A Nile Red/BODIPY-based bimodal probe sensitive to changes in the micropolarity and microviscosity of the endoplasmic reticulum

Article abstract of DOI:10.1039/c4cc04915b

We herein report a fluorescent bimodal probe (1) capable of determining ER viscosity and polarity changes using FLIM and fluorescence ratiometry, respectively; during ER stress caused by tunicamycin, the viscosity was increased from ca. 129.5 to 182.0 cP

Full text of DOI:10.1039/c4cc04915b

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A Nile Red/BODIPY-based bimodal probe sensitive
to changes in the micropolarity and
microviscosity of the endoplasmic reticulum†
Cite this: Chem. Commun., 2014,
50, 11672
Received 27th June 2014,
Accepted 8th August 2014
Zhigang Yang,a Yanxia He,a Jae Hong Lee,a Weon-Sik Chae,b Wen Xiu Ren,a
Joung Hae Lee,c Chulhun Kang*d and Jong Seung Kim*a
DOI: 10.1039/c4cc04915b
www.rsc.org/chemcomm
We herein report a fluorescent bimodal probe (1) capable of deter- Subsequently, our group reported a molecular rotor based on
mining ER viscosity and polarity changes using FLIM and fluorescence through-bond energy transfer, containing a coumarin unit and a
ratiometry, respectively; during ER stress caused by tunicamycin, the BODIPY moiety, to measure mitochondrial viscosity in live cells.12
viscosity was increased from ca. 129.5 to 182.0 cP and the polarity of
the ER (dielectric constant, e) enhanced from 18.5 to 21.1.
On the other hand, for polarity measurements in biological
systems, fluorophores with an intramolecular charge transfer
(ICT) (e.g., Nile Red) have been widely adopted.13 However, to
Viscosity and polarity, two significant environmental factors, understand cell-based biological events such as ER stress, it
markedly influence the behaviour of the surrounding targets1 in would be desirable to have a bimodal fluorescent probe that
various chemical and biological processes, including intracellular can be used to measure viscosity and polarity simultaneously
diffusions (of nutrients, metabolites, signalling proteins, etc.) and and that can also be localised in the cell. To our knowledge,
changes in the configurations of proteins and cell membranes.2,3 such a probe has not been reported yet.
Endoplasmic reticulum (ER) is a cellular organelle involved in the
In this context, we synthesized a new bimodal fluorescent
synthesis of secretory and membrane proteins and their post- probe (1) composed of BODIPY and Nile Red linked with a
translational modification, including glycosylation. The accumu- –(CH2)6– spacer (Scheme 1). The BODIPY moiety, designed as a
lation of large amounts of unfolded or misfolded proteins in the fluorescent molecular rotor, can be used to quantify local
ER activates the ER stress response,4 which has been implicated cellular viscosity by fluorescence lifetime imaging microscopy
in various human diseases such as diabetes and Alzheimer’s (FLIM), and Nile Red is a polarity-sensitive fluorophore.14 We
disease.4,5 The accumulation of partially unfolded or glycosylated report herein that the newly synthesized probe localizes in the
proteins in the ER would certainly alter the viscosity and polarity ER and can be used to quantify distinct changes in both local
of the ER.6 Therefore, it is reasonable to anticipate alteration of viscosity and local polarity of ER-stressed HeLa cells.
viscosity and polarity during ER stress, and it is of interest to
monitor the corresponding changes.
The probe (1) was prepared by conjugation of Nile Red and
BODIPY with a –(CH2)6– group. Initially, m-N,N-diethylamino-
With the emergence of biosensing technology, various fluores- phenol was subjected to nitrosation to provide intermediate 4,
cent chemosensors have been introduced to measure biological
viscosity7 and polarity.8 For viscosity measurements, ‘‘molecular
rotors’’ have been developed for use with ratiometric9 or fluores-
cence lifetime imaging techniques.10 A bimodal molecular rotor
that could be used for dual fluorescence ratiometry and fluores-
cence lifetime imaging was developed by Peng and coworkers.11
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
E-mail: jongskim@korea.ac.kr
b Gangneung Center, Korea Basic Science Institute, Gangneung 210-702, Korea
c Korea Research Institute of Standards and Science, Daejon 305-600, Korea
d The School of East-West Medical Science, Kyung Hee University, Yongin 446-701,
Korea. E-mail: kangch@khu.ac.kr
† Electronic supplementary information (ESI) available: Limited experimental
and spectral data, cell imaging figures, and 1H and 13C NMR. See DOI: 10.1039/
c4cc04915b
Scheme 1 Chemical structure of the target compound (1).
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intensity of BODIPY was enhanced ca. 10-fold with increasing
solvent viscosity from 0.6 (methanol) to 950 cP (99% glycerol) at
room temperature (25 1C) (Fig. 2A). For fluorescence lifetime
measurements, upon extrapolation of the fluorescence decay of
BODIPY, we noticed that two factors (BODIPY, t1 and Nile Red, t2)
contributed to the emission at 516 nm. The fluorescence lifetime
of BODIPY (t1) markedly increased from ca. 0.6 ns in MeOH to 10
ns in 99% glycerol because free rotation around the C–C bond
between BODIPY and the phenyl unit was gradually suppressed by
increasing solvent viscosity. The linear relationship (R2 = 0.98) of
t1 vs. viscosity (Fig. 2B) could be used to quantify the local viscosity.
For Nile Red (Fig. 2A), the maximum emission wavelength red
shifted and the fluorescence was gradually quenched as the
proportion of glycerol in a methanol–glycerol mixture increased.
This is probably because the solvent polarity changed (dielectric
constants at 25 1C: methanol, e = 32.6; glycerol, e = 45.8) or because
solubility in a viscous medium is considerably reduced. Further-
more, we found that the fluorescence lifetime of Nile Red (ca. 5 ns,
mono-exponential decay) in the probe rarely changed even with
markedly increased solvent viscosity (Fig. S1, ESI†).
Second, we investigated the changes in fluorescence spectra
and the fluorescence lifetime of the probe in nonviscous solvents
with different polarities. Very sharp changes in the fluorescence of
Nile Red in 1 were observed in solvents of different polarities and
low viscosity (Fig. 1B; e.g., 1,4-dioxane, e = 2.25, Z = 1.3 cP; MeOH,
e = 32.6, Z = 0.6 cP at 25 1C). As the proportion of the polar solvent
(water, e = 78.3 at 25 1C) in the binary mixture (1,4-dioxane–water)
increased from 0% to 70%, corresponding to an increase in
solvent mixture polarity,15 the maximum emission wavelength
of Nile Red shifted from 565 to 635 nm because of emission from
the ICT excited state, and its fluorescence intensity markedly
decreased (Fig. 2C). From a plot of the fluorescence intensity ratio
I575/I625 vs. dielectric constant (fitting with an exponential relation-
ship with R2 = 0.98) (Fig. 2D), we could estimate the local polarity
of certain media and even live cells.13 As shown in Fig. 2D, the
I575/I625 ratio decreases drastically, from ca. 6.0 to 0.5, with
increasing solvent polarity when the dielectric constant is below
20; this change becomes more gradual when the solvent dielectric
constant is over 20, indicating that Nile Red is very sensitive to
nonpolar environments. The corresponding fluorescence lifetime
(mono-exponential decay) is shortened from ca. 9 to 5 ns with
increasing solvent polarity. Since the BODIPY moiety in 1 rarely
responds to polarity (Fig. S1, ESI†), it exhibits very low fluores-
cence intensity and an invariable fluorescence lifetime. Therefore,
1 is a dual-function probe that can be used to quantify the local
viscosity and polarity of certain media through changes in the
fluorescence of BODIPY and Nile Red, respectively.
Fig. 1 Absorption (A) and emission (B) spectra of 1 (5.0 mM) in various
nonviscous solvents, excited at 488 nm.
which was subsequently reacted with naphthalene-1,6-diol to afford
Nile Red (3); this was followed by attachment to the –(CH2)6– spacer
to produce 2. Reaction of 2 with 4-hydroxylphenyl BODIPY (6)
afforded 1 in moderate yield. Detailed procedures and physical
properties of compounds are provided in the ESI.†
To characterize the spectral properties of the probe, we first
studied its solvent dependency. As shown in Fig. 1, the probe exhibits
two absorption and two emission bands mainly contributed by its
BODIPY and Nile Red moieties. The peaks centred at 490 and 516 nm
correspond to the maximum absorption and emission wavelengths
of BODIPY, which is almost nonfluorescent in nonviscous solvents
because of the free rotation of BODIPY.12 The maximum absorption
and emission wavelengths of Nile Red shifted in solvents of varying
polarities (labs, 525–575 nm and lem, 565–635 nm for 1,4-dioxane to
MeOH), and the fluorescence intensity gradually decreased with
increasing solvent polarity, owing to the ICT process that enables
this dye to respond to environmental polarity.
To prove that the probe can be used to determine the local
viscosity and polarity of certain media, we investigated fluores-
cence responses of the sensor towards arbitrary changes in
viscosity or polarity. First, fluorescence spectra and the fluores-
cence lifetime of the probe were measured in solvents of different
viscosities. As shown in Fig. 2 and Fig. S1, ESI† the fluorescence
Before 1 could be used as a bimodal probe to measure
viscosity and polarity in the ER, the intracellular localisation of
the probe had to be determined. In the preparation for colocal-
Fig. 2 Fluorescence spectra and fluorescence lifetime of 1 (1.0 mM) in isation experiments, we studied the fluorescence intensity of the
solvents of varying viscosity (A) and polarity (C); fluorescence lifetime of
probe in HeLa cells incubated for different periods of time. We
found that the fluorescence of the probe (5.0 mM) inside HeLa
cells was saturated upon incubation for 25 min (Fig. S2 and S3,
the probe (BODIPY moiety at 516 nm and Nile Red moiety at 635 nm) was
measured using a time-correlated single photon counting (TCSPC) system,
excited at 375 nm (B); and the fluorescence intensity ratios for Nile Red
ESI†). Then the probe and different fluorescent markers specific
(I575/I625) were determined in solvents of varying polarity, using an excita-
tion wavelength of 488 nm (D).
for intracellular organelles (mitochondria, ER, and lysosomes)
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Fig. 5 Fluorescence lifetime imaging (FLIM) of the BODIPY moiety in 1
(5.0 mM) in HeLa cells; (A) FLIM in HeLa cells untreated by tunicamycin;
(B) FLIM in HeLa cells treated with tunicamycin (10 mg mLÀ1) for 24 hours
before incubation with 1; excited at 375 nm and emission was detected at
500–530 nm using an inverted-type scanning confocal microscope
(MicroTime 200, PicoQuant, Germany) with a 60Â objective.
Fig. 3 Colocalisation experiment of 1 (5.0 mM) with organelle-specific
markers in HeLa cells; (A, E), fluorescence imaging of Nile Red (collected
at 560–590 nm), excited at 488 nm; (B), MitoTracker, excited at 514 nm;
(C), merged image of A and B; (D), ER-Tracker, excited at 405 nm; (F), merged
image of D and E.
When the probe was initially incubated with HeLa cells, the
average Nile Red ratio was measured to be ca. 1.09, corresponding
to an e value of ca. 18.5. Upon treatment of HeLa cells with
were incubated with HeLa cells for 30 min. The results of tunicamycin (10 mg mLÀ1) for 24 h, the average ratio decreased
to ca. 0.86 (e = 21.1), indicating a detectable increase in the average
polarity of the ER (detection sensitivity is ca. 0.6). This increased
polarity of the ER could be due to the substantial accumulation of
polar proteins in the ER, resulting in ER stress.6
colocalisation experiments using a confocal laser microscope
with the probe and the organelle-specific dyes are shown in
Fig. 3 and Fig. S4, ESI.† The merged colours in panels C and F
represent the colocalisation of 1 with MitoTracker and ER-
Tracker, respectively. Panel F shows the most overlap between
FLIM was used to obtain further insight into ER viscosity
the probe and the organelle-specific dye, indicating that the changes during ER stress in HeLa cells. The probe displayed
probe is located mainly in the ER.
two exponential decays in HeLa cells before and after treatment
To validate the use of 1 for ER polarity imaging, confocal with tunicamycin (Fig. S8 and S9, ESI†), which are attributable
to the BODIPY and Nile Red moieties. As shown in Fig. 2B and
Fig. S1, ESI† the BODIPY moiety was sensitive to viscosity with a
viscosity sensitivity of ca. 2.3 cP (determined by sensitivity
measurements). Therefore, we measured the fluorescence lifetime
of BODIPY to calculate the average local viscosity in the ER. As
laser fluorescence images of HeLa cells were acquired with or
without tunicamycin treatment. And the tunicamycin is known
to inhibit glycosylation during protein or glycolipid synthesis,
causing the accumulation of proteins or lipids in the ER
(ER stress).16 Confocal laser fluorescence images for BODIPY
emissions (515 Æ 15 nm), Nile Red I (575 Æ 15 nm), and Nile shown in Fig. 5A and Fig. S8, ESI† the average fluorescence
Red II (635 Æ 15 nm) are shown in Fig. 4; Fig. S5–S7, ESI.† The lifetime of 1 in untreated HeLa cells was ca. 2.07 Æ 0.74 ns,
fluorescent ratiometric images (Fig. 4C and D) of the probe in corresponding to a viscosity of ca. 129.5 Æ 7.2 cP. After the cells
had been treated with tunicamycin (10 mg mLÀ1) for 24 h and
incubated with 1 for 20 min at 37 1C, the fluorescence lifetime
HeLa cells were obtained by dividing one emission channel of Nile
Red by the other (I(575Æ15)/I(635Æ15)) (B/C and F/G, Fig. S5, ESI†).13
increased to ca. 2.58 Æ 1.1 ns (Z = 182 Æ 29.8 cP), confirming that
the viscosity of the ER was marginally increased because of
accumulation of nascent proteins in the ER.
In conclusion, we have synthesized a novel, microenvironment-
sensitive, bimodal fluorescent probe (1) for the simultaneous
determination of local polarity and viscosity of the ER in live cells.
The probe is composed of Nile Red and BODIPY linked with a
–(CH2)6– spacer, showing low cytotoxicity (Fig. S10, ESI†). Using
this probe, the local polarity and viscosity of media could be
determined using the ratio of fluorescence intensities at 575 and
625 nm (I575/I625) and the fluorescence lifetime at 516 nm, respec-
tively. We also found that the probe was mainly located in the
ER in live cells. Upon treatment of HeLa cells with tunicamycin
to cause ER stress, the polarity (e) of the cells increased from
Fig. 4 Fluorescence imaging of 1 (5.0 mM) in HeLa cells; (A), fluorescence ca. 18.5 to 21.1, based on the ratiometric imaging of Nile Red
of the Nile Red moiety (I) (collected at 560–590 nm); (B), fluorescence of
(I(575Æ15)/I(635Æ15)). Furthermore, the fluorescence lifetime of
Nile Red (II) (collected at 625–650 nm); (C), ratiometric image of Nile Red
BODIPY, as determined by FLIM, increased from ca. 2.07 Æ 0.74
obtained from panels A and B untreated by tunicamycin; (D), ratiometric
image of Nile Red in HeLa cells treated by tunicamycin for 24 hours;
to 2.58 Æ 1.1 ns upon tunicamycin treatment, which corresponded
to an increase in the viscosity of the ER from ca. 129.5 Æ 7.2 to
excited at 488 nm, and fluorescence images were obtained using a Leica
TCS SP2 confocal laser microscope.
182 Æ 29.8 cP. Therefore, the newly designed bimodal probe
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This work was supported by the CRI project (No. 2009-0081566,
JSK) and the Basic Science Research Program (2012R1A1A2006259,
CK) through the National Research Foundation of Korea, funded
by the Ministry of Science, ICT and Future Planning.
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