An easily accessible aggregation-induced emission probe for lipid droplet-specific imaging and movement tracking

Article abstract of DOI:10.1039/c6cc09471f

Lipid droplets (LDs) as dynamic organelles are associated with many metabolic processes. Ideal fluorescent probes for LD-specific imaging require excellent specificity, superior brightness, fast cell permeability, and easy preparation. However, convention

Full text of DOI:10.1039/c6cc09471f

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An easily accessible aggregation-induced emission
probe for lipid droplet-specific imaging and
movement tracking†
Cite this:DOI: 10.1039/c6cc09471f
Received 28th November 2016,
Accepted 15th December 2016
Meng Gao,‡a Huifang Su,‡b Shiwu Li,a Yuhan Lin,a Xia Ling,a Anjun Qin*a and
Ben Zhong Tang*ab
DOI: 10.1039/c6cc09471f
www.rsc.org/chemcomm
Lipid droplets (LDs) as dynamic organelles are associated with many for LD-specific imaging with TPEF ability to investigate their
metabolic processes. Ideal fluorescent probes for LD-specific imaging biological functions.
require excellent specificity, superior brightness, fast cell permeability,
Ideal LD-specific two-photon fluorescent probes need to tackle
and easy preparation. However, conventional fluorophores for LD the following challenges: (1) selective accumulation in LDs at high
imaging suffer from drawbacks of aggregation-caused quenching concentration to achieve superior brightness; (2) small molecular
(ACQ), poor photostability, and difficulty of preparation. To tackle these size with fast cell permeability; (3) easy and economic preparation
challenges, herein, we develop an easily accessible aggregation- from commerically available substrates; (4) high two-photon absorp-
induced emission (AIE) fluorescent probe for LD-specific imaging and tion cross-sections in the NIR range; and (5) strong photostability for
dynamic movement tracking. This AIE probe has significant advantages long-term real-time monitoring. Although several LD-specific
in terms of fast cell permeability, low cytotoxicity, strong photostability, fluorescent dyes have been developed, including commercially
and high two-photon absorption cross-sections in the near infra-red available Nile red, BODIPY493/503 Green, and LipidTox Red,
(NIR) range. It is thus expected to have broad applications in the study their applications are still restricted due to one or more of the
of LDs’ biological functions.
following disadvantages: aggregation-caused quenching (ACQ),
suitability only for fixed cells, high background noise, small
Lipid droplets (LDs) as reservoirs of lipids and proteins are dynamic Stokes shifts (less than 40 nm), and difficulty of preparation.11
organelles,1 which are closely related with many metabolic processes
Recently, aggregation-induced emission (AIE) provided a
and diseases, such as inter-membrane lipid traffic,2 virus infection,3 revolutionary solution to solve the self-quenching problem at high
inflammation,4 cancer,5 and obesity.6 The real time monitoring of concentration or in the aggregate state.12 We have previously
LDs’ distribution and movement is critical for understanding their developed several AIE fluorogens (AIEgens) for LD-specific
biological functional roles. Recently, various imaging techniques imaging with advantages of excellent photostability and high
have been adopted for LDs’ visualization, such as color staining,7 emission efficiency after accumulating in LDs.13 However, these
Raman microscopy,8 and transmission electron microscopy (TEM).9 AIEgens were only investigated via one-photon microscopy and
Compared with these imaging methods, near infra-red (NIR) their preparation requires multi-synthetic steps. Therefore,
two-photon excited fluorescence (TPEF), which employs two easily accessible AIE probes with high two-photon absorption
near-infrared photons for the excitation, has significant advantages cross-sections are highly desirable for LD-specific imaging.
in low photo-damage and high-resolution imaging ability due to
Triphenylamine (TPA) and indane-1,3-dione (IND) are classic
focused excitation and reduced autofluorescence.10 Thus, there electron-donating and -withdrawing groups, respectively, and
is a high demand for robust and reliable fluorescent bioprobes they have been broadly applied in the preparation of nonlinear
optical materials.14 Herein, we found that their Knoevenagel
condensation product 2-(4-(diphenylamino)benzylidene)-1H-indene-
1,3(2H)-dione (IND-TPA) has both twisted intramolecular charge
transfer (TICT) and aggregation-induced emission (AIE) proper-
ties. This can be due to its donor (D)–acceptor (A) structure and
the restriction of intramolecular motion in the aggregate state
(Scheme 1). Based on its AIE and TICT properties, it can be used
for LD-specific imaging and movement tracking with the advan-
tages of easy preparation, NIR two-photon excited red emission,
and the excellent signal-to-noise ratio.
a State Key Laboratory of Luminescent Materials and Devices, South China
University of Technology, Guangzhou 510640, China. E-mail: msqinaj@scut.edu.cn
b Department of Chemistry and Hong Kong Branch of Chinese National Engineering
Research Center for Tissue Restoration and Reconstruction,
The Hong Kong University of Science & Technology, Clear Water Bay,
Kowloon, Hong Kong, China. E-mail: tangbenz@ust.hk
† Electronic supplementary information (ESI) available: Experimental details and
video for fluorescence monitoring of LDs’ movement. See DOI: 10.1039/c6cc09471f
‡ These authors contributed equally to this work.
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Table 1 Photophysical properties of IND-TPA in THF/water mixture with
different water fractions (fw)
fw (%) lab [nm] lem [nm] Ffc [%] td (ns) kre [107
S
À1] knr [108 SÀ1
]
a
b
f
0
70
99
478
489
502
594
609
612
6.9
0.5
10.2
1.30
1.16
2.60
5.31
0.43
3.92
7.16
8.58
3.45
a
b
Maximum absorption wavelength. Maximum emission wavelength.
Absolute quantum yield. d Average fluorescence lifetime. e Radiative decay
c
rate kr = F/t. f Non-radiative decay rate knr = (1 À F)/t. [IND-TPA] = 10 mM.
Scheme 1 IND-TPA with TICT and AIE characteristics for LD-specific
two-photon excited fluorescence imaging.
the 2.2-fold increase in fluorescence lifetime, 9.1-fold increase in
radiative decay rates, and 2.5-fold decrease in non-radiative
decay rates from fw = 70% to 99%.
2-(4-(Diphenylamino)benzylidene)-1H-indene-1,3(2H)-dione
(IND-TPA) is easily accessible from commercially available
1,3-indanedione and 4-(N,N-diphenylamino)benzaldehyde via a
one-step Knoevenagel condensation reaction.15 The structure of
IND-TPA was well elucidated by NMR spectroscopy (Fig. S1, ESI†)
and high-resolution mass spectrometry. We first examined the
photophysical properties of IND-TPA. The photo-luminescence
(PL) spectra of IND-TPA in the THF/water mixture are shown in
Fig. 1; the fluorescence quantum yields (Ff), fluorescence life-
time (t), calculated radiative (kr) and non-radiative decay (knr) rates
are shown in Table 1. The maximum absorption and emission
peaks at 478 and 594 nm were respectively observed for IND-TPA in
THF solution. Increasing water fractions (fw) from 0 to 70% in the
THF/water mixture resulted in a 13.8-fold decrease in the fluores-
cence quantum yield from 6.9% to 0.5% and a red-shifted emission
from 594 to 609 nm. This phenomenon can be due to the TICT
effect,16 which was also confirmed by the red-shifted absorption
from 478 to 489 nm (Table 1 and Fig. S2, ESI†). Further addition of
water from fw = 70% to 99% led to a red-shifted emission to 612 nm
and a 20.4-fold increase in fluorescence quantum yield from 0.5%
to 10.2%. This can be explained by the aggregate formation caused
by poor solubility of IND-TPA in aqueous solution. The formed
nanoaggregates at fw = 99% were then analyzed by dynamic light
scattering analysis, and an average diameter of 119.6 nm with a low
polydispersity index of 0.164 was observed (Fig. S3, ESI†). In
the aggregate state, the restriction of intramolecular motion will
facilitate the radiative release of the photo-excited energy and
inhibit the non-radiative decay, which was clearly illustrated by
To verify that the aggregation-induced emission phenomenon
is caused by the restriction of intramolecular motion, we then
measured the emission of IND-TPA in polar solvents of ethylene
glycol and glycerol with low (Z = 13.5 mPa S) and high viscosity
(Z = 945 mPa S), respectively.17 A 4-fold emission intensity
enhancement and a 1.44-fold fluorescence lifetime increase were
observed in glycerol compared with those in ethylene glycol,
which clearly verified that the AIE phenomenon was caused by
the restriction of intramolecular motion (Fig. S4, ESI†). IND-TPA
also exhibited a large Stokes shift of 3580 cmÀ1 in the aggregate
state (fw = 99%), which can minimize the self-absorption and
auto-fluorescence interference. The AIE properties of IND-TPA,
coupled with its minimum fluorescence in aqueous solution due
to the TICT effect, make it ideal for LDs’ imaging with a high
signal-to-noise ratio through high concentration accumulation
in hydrophobic LDs and a very low background noise in polar
cytoplasm.
Based on the MTT assay, the cytotoxicity of IND-TPA was
then evaluated for lung cancer HCC827 and A549 cells, which
express high contents of LDs.18 As shown in Fig. 2, no significant
change for the cell viability was observed at different concentra-
tions of IND-TPA (0, 5, 10, 20, 40, 60, 80, and 100 mM), suggesting
that IND-TPA has a very low cytotoxicity and can be used as a
biocompatible fluorescent probe for long-term monitoring of the
dynamic processes of live cells.
We then conducted the cell imaging experiment. A bright
fluorescence was observed for both HCC827 and A549 cells
after incubating with IND-TPA for only 15 min at 37 1C,
Fig. 1 (A) Normalized UV-Vis absorption spectra of IND-TPA in THF
(dashed line); PL spectra of IND-TPA in THF and THF/water mixture with
different water fractions (fw). (B) Plots of relative maximum emission
intensity (I/I0) of IND-TPA versus the solvent composition of THF/water
mixture. [IND-TPA] = 10 mM; lex = 478 nm.
Fig. 2 Cell viabilities of lung cancer HCC827 and A549 cells after incuba-
tion with different concentrations of IND-TPA (0, 5, 10, 20, 40, 60, 80 and
100 mM) for 24 h.
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Fig. 3 CLSM images of HCC827 (A–E) and A549 (F–J) cells after incubation
with IND-TPA (5 mM) and BODIPY493/503 (100 nM) at 37 1C for 15 min.
(A and F) Bright-field images. (B and G) Fluorescence image from IND-TPA.
(C and H) Fluorescence image from BODIPY493/503. (D and I) The merged
images. (E and J) The intensity profile of ROI lines. Scale bar = 20 mm.
Fig. 5 CLSM images of HCC827 cells stained with 5.0 mM IND-TPA. (A–D)
Different pseudo-colors are used to illustrate the fluorescence images at
different times of 0, 1, 3, and 5 min. Merging images at two different times:
(E) 0 and 1 min, (F) 1 and 3 min, (G) 3 and 5 min, and (H) bright-field image.
Scale bar = 20 mm.
suggesting IND-TPA has a fast permeability for living cells
(Fig. 3B and G). To test the specificity of IND-TPA for LDs, the
co-localization experiment with the lipid dye BODIPY493/503
was conducted and a high overlap coefficient of 0.96 was
observed for both HCC827 and A549 cells. The intensity profiles
of IND-TPA and BODIPY493/503 for the region of interest (ROI)
lines across HCC827 and A549 cells also varied in close synchrony
(Fig. 3D, E, I and J). Moreover, a high correlation was found in the
intensity scatter plot of IND-TPA and BODIPY493/503 (Fig. S5,
ESI†), suggesting that IND-TPA specifically exists in the core of
LDs. The excellent selectivity of hydrophobic IND-TPA for LDs
can be due to its selective accumulation in the non-polar core of
LDs and the high sensitivity to polar environments, which
eliminates the possible interference from phospholipid membranes
with higher polarity.11b Moreover, the large Stokes shift and the
red emission of IND-TPA can spectrally be resolved from blue
and green fluorophores. For example, the co-staining with the
blue emissive nucleic acid dye Hoechst 33342 further confirmed
the excellent selectivity of IND-TPA for LDs and no labeling for
nucleus (Fig. S6, ESI†).
IND-TPA has a strong photostability and is favorable for long-
term tracking of the dynamic movements of LDs.11f
The excellent specificity, high brightness, and strong photo-
stability of IND-TPA suggest its suitability for monitoring LDs’
movement, which is closely related with the metabolism of fatty
acids and interaction with other organelles.19 We then monitored
the dynamic movements of LDs stained with IND-TPA by confocal
microscopy. As shown in Fig. 5, the spatial distribution of LDs
could be easily identified by IND-TPA in the merged images
collected at different times. Moreover, Movie S1 (ESI†) shows a
series of 40 fluorescence image frames of LDs taken at intervals of
7.75 seconds, and the movement of LDs can be clearly observed
and monitored with high signal-to-noise ratios.
We then measured the two-photon absorption cross-sections
of IND-TPA in the 800–1000 nm NIR range at 20 nm intervals.
Compared with classic small two-photon fluorophores based on
naphthalene derivatives,10a,b IND-TPA shows a relatively high
two-photon absorption cross-section of 119 GM (1 GM =
10À50 cm4 s photonÀ1) upon excitation at 920 nm (Fig. 6A). This
can be due to its intramolecular charge transfer effect and the
conjugated p system. Live cell imaging experiments were then
conducted using a two-photon scanning confocal microscope for
HCC827 cells. As shown in Fig. 6B–D, LDs in HCC827 cells stained
with IND-TPA can be clearly observed with high signal-to-noise
ratios, suggesting its potential applications for in vivo studies.
The photostability of IND-TPA in HCC827 cells was then
measured under irradiation at 514 nm with 7% laser power.
After continuous irradiation for over 10 min, the fluorescence
intensity loss was less than 20% (Fig. 4), which suggests that
Fig. 4 I/I0 (%) of fluorescence intensity of HCC827 cells stained with
IND-TPA (5 mM) with increasing time of irradiation at 514 nm with 7% laser
power. Inset: Fluorescence images of HCC827 cells with increasing time of
irradiation.
Fig. 6 (A) Two-photon absorption cross-sections of IND-TPA with fluorescein
as the reference. (B) Bright field image of HCC827 cells. (C) Two-photon excited
fluorescence image. (D) The merged image. Scale bar = 10 mm. lex = 920 nm.
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In conclusion, we have developed an easily accessible AIE
probe IND-TPA for LD-specific live cell imaging with significant
advantages of superior brightness in the aggregate state, fast
cell permeability, low cytotoxicity, and high NIR two-photon
absorption cross-sections. Moreover, IND-TPA can be used for
real-time monitoring of the dynamic movement of LDs with
high spatiotemporal resolution. It is anticipated that IND-TPA
can serve as a convenient and practical tool for the investigation
of LDs’ biological functions.
This work was financially supported by the Science and
Technology Planning Project of Guangzhou (Project No.
201607020015); the Key Project of the Ministry of Science and
Technology of China (2013CB834702); the National Natural
Science Foundation of China (51620105009 and 21602063); Natural
Science Foundation of Guangdong Province (2016A030313852 and
2016A030312002); China Postdoctoral Science Foundation Grant
(2015M580716 and 2016T90778); the Fundamental Research Funds
for the Central Universities (2015ZY013 and 2015ZZ104);
the Innovation and Technology Commission of Hong Kong
(ITC-CNERC14SC01); and Guangdong Innovative Research
Team Program (201101C0105067115).
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