. Moreover, cells contain various reactive oxygen
species (ROS), reactive sulphur species (RSS), and ions, which may
influence the probe RV-1 in the complex biological system. Thus, we
investigated the selectivity of RV-1 for viscosity over DNA, BSA, ROS,
RSS and various ions by fluorescence spectrometry (Fig.5a). The
addition of ROS, RSS and various ions didn’t cause observable
changes in the fluorescence emission of the probe RV-1, and
4
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Green (Fig. S7, ESI†), suggesting that the probe RV-1 specifically
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DOI: 10.1039/C8TB00298C
exists on the mitochondria in the cells.
Fig.7 (a-c) Confocal fluorescence images of the HeLa cells after incubation with 10 µM
probe RV-1 + 10 µM Nystatin and Mito-Tracker Green (10 µM) at 37 °C for 30 min.(a)
Fluorescence image from probe RV-1 + 10 µM Nystatin (b) Fluorescence image from
Mito-Tracker Green; (c) The merged images; (d) The intensity profile of ROI lines. Scale
bar = 20 µM.
Fluorescence imaging in zebra fishes
Furthermore, we applied the probe RV-1 for detection
29
viscosity changes in zebra fishes. As shown in Fig.8, the zebra
fishes only treated with the probe RV-1 exhibited essentially
no fluorescence in the TRITC channel (570-620 nm). When
added Monensin or nystatin to the zebra fishes and stayed for
Fig.6 (A) (a-c) Confocal fluorescence images of the Hela cells incubated with 10 µM
free probe RV-1 for 30 min; (d-f) Confocal fluorescence images of the Hela cells
incubated with 10 µM probe RV-1 + 10 µM Monensin for 30 min. (g-i) Confocal
fluorescence images of Hela cells incubated with 10 µM probe RV-1+10 µM nystatin for
3
0 min, and then were treated with the probe RV-1 for
another 30 min, strong red fluorescence from the TRITC
channel and was observed. These phenomena were consistent
3
0 min. ; (B) Fluorescence intensity quantification. The images were collected in 570- with the observations in the living cells, which further
620 nm, excited at
λ
ex=561 nm, Scale bar = 20 µm, Data are mean S.D. (bars) (n= 3).
confirmed that the probe RV-1 could be applied for detection
of the viscosity changes in the living zebra fishes.
Because the ring-open forms of the rhodamine dyes which
possessed a positive charge delocalized through the whole
2
8
molecular system often had the mitochondria-selectivity, we
expected that the probe RV-1 also could be specifically localized on
the mitochondria. To test the specificity of the probe RV-1 for
mitochondria, we carried out the co-localization experiment with
the commercial mitochondria staining dye Mito-Tracker Green. As
shown in Fig. 7a, the cells exhibited red fluorescence in the TRITC
channel due to the emission of RV-1. At the same time, the cells
showed green fluorescence in FITC channel owing to the emission
of Mito-Tracker Green (Fig. 7b). The merged image (Fig.7c) indicate
that the two channels overlapped very well, and a high overlap
coefficient of 0.935 was observed. The intensity profiles of RV-1 and
Mito-Tracker Green for region of interest (ROI) lines in HeLa cells
also varied in close synchrony (Fig.7d). Moreover, a high correlation
was found in the intensity scatter plot of RV-1 and Mito-Tracker
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Fig.9 (a) In vivo fluorescence imaging of viscosity in the living mice using RV-1. (a)
00μL of 10μM RV-1 was injected to healthy mice; (b) 100μL of 10μM RV-1 was
1
injected to mice; (c) The relative average fluorescence intensity of ROI，Error bars
represent standard deviation (±S.D.). n = 3，the statistical analysis was performed
from three separate biological replicates. λex= 580 nm，λem= 660 nm
Conclusions
In summary, we have rationally designed phenyl-substituted
imidazole fused rhodamine RV-1 as a new viscosity probe. The
novel probe RV-1 showed a turn-on response to the viscosity
with a long absorption wavelength at 573 nm and a large
Stokes shift of 82 nm. RV-1 exhibited intense fluorescence
emission at about 655 nm in 99% glycerol, with a very large, up
to 48.5-fold enhancement of fluorescence intensity from
Fig.8 (A) (a-c) Confocal fluorescence images of the zebra fishes incubated with 10 µM
probe RV-1 for 30 min; (d-f) Confocal fluorescence images of the zebra fishes incubated
with 10 µM probe RV-1+10 µM Monensin for 30 min；(g-i) Confocal fluorescence
images of zebra fishes treated with 10 µM probe RV-1+10 µM nystatin for 30 min.
Scale bar =1000 µM. (B) Fluorescence intensity quantification. The images were
collected in 570-620 nm, excited at
bars) (n= 3).
λ
ex=561 nm, Scale bar = 20 µm, Data are mean S.D. methanol to 99% glycerol. Moreover, the probe RV-1 has been
successfully applied for detection of the viscosity change in
(
living cells and zebra fishes. More importantly, we used the
Fluorescence imaging in living mice
probe RV-1 to monitor viscosity changes in living mice. We
expect that the new probe RV-1 could be exploited as a
powerful molecular tool for studying the critical role of
viscosity changes in biological systems.
Lipopolysaccharide(LPS) has been reported that it could
30
increase the blood viscosity of the rat .Therefore we used
LPS-injected mouse for imaging the blood viscosity changes in
vivo. As shown in Fig.9, The healthy mice displayed essentially
no fluorescence treaded with RV-1. By contrast, the LPS-
injected mice showed a very strong fluorescence emission,
Experimental
which displayed that the probe RV-1 could detection of blood Preparation of the test solutions
viscosity changes in the LPS-injected mice.
Hydroxyl radical was generated by in situ by the Fenton
31
reactions. To 3 mL solution of RV-1 in H
2
O, hydrogen
peroxide stock solution (120 µL, 100 mM) was added. Aqueous
2+
2 2
Fe (60 µL ,100 mM) was then added to the probe RV-1/H O
solution to generate hydroxyl radical (200 µM).The
6
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concentration of the solutions of NaClO, H
ARTICLE
2 2
O
, cysteine(Cys), 6H). 13C NMR (101 MHz, Chloroform-d) δ 170.13 , 152.96 , 151.94 ,
View Article Online
DOI: 10.1039/C8TB00298C
2+
2+
2+
3+
homocysteine(Hcy), glutathione(GSH), Cu , Zn , Mg , Fe , 149.82 , 144.75 , 135.00 , 131.88 , 129.63 , 128.70 , 128.39 , 128.29 ,
+
+
2+
Na , K , Ca were 200 µM, the concentration of the solutions 128.00 , 127.36 , 127.11 , 124.98 , 124.26 , 120.36 , 119.00 , 114.06 ,
of BSA and DNA were 200 µL, 100 mg/L.
108.59 , 104.75 , 97.72 , 44.45 , 12.53 . HRMS (ESI): m/z calculated
+ [M+], 590.2438, found 590.2438.
32 3 3
for C39H N O
Measurement of Fluorescence lifetime
The fluorescence lifetime of the probe RV-1 was measured by
Edinburgh FLS920 Fluorescence
concentration of the dye was 10.0 µM in various solvent
mixture (methanol-glycerol solvent systems).
Spectrometer. The
Conflicts of interest
There are no conflicts of interest to declare.
Measurement of fluorescence quantum yield (Φ)
Fluorescence quantum yield was determined by the relative
comparison with Rhodamine B (Φs = 0. 97 in ethanol) as
standard for RV-1 in different solvents. And they were
Acknowledgements
32
calculated by equation 1.
퐼퐴푠 휂2
This work was financially supported by NSFC (21472067 and
훷 = 훷푆 퐼푠퐴
∙
(1)
2
휂푠
21672083), Taishan Scholar Foundation (TS 201511041), and the
startup fund of the University of Jinan (309-10004).
In which, A is the absorbance, I is the integrated fluorescence
intensity, and η is the refractive index of the solvent.
The Förster-Hoffmann equation
Notes and references
The relationship between the fluorescence emission intensity
1
2
A. S. Verkman, Trends Biochem. Sci, 2002, 27, 27-33.
of the probe RV-1 and the solvent viscosity could be
G. Guigas, C. Kalla and M. Weiss, Biophys. J., 2007, 93, 316-
2
0
formulated by the Förster-Hoffmann equation:
3
23.
3
4
K. Luby-Phelps, Mol. Biol. Cell, 2013, 24, 2593-2596.
log I = C+ xlogη
log τ =C+ xlogη
(2)
(3)
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.0 ml 98% H SO ，and was stirred at 90°C for 12h. Then, the
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mixture was cooled to room temperature, poured onto ice (200 g).
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afford compound RV-1 as a dark purple solid (0.696g, 59%). H NMR 15 L. D. Lavis, Annu. Rev. Biochem., 2017, 86, 825-843.
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dt, J = 20.2, 7.3 Hz, 2H), 7.55 – 7.43 (m, 5H), 7.37 – 7.22 (m, 6H),
1
7
1
.20 (d, J = 7.6 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 6.49 (d, J = 8.8 Hz,
H), 6.37 – 6.26 (m, 2H), 3.30 (q, J = 7.1 Hz, 4H), 1.14 (t, J = 7.0 Hz,
This journal is © The Royal Society of Chemistry 20xx
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Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB00298C
We designed a novel mitochondria-targeted rhodamine analogue for detection of viscosity
changes in living cells, zebra fishes, and living mice.