Ratiometric detection of viscosity using a two-photon fluorescent sensor

Article abstract of DOI:10.1002/chem.201202646

In the thick of it: A carbazole-based cyanine derivative has been used as a ratiometric fluorescent sensor to detect mitochondrial viscosities over the range 1-950 cP within live cells and living tissues by using two-photon microscopy. Its success is based on the fast- and low-barrier rotation around its methine bond (see figure) and the huge increase of the fluorescence quantum yield (QY) from the loss of mobility in viscous media. Copyright

Full text of DOI:10.1002/chem.201202646

DOI: 10.1002/chem.201202646
Ratiometric Detection of Viscosity Using a Two-Photon Fluorescent Sensor
Fei Liu,[a] Tong Wu,[a] Jianfang Cao,[a] Shuang Cui,[b] Zhigang Yang,[a] Xinxin Qiang,[a]
Shiguo Sun,[a] Fengling Song,[a] Jiangli Fan,[a] Jingyun Wang,*[b] and Xiaojun Peng*[a]
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Cellular viscosity is known to influence how biomolecules
and chemical signals interact and are transported within live
cells, and consequently abnormal changes in cellular viscosi-
ty are related to cellular aspects of many diseases and mal-
functions.[1,2] At this time, however, the existing mechanical
methods for the measurement of viscosity cannot be applied
at the cellular level.
living tissues. In the fields of biological research and pathol-
ogy, using the TPM technique and chemicals to visualize vis-
cosity are very significant.
In the present investigation, a carbazole-based cyanine
derivative Caz-Cy2 (labs =485 nm; lemACHTUNRGTNEUNG(blue)=380 nm,
As the rotation of a molecular rotor is an efficient
quenching pathway in nonviscous media, a fluorescent com-
pound coupled with a rotor moiety can result in weak intrin-
sic fluorescence.[3–8] However when the rotation is restricted,
as in viscous media, emission is enhanced. Based on this
principle, Theodorakis et al.[9] produced a ratiometric viscos-
ity sensor comprising a dye with two distinct fluorescent
units. A coumarin acted as a donor fluorophore to induce
excitation of the rotor moiety (CMAM) by means of reso-
nance energy transfer (RET), thereby allowing rapid meas-
urement of the viscosity. Suhling et al.[10] reported a dipyrrin
fluorescent sensor in which restricted rotation of a phenyl
group permitted the imaging of the local microviscosity. Our
group subsequently reported a fluorescent sensor RY3, in
lemACTHUNRTGNEUNG(red)=580 nm) was synthesized by linking the Caz and
semi-Cy2 moieties to give rise to a ratiometric two-photon
(TP) viscosity sensor. The spectrum of Caz has a short wave-
length peak similar to Caz-Cy2 (labs =345 nm, lemACHTUNGTRENNUNG(blue)=
368 nm; Figures S1 and S2 in the Supporting Information).
Semi-Cy2, the conjugated electron acceptor, was hypothe-
sized to be rotated around bond f1 (labeled red in the for-
mulae) with Caz in unconstrained environments, and so
gave rise to the red emission. Compared with traditional cy-
anine dyes such as Cy3 and Cy5, Caz-Cy2 showed unique
spectroscopic characteristics, such as very low fluorescence
in nonviscous solvents and a large Stokes shift (>100 nm).
As shown in Figure 1, the quantum yield (QY) of Caz-Cy2
ꢀ
which an aldehyde group ( CH=O) acted as a rotor to
measure the intracellular viscosity.[11] RY3 was the first mo-
lecular rotor capable of dual-mode fluorescence imaging,
potentially permitting investigation of the pathologies of dis-
eases.
Recently, two-photon microscopy (TPM), which employs
two near-infrared photons as the excitation source, has
become important in bioimaging applications.[12,13] Thus,
TPM has the advantage of offering intrinsic 3D resolution
combined with reduced phototoxicity, increased specimen
penetration (>500 mm), localized excitation, and prolonged
observation time.[14–17] In addition, the long-wavelength exci-
tation (>700 nm) effectively avoids background interference
owing to the autofluorescence of the biomatrix. Recently,
Choꢁs[18,19] group developed a series of two-photon fluores-
cent sensors based on the naphthalene framework that can
be used in live cells and intact tissues and is visualized by
TPM. However, although the TPM technique and TPM sen-
sors have been applied in the detection of metal ions and
small molecules, and for the labeling of proteins and DNA,
to our knowledge, no TPM sensor has yet been found that
detects cellular microenvironmental viscosity, especially in
Figure 1. A histogram showing the fluorescence quantum yields of Caz-
Cy2 (1 mm) in different solvents (lem =580 nm): a) dichloromethane,
b) DMSO, c) ethanol, d) dioxane, e) methanol, f) water, g) water/glycerol
(8:2 v/v), h) water/glycerol (6:4 v/v), i) water/glycerol (4:6 v/v), j) water/
glycerol (2:8 v/v), k) glycerol (99%). Rhodamine B was used as a stand-
ard reference with a fluorescence quantum yield of 0.97 in ethanol.
[a] F. Liu, Dr. T. Wu, J. Cao, Dr. Z. Yang, Dr. X. Qiang, Dr. S. Sun,
Dr. F. Song, Dr. J. Fan, Prof. X. Peng
State Key Laboratory of Fine Chemicals
Dalian University of Technology
No. 2 Linggong Road, High-Tech District
Dalian 116024 (P.R. China)
Fax : (+86)411-84986306
was very low, approximately 0.012–0.04, in low-viscosity sol-
vents, and was not apparently affected by solvent polarity.
This is crucial to permit a molecular rotor to sense environ-
mental viscosity.[20] On the other hand, Caz-Cy2 showed a
dramatic fluorescence enhancement with increases of the
solvent viscosity. As glycerol was gradually added to water,
the viscosity of the solutions increased from 1.0 cP (water)
to approximately 950 cP (99% glycerol), and paralleling
this, the fluorescence of Caz-Cy2 increased 23-fold (QY=
0.28) in 99% glycerol (Figure 1g–k).
E-mail: pengxj@dlut.edu.cn
[b] S. Cui, J. Wang
School of Life Science and Biotechnology
Dalian University of Technology
No. 2 Linggong Road, High-Tech District
Dalian 116024 (P.R. China)
E-mail: wangjingyun67@126.com
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202646.
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Figure 2. a) Changes in the fluorescence emission spectra of Caz-Cy2 as a
function of the solvent viscosity (excited at 335 nm); b) the linear re-
sponse between the log(I385nm/I580nm) and the log(viscosity) in the water/
glycerol solvent (excited at 335 nm); c) the red-fluorescence-emission
spectra of Caz-Cy2 as a function of solvent viscosity (excited at 480 nm);
d) the linear response between log(I/I0) and log(viscosity) in the water/
glycerol solvent (excited at 480 nm).
Figure 3. Two-photon (TP) action spectra of Caz-Cy2 in solvents of vary-
ing viscosity. TP excitation: 720–920 nm.
Carbazole and its derivatives have been reported to have
excellent two-photon properties.[22–24] As shown in Figure 3,
the two-photon absorption spectra of Caz-Cy2 increased
dramatically with a rise in solvent viscosity, which was ach-
ieved by increasing the proportion of glycerol in the water.
From 1.0 P (water) to approximately 950 cP (99% glycerol),
the two-photon action cross-section (dF) at 720 and 920 nm
increased from 3.9 to 81.2 GM and 5.3 to 29.6 GM, respec-
tively (Figure 3; 1 GM=10ꢀ50 cm4 sphotonꢀ1).
As illustrated in Figure 2, the fluorescence spectrum of
Caz-Cy2 possesses two emission bands in aqueous solution.
The red-emission band (lem
much faster than the blue-emission band (lem
380 nm) as the solvent viscosity was increased. Moreover,
the logarithm of the fluorescence ratio thereof (I580nm/I380nm
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
)
had a linear relationship with that of the viscosity (h) of the
solution, which was identical to that measured for the TP
process (see Figure S3 in the Supporting Information). This
is as expected from the Fçrster–Hoffmann equation
[Eq. (1)]:[21]
Possible effects of intracellular biochemicals on the use of
Caz-Cy2 as a viscosity sensor were evaluated by adding vari-
ous compounds to Caz-Cy2 solutions. As seen in Figures S6
and S7 in the Supporting Information, the spectrum of the
dye was influenced more by viscosity than by these biologi-
cal species.
Cell-staining experiments showed that Caz-Cy2 could
readily enter live HeLa cells and give clear fluorescence
images. To investigate the localization of Caz-Cy2, dual
staining with two commercially available organelle sensors
was carried out. As seen in Figure 4, the dye colocalized
with the commercially available mitochondrial sensor, Mito
Tracker Deep Red FM, with weighted colocalization coeffi-
cient values to 0.98 (analyzed using Olympus Micro FV10-
ASW software). Dual staining of Caz-Cy2 with the nuclear
log If ¼ C þ x log h
ð1Þ
in which C is a concentration- and temperature-dependent
constant and x is a dye-dependent constant. Therefore,
log(I580nm/I385nm) of Caz-Cy2 and logh were fitted according-
ly. The R2 of 0.99 and the slope x of 0.27 indicate that Caz-
Cy2 could be applied as a ratiometric sensor to quantitative-
ly detect the solution viscosity (Figure 2b).
As shown in Figure 2c and d, we investigated the red-
emission spectra of Caz-Cy2 in mixtures of water and glycer-
ol. The emission of rotor Caz-Cy2 was greatly enhanced and
exhibited a stable spectrum in its fluorescence intensity
within one hour (Figure S5a in the Supporting Information)
in solvents of high viscosity when excited at 480 nm. The
emission intensity at 580 nm in glycerol was 80-fold that
seen in water, and the logarithm of the fluorescence ratio
thereof (I/I0) at 580 nm showed a good linear relationship
with that of the viscosity of the solution. The R2 of 0.98 and
the slope x of 0.52 indicate that Caz-Cy2 could be applied
with the fluorescence-enhancement method that senses the
viscosity change by the rotor Caz-Cy2.
Figure 4. Live-cell counterstain of Caz-Cy2 with MitoTracker Deep Red
FM (MitoTracker for short). One- and two-photon excitation were at 488
and 720 nm, respectively: a) TPM image of Caz-Cy2 (530-580 nm);
b) OPM image of MitoTracker (690–740 nm); c) overlap of (a) and (b);
d) overlap of (c) with the white-light image.
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Ratiometric Detection of Viscosity
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dye DRAQ 5, however, showed no colocalization (see Fig-
ure S8 in the Supporting Information).
the small intracellular red zones represent high viscosity mi-
croenvironments (>900 cP) in cells. It is worth noting that
the changes in the emission ratios measured deep inside the
tissue slice are comparable to those in the cells. Further-
more, it demonstrates that Caz-Cy2 is capable of detecting
viscosity at depths of 60–130 mm in live tissues using TPM
(see Figure S9 in the Supporting Information). In addition,
the two-photon excitation fluorescence (TPEF) stability of
Caz-Cy2 could be visualized for more than 44 min without
appreciable decay in the tissue (see Figure S5b in the Sup-
porting Information); this means that Caz-Cy2 has a high
photostability for deep-tissue imaging.
To clearly understand the rotor mechanism occurring with
Caz-Cy2, the molecular geometries and photophysical prop-
erties of Caz-Cy2 were investigated computationally (see
the Supporting Information). A DFT study of Caz-Cy2 con-
firmed that the nonradiative deactivation is mainly accessed
by rotating the monomethine bridge. When this free rota-
tion is restricted, it leads to an increase in the fluorescence
quantum yield. As shown in Figure 7a, there are several me-
thine bonds in Caz-Cy2 that may be involved in the rotation.
However, the results of the quantum chemical calculations
were able to explain which of these caused the mainly non-
radiative deactivation (see Figure S11 in the Supporting In-
formation). The calculated activation energy and energy gap
(the energy difference between the initial and final electron-
A wide range of human diseases, such as cancers, Alz-
heimerꢁs disease (AD), and diabetes, are closely related to
mitochondrial dysfunction, which is caused by pathological
processes, such as overproduction of reactive oxygen species
(ROS), reactive nitrogen species (RNS), and amyloid beta
peptide (Ab).[25–27] Such pathological processes may involve
changes in viscosity, and consequently Caz-Cy2 may offer a
new approach to investigating such processes.
Since the fluorescence signal ratio is independent of the
dye concentration or intracellular distribution, it is consid-
ered superior to using the intensity signal. So Caz-Cy2 with
two emission peaks can be also applied to the ratiometric
fluorescence imaging of viscosity in live cells. Excited at
720 nm, the fluorescence images were obtained by collecting
the blue emission of (375–440 nm) (green channel: Fig-
ure 5a) and the red emission of (575–645 nm) (red channel:
Figure 5. Live HeLa cell staining with Caz-Cy2 (10 mm, 40 min incuba-
tion) excited at 720 nm. A 1000ꢂ magnification was utilized in the imag-
ing: a) blue channel (375–440 nm); b) red channel (575–640 nm); c) over-
lap of images (a) and (b); d) the ratio image of Caz-Cy2 obtained by the
Image Pro-plus software.
Figure 5b). The ratio image (I580 nm/I380 nm) clearly displayed
the distribution of viscosity in the cells. In Figure 5d, the
“blue” imaging areas of HeLa cells indicate low viscosity
with small ratio values (<100 cP); the “green” regions indi-
cate an intermediate viscosity with the ratio value of 100–
500 cP; and the small intracellular red zones represent high
viscosity microenvironments (>900 cP) in cells.
We further investigated the application of this sensor in
tissue imaging (Figure 6). A fresh rat hepar slice, incubated
with 100 mm Caz-Cy2 for 40 min, shows the viscosity distri-
bution at a 110 mm depth. In Figure 6d, the “blue” imaging
areas of the tissue slice indicate low viscosity with small
ratio values (<100 cP); the “green” regions indicate an in-
termediate viscosity with the ratio value of 100–500 cP; and
Figure 6. Images of a fresh rat hepar slice stained with Caz-Cy2 (100 mm,
40 min incubation) at 110 mm depth, excited at 720 nm: a) blue channel
(375–440 nm); b) red channel (575–640 nm); c) overlap of images (a),
(b), and the white-light image; d) the ratio image of Caz-Cy2 obtained by
the Image Pro-plus software.
Figure 7. a) The probable rotation of different chemical bonds. b) Poten-
tial-energy curves of Caz-Cy2 at the energy levels of the S0, S1, S2, and S3
states with torsion angles around bond f1; ic=intersystem crossing.
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X. Peng et al.
in which the subscripts s and r stand for the sample and reference mole-
cules, respectively. The intensity of the signal collected by a charged-cou-
pled device (CCD) detector was denoted as S. F is the fluorescence
quantum yield. f is the overall fluorescence collection efficiency of the
experimental apparatus. The number density of the molecules in solution
was denoted as c. dr is the TPA cross-section of the reference molecule.
ic states) for rotations around different chemical bonds in
Caz-Cy2 in the S0 and S1 states are listed in Table S1 in the
Supporting Information. The data suggest that rotation
about the central double bond (f2) is most difficult, since
there is a higher energy barrier to rotation in the S1 state.
The rotation around the indolium–ethylene bond (f3) is also
impossible, since there is a large energy gap to the ground
state, and the decay rate of excited electronic states depends
exponentially on the energy gap. The state formed by twist-
ing of the f1 bond possesses a remarkably low energy gap to
the ground state, and a lower barrier to rotation in the S1
state, which causes mainly nonradiative deactivation (Fig-
ure 7b).
Live-cell incubation: HeLa cells were cultured in Dulbeccoꢁs modified
Eagleꢁs medium (DMEM; Invitrogen) supplemented with 10% fetal calf
serum (FCS; Invitrogen). One day before imaging, the cells were seeded
into 24-well flat-bottomed plates. The next day, the cells were incubated
with 8.0 mm dye for 40 min at 378C under 5% CO2 and washed with
phosphate-buffered saline (PBS) three times.
Preparation and staining of the tumor slices: Tumor slices were prepared
from nude mice, which were seeded S180 cells. Normal tissue slices were
prepared from the liver of nude mice. Slices were incubated with Caz-
Cy2 (20 mm) in artificial cerebrospinal fluid (ACSF; 138.6 mm NaCl,
3.5 mm KCl, 21 mm NaHCO3, 0.6 mm NaH2PO4, 9.9 mm d-glucose, 1 mm
CaCl2, and 3 mm MgCl2) bubbled with 95% O2 and 5% CO2 for 30 min
at 378C. Slices were then washed three times with ACSF and transferred
to a glass-bottomed dish (MatTek, 35 mm dish with 20 mm bottom well)
and observed in a spectral confocal multiphoton microscope.
In conclusion, a carbazole-based cyanine derivative Caz-
Cy2 (labs =485 nm; lemACHTNUTGRENN(UG blue)=380 nm, lemACHTUNTGREN(NUNG red)=580 nm)
was developed as a ratiometric TP viscosity sensor. Caz-Cy2
was able to quantitatively detect the solution viscosity with-
out any apparent influence from the environmental polarity
or biomacromolecules. Caz-Cy2 is an excellent TP rotor
sensor candidate that appears able to report on the viscosity
of mitochondria in live cells as well as in living tissues at
depths of 60–130 mm by ratiometric fluorescence imaging.
Fluorescence imaging: One-photon fluorescence imaging and two-photon
fluorescence ratio imaging of Caz-Cy2 in cells were obtained with spec-
tral confocal multiphoton microscopes (Leica TCS SP2). Two-photon flu-
orescence microscopy images of Caz-Cy2 and cells were obtained with a
DM IRE2 microscope (Leica) by exciting Caz-Cy2 with a mode-locked
titanium–sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs)
set at a wavelength of 720 nm and output power of 1230 mW.
The photostability test: The two-photon photostability of Caz-Cy2 was
established as described in the literature.[30] A microscope was used and
Caz-Cy2 was excited with a mode-locked titanium–sapphire laser source
(Coherent Chameleon, 90 MHz, 200 fs) set at a wavelength of 720 nm
and an output power of 1230 mW, which corresponded to an approxi-
mately 10 mW average power in the focal plane. In this process, the laser
has been turned on and is continuously exciting the sample. A CCD re-
corded a set of intensities at various time intervals (2 min) during 44 min.
Moreover, the average intensity of every set of intensities data was calcu-
lated by using Olympus Fluoview, version 3.0, software.
Experimental Section
Viscosity determination and fluorescence spectral measurement detec-
tion: The solvents were obtained by mixing a water/glycerol system.
Measurements were carried out with a NDJ-7 rotational viscometer, and
each viscosity value was recorded. The solutions of Caz-Cy2 of different
viscosity were prepared by adding the stock solution (1.0 mm) to the sol-
vent mixture (10 mL; water/glycerol) to obtain the final concentration of
the dye (1.0 mm). These solutions were sonicated for 5 min to eliminate
air bubbles. After standing for 1 h at a constant temperature, the solu-
tions were measured in a UV spectrophotometer and a fluorescence
spectrophotometer.
Synthesis of Caz-Cy2: Quaternized salt c (1.58 g, 5.0 mmol) and aldehyde
b (1.00 g, 4.5 mmol) were added to a 100 mL flask with ethanol (50 mL),
followed by piperidine catalyst (1.0 mL). The mixture was stirred for 12 h
at 808C. On standing at room temperature, a residue precipitated out
from ethanol; further recrystallization in ethanol gave the desired prod-
uct. M.p. 120–1228C; 1H NMR (400 MHz, CDCl3): d=9.11–9.05 (m,
1H), 8.54 (s, 1H), 8.47 (d, J=8.7 Hz, 1H), 8.41 (d, J=15.8 Hz, 1H), 7.91
(d, J=15.8 Hz, 1H), 7.52 (d, J=12.6 Hz, 5H), 7.42 (s, 2H), 7.33 (s, 1H),
5.01 (d, J=7.3 Hz, 2H), 4.32 (d, J=7.2 Hz, 2H), 1.85 (s, 6H), 1.62 (s,
3H), 1.43 ppm (t, J=7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3): d=
157.27, 144.20, 140.45, 130.79, 129.43, 128.70 127.02, 125.43, 124.39,
121.14, 113.95, 108.80, 77.78, 77.05, 76.73, 38.16, 27.68 ppm; HR-TOF-
Absorption and fluorescence-quantum-yield measurements: Absorption
spectra were measured on a Lamda LS35 spectrophotometer. Fluores-
cence spectra were obtained with a FP-6500 spectrophotometer (Jasco,
Japan). The relative fluorescence quantum yields were determined with
rhodamine B as a standard and were calculated using Equation (2):
2
Fx ¼ FsðFx=FsÞðAs=AxÞðlexs=lexxÞðnx=nsÞ
in which F represents the quantum yield, F stands for the integrated area
under the corrected emission spectrum, A is the absorbance at the excita-
tion wavelength, lex is the excitation wavelength, n is the refractive index
+
MS: m/z calcd for C28H29N2 [M]+: 393.2325; found: 393.2338.
of the solution (because of the low concentrations of the solutions (10ꢀ7
–
10ꢀ8 molLꢀ1), the refractive indices of the solutions were replaced with
those of the solvents), and the subscripts x and s refer to the unknown
and the standard, respectively.
Measurement of the TP cross-section: The TP cross-section (d) was de-
termined by using a femtosecond (fs) fluorescence measurement tech-
Acknowledgements
ACHTUNGTRENNUNG
nique as described in the literature.[28] The TP-induced fluorescence in-
tensity was measured at 720–900 nm by using rhodamine 6G as the refer-
ence, the two-photon property of which has been well characterized in
the literature.[29] The intensities of the two-photon-induced fluorescence
spectra of the reference and sample emitted at the same excitation wave-
length were determined. The TPA cross-section was calculated by using
Equation (3):
This work was supported by the National Science Foundation of China
(21136002, 21076032, 20923006), the National Basic Research Program of
China (2009CB724706, 2013CB733702), the High Technology Research
and Development Program of China (863 program) (2011AA02A105).
Keywords: cells
microscopy · viscosity
· fluorescence · sensors · two-photon
d ¼ drðSsFrꢀrcrÞ=ðSrFsꢀscsÞ
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Received: July 25, 2012
Revised: November 12, 2012
Published online: December 19, 2012
Chem. Eur. J. 2013, 19, 1548 – 1553
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