In situ generation of photoactivatable aggregation-induced emission probes for organelle-specific imaging

Article abstract of DOI:10.1039/C8SC01887A

Photoactivatable fluorescent probes are ideal tools for organelle study with a significant advantage of high spatiotemporal resolution. However, conventional photo-caged fluorophores for organelle-specific imaging suffer from several drawbacks, such as aggregation-caused quenching (ACQ), instability under ambient light, low photoactivation efficiency, and toxic photo-cleavage byproducts. Herein, we propose a strategy for in situ generation of photoactivatable aggregation-induced emission (AIE) probes of 2-(2-hydroxyphenyl)-benzothiazolines from easily available disulfide and thiol substrates through tandem S-S bond reduction and intramolecular cyclization reaction. Because the photoactivatable AIE probes can be in situ generated in a quantitative yield, they can be directly used for bio-imaging without complicated separation steps. Under both one- and NIR two-photon irradiation, excellent spatiotemporal resolution and high photoactivation efficiency were achieved for specific imaging of lipid droplets and lysosomes, respectively. Based on their in situ generation and adjustable organelle-targeting ability, the photoactivatable AIE probes could become an easy-to-use imaging tool in the study of the biological functions of organelles.

Full text of DOI:10.1039/C8SC01887A

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In situ generation of photoactivatable aggregation-induced
emission probe for organelle-specific imaging
Shiwu Li,a,b Xia Ling,a Yuhan Lin,a Anjun Qin,a Meng Gao,*c Ben Zhong Tang*a,d
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Photoactivatable fluorescent probes are ideal tools for organelle study with significant advantage of high spatiotemporal
resolution. However, conventional photo-caged fluorophores for organelle-specific imaging suffer from several drawbacks,
such as aggregation-caused quenching (ACQ), instability under ambient light, low photoactivation efficiency, and toxic
photo-cleavage byproducts. Herein, we propose a strategy for in situ generation of photoactivatable aggregation-induced
emission (AIE) probes of 2-(2-hydroxyphenyl)-benzothiazolines from easily available disulfide and thiol substrates through
tandem S–S bond reduction and intramolecular cyclization reaction. Because the photoactivatable AIE probes can be in
situ generated in a quantitative yield, they can be directly used for bio-imaging without complicated separation steps.
Under both one- and NIR two-photon irradiation, an excellent spatiotemporal resolution and a high photoactivation
efficiency were achieved for specific imaging of lipid droplets and lysosomes, respectively. Based on their in situ
generation and adjustable organelle-targeting ability, the photoactivatable AIE probes could become an easy-to-use
imaging tool in study of biological functions of organelles.
www.rsc.org/
suffer from several drawbacks in terms of difficult synthesis,
low photoactivation efficiency, inadvertent activation under
ambient light, and generation of photocleavage toxic
byproducts.6 Moreover, conventional fluorophores can easily
undergo serious selfꢀquenching after accumulation in
organelles, which further restricted their applications for
organelle study.7
Introduction
The intracellular locations and motilities of organelles are
closely related with their biological functions.1 It's thus highly
desirable to track organelle distribution and movement within
living cells to reveal their functions in different biological
processes.2 To date, many “alwaysꢀon” fluorescent probes have
been reported for organelleꢀspecific imaging, but the lack of
spatiotemporal controlling ability has seriously restricted their
applications.3 In contrary, photoactivatable fluorescent probes
can transform from nonꢀemissive state to highly emissive state
under light irradiation with high spatiotemporal resolution,
which are powerful imaging tools for organelle study.4 In the
past few years, a series of caged proteins and fluorophores have
been developed for photoactivatable imaging,5 however, they
Recently, aggregationꢀinduced emission (AIE) has been
proposed as a fundamental solution to tackle the challenge of
fluorescence selfꢀquenching.8 Based on their unique advantages
of high brightness in the aggregate state, strong photostability,
and low cytotoxicity,9 the AIEꢀactive probes have found broad
applications in bioꢀimaging and sensing.10 Recently, several
photoactivatable or photoswitchable AIE fluorogens (AIEgens)
have
been
developed,
including
tetraphenylethene,
cyanostilbene, dihydroꢀ2ꢀazafluorenone, and distyrylanthracene
derivatives.11 However, these AIEgens need several synthetic
steps for preparation and tedious column chromatography
procedures for separation. To make photoactivatable probes
easyꢀtoꢀuse for organelle study, the following requirements
need be satisfied: (1) the photoactivatable probes could be in
situ generated in a quantitative yield from easily available
substrates and directly used for bioꢀimaging without
complicated separation steps; (2) the targeting groups for
different organelles could be easily introduced; (3) the
photoactivation process could be conducted under NIR twoꢀ
photon irradiation to reduce the photodamage effect and
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guarantee a high spatiotemporal resolution; (4) the fluorescence Photoactivatable Imaging of Lipid Droplets
selfꢀquenching effect for organelle imaging should be
The HeLa or MCFꢀ7 cells were first treated with oleic acid (50
overcome.
Herein, we unexpectedly found that the bis(2ꢀ(2ꢀ
hydroxybenzylidene)amino)aryl disulfides and ethyl
mercaptoacetate can easily undergo S–S bond reduction to
afford Schiff base intermediate , which then underwent
intramolecular cyclization reaction to in situ quantitatively
ꢁM) for 6 h to induce the formation of lipid droplets. The cell
culture media was removed and washed twice by HBSS buffer.
Then the in situ generated compound 3a in DMSO solution (5.0
µL, 10 mM) was directly added into the HBSS buffer (1.0 mL)
and incubated with the cells for 5 min. The fluorescence images
were then taken under confocal microscope through continuous
irradiation at 405 nm with 10% laser power, λem = 500–700 nm.
1
2
generate
2ꢀ(2ꢀhydroxyphenyl)ꢀbenzothiazolines
3.
The
compounds
3
can further undergo photooxidative
Photoactivatable Imaging of Lysosomes
dehydrogenation reaction to afford the AIEꢀactive compound 2ꢀ
(2ꢀhydroxyphenyl)ꢀbenzothiazoles under both oneꢀ and twoꢀ
photon irradiation (Scheme 1). The in situ generated
compounds with a methoxy or morpholinomethyl substituent
can be respectively used for photoactivatable imaging of lipid
droplets (LDs) and lysosomes with a high specificity and an
excellent photoactivation efficiency. Because the disulfide and
thiol substrates are easily available and the photoactivatable
The in situ generated compound 3b in DMSO solution (2.0 µL,
10 mM) was directly added into the HBSS buffer (1.0 mL) and
incubated with the cells for 5 min. The fluorescence images
were then taken under confocal microscope through continuous
irradiation at 405 nm with 5% laser power, λem = 450–657 nm.
4
3
Colocalization Experiment
For coꢀstaining of compound 3a and BODIPY493/503 Green,
the HeLa or MCFꢀ7 cells in HBSS buffer were first incubated
with BODIPY493/503 Green (1.0 µg/mL) for 10 min, then
probes
3 could be in situ generated in a quantiative yield, they
would be easyꢀtoꢀuse tools for organelle study.
incubated with 3a (50 µM) for 5 min. The fluorescence images
of 3a after photoactivation were taken with λex = 405 nm and
λem = 550–700 nm. For BODIPY493/503 Green, λex = 488 nm,
λem = 500–550 nm.
For coꢀstaining of compound 3b and LysoTracker Red, the
HeLa or MCFꢀ7 cells in HBSS buffer were first incubated with
3b (20 µM) for 5 min, then incubated with LysoTracker Red
(100 nM) for 5 min. The fluorescence images of 3b after
photoactivation were taken with λex = 405 nm and λem = 410–
539 nm. For LysoTracker Red, λex = 543 nm, λem = 576–657
nm.
Photoactivatable Imaging under Two-photon Irradiation
The HeLa or MCFꢀ7 cells stained with the in situ generated
3
were irradiated in a bleach model with twoꢀphoton femtosecond
laser at 780 nm (1.0% power). The emission filter was 575–630
nm and 495–540 nm for 3a and 3b, respectively.
Spatiotemporal Imaging with Two-Photon Irradiation
The fluorescence image of multiꢀcells was first taken under
twoꢀphoton irradiation (780 nm, 0.5% laser power). The
Scheme 1 The in situ generation of photoactivatable AIE probe
imaging of lipid droplets and lysosomes.
3 for specific
selected cells were then continuously irradiated with
a
bleaching model (780 nm, 1.0% laser power) to activate
fluorescence. Then, the whole observation window of multiꢀ
cells was imaged. This process was repeated to lightꢀup all the
selected cells. The emission filter was 575–630 nm and 495–
540 nm for 3a and 3b, respectively.
Experimental section
The In Situ Preparation of Photoactivatable AIE Probe 3
The disulfide and ethyl mercaptoacetate were first dissolved
1
Results and Discussion
in DMSO solutions at a concentration of 10 mM and 20 mM,
respectively. Their DMSO solutions were then mixed with an
equal volume (100 µL) to in situ generate the photoactivatable
The compounds
condensation
1
or
,
3
, and
cyclization
derivatives
4 can be easily prepared by
reaction
between
and
2ꢀ
probe
3 (10 mM, 200 µL) in a quantitative yield within 5 min.
hydroxybenzaldehyde
2,2'ꢀ
disulfanediyldianiline or 2ꢀaminobenzenethiol (For detailed
synthetic procedures, see Scheme S1).12 Structures of all these
compounds were unambiguously verified by NMR and HRMS
analysis (Fig. S1–S6).
Cell Culture
HeLa and MCFꢀ7 cells were cultured in DMEM containing 1%
penicillinꢀstreptomycin and 10% FBS at 37 °C in a humidity
atmosphere with 5% CO2.
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The AIE property of compounds
ARTICLE
Fig. 1 The 1H NMR stacking spectra of (A) 1a, (B) "1a + ethyl thioglycolate", and
(C) purified 3a. (* represents diethyl 2,2'-disulfanediyldiacetate)
4
was first investigated.
For 4ꢀmethoxyꢀsubstituted compound 4a, a weak "enol"
emission at 404 nm was observed in THF solution, while a
strong "keto" emission at 570 nm was observed in the solid
state (Fig. S7). Compared with in THF solution, the
fluorescence quantum yield and lifetime of 4a showed a 33.4ꢀ
fold increase from 1.2 to 40.1% and a 5.2ꢀfold increase from
0.94 to 4.88 ns, respectively (Table S1). For 4ꢀ
morpholinomethylꢀsubstituted compound 4b, a weak "enol"
emission at 446 nm in THF solution and a strong "keto"
emission at 523 nm in the solid state were observed. The
fluorescence quantum yield and lifetime of 4b respectively
showed a 25.8ꢀfold increase from 1.1% to 28.4% and a 4.1ꢀfold
increase from 1.56 to 6.39 ns (Table S1). The AIE property of
The photoactivation ability of the in situ generated
compound
3 was then investigated. Under UV irradiation at 365
nm, the PL intensity of the in situ generated 3a increased
quickly with a maximum emission wavelength at 576 nm (Fig.
2AꢀB). Meanwhile, the absorption intensity of 3a at 353 nm
also increased rapidly (Fig. 2CꢀD). The PL and UVꢀVis spectra
changes can be owing to the generation of compound 4a
through photooxidative dehydrogenation of the C–N single
bond to C=N double bond. The 1H NMR spectra analysis
further
verified
the
photooxidative
dehydrogenation
compounds
4 can be owing to the synergistic effects of
transformation by disappearance of the hydrogen peaks of the
C–N single bond (Fig. S12). Moreover, a gradually lightꢀup
fluorescence was observed for compound 3a in the solid state
under UV irradiation (365 nm), which suggests that the
photooxidative dehydrogenation reaction can also smoothly
proceed in the aggregate or solid state (Fig. 2E). For compound
restriction of intramolecular motion (RIM) and excited state
intramolecular proton transfer (ESIPT) mechanisms.13 The
excellent "keto" emission efficiency of
with large Stokes shifts (up to 208 nm) would greatly favor
their bioꢀimaging applications.
4 in the aggregate state
3b
,
it can also smoothly undergo photooxidative
We then investigated the in situ generation ability of
dehydrogenation reaction to efficiently afford the AIEꢀactive
compound 4b both in solution and in the solid state (Fig. S13–
15).
compounds
to was achieved by a simple mixing of the DMSO solutions
of disulfides and ethyl mercaptoacetate within 5 min. This
quantitative transformation was clearly verified by the NMR
analysis of the reaction mixtures. As shown in Fig. 1, the ꢀHa
and ꢀHb peaks of 1a at 12.0 and 9.0 ppm completely
disappeared after reaction with ethyl mercaptoacetate, and new
ꢀHa′ and ꢀHb′ peaks appeared at 9.4 and 6.4 ppm overlap well
with the purified compound 3a Meanwhile, the ethyl
mercaptoacetate was oxidized into diethyl 2,2'ꢀ
3. To our satisfaction, a quantitative transformation
3
1
O
C
O
C
.
disulfanediyldiacetate (Fig. S8). The in situ generation of
compound 3a was also verified by the UVꢀVis absorption
measurement. The main absorption peak of 1a at 380 nm
disappeared after reaction with ethyl mercaptoacetate, and a
new absorption peak at 309 nm was observed, which overlaps
well with the absorption spectrum of purified compound 3a
(Fig. S9). Similarly, 3b could also be in situ generated in a
quantitative yield by the simple mixing of 1b and ethyl
mercaptoacetate in DMSO solution (Fig. S10). The quantitative
transformation to compound
experiment (Fig. S11).
3 was also verified by the HPLC
Fig. 2 (A) The PL spectra of in situ generated 3a in aqueous solution under
irradiation at 365 nm for 0–20 min. (B) Plot of relative PL intensity I/I0 at 576 nm
versus the irradiation time. (C) The UV-Vis spectra of in situ generated 3a in
aqueous solution under irradiation at 365 nm for 0–20 min. (D) Plot of relative
UV-Vis absorption intensity I/I0 at 353 nm versus the irradiation time. [3a] = 100
µM. (E) Bright field and fluorescence images of 3a in the solid state taken under
white light and UV irradiation (365 nm).
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The cytotoxicities of ethyl mercaptoacetate and the in situ situ generated 3b with a basic morpholine group could be used
generated 2,2'ꢀdisulfanediyldiacetate and compounds were for
3
then investigated based on MTT assay and no significant
decrease in cell viability was observed for both HeLa and
MCFꢀ7 cells (Fig. S16ꢀ19). Because compounds
3 can be in situ
generated in a quantitative yield, they were then directly used
for cell imaging experiment, which was conducted with HeLa
cells as a model cell line. After incubation with the in situ
generated 3a for 5 min, a fast lightꢀup fluorescence was
observed inside the cells under light irradiation at 405 nm,
while almost no fluorescence was observed outside the cells
even without washing (Fig. 3AꢀG). These results suggest that
the in situ generated 3a can be quickly uptaken by HeLa cells
and the photooxidative dehydrogenation reaction can smoothly
proceed inside the cells. Through statistical analysis of the
intracellular fluorescence intensity, a nearly 53ꢀfold lightꢀup
ratio was obtained (Fig. 3H). The PL spectra of 3a after
photoactivation overlaps well with the "keto" emission spectra
of compound 4a, which suggests the smooth proceeding of
Fig. 4 (A-G) Bright field and fluorescence images of live HeLa cells taken under
ESIPT process after photoactivation (Fig. S20). Furthermore, white light and prolonged irradiation at 405 nm. (H) Plot of fluorescence
enhancement (I/I0) of HeLa cells with increasing irradiation time at 405 nm. (I-K)
Fluorescence images of HeLa cells stained with the in situ generated 3b after
photoactivation (green), LysoTracker Red (red), and the merged image. (L) The
the coꢀlocalization experiment of photoactivated 3a with lipid
dye BODIPY493/503 Green showed a high overlap coefficient
of 0.89, which suggests that the probe could selectively intensity profile of ROI lines. [3b] = 20 μM.
accumulate in LDs (Fig. 3IꢀK, Fig. S21A). Moreover, the
intensity profile of photoactivated 3a and BODIPY493/503
showed a close synchrony for the region of interest (ROI) line
across HeLa cells (Fig. 3L), which further verified the specific
targeting ability of 3a for LDs.
Fig. 5 The temporal and spatial photoactivation of LDs (A, B) and lysosomes (C, D)
in HeLa cells under two-photon irradiation at 780 nm. [3a] = 50 μM; [3b] = 20 μM
Fig. 3 (A-G) Bright field and fluorescence images of live HeLa cells taken under
white light and prolonged irradiation at 405 nm. (H) Plot of fluorescence
enhancement (I/I0) of HeLa cells with increasing irradiation time at 405 nm. (I-K)
Fluorescence images of HeLa cells stained with the in situ generated 3a after
photoactivatable imaging of acidic lysosomes with a high
photoactivation (artificial red color), BODIPY493/503 (green), and the merged
image. (L) The intensity profile of ROI lines. [3a] = 50 μM.
specificity. To further verify the organelleꢀspecific imaging
ability of the in situ generated 3, the cell imaging experiment
was then conducted for MCFꢀ7 cells. A high lightꢀup ratio of
18ꢀfold was observed for 3a and an excellent overlap
coefficient of 0.93 was observed with BODIPY493/503 Green
(Fig. S22). Meanwhile, a 4ꢀfold lightꢀup ratio was observed for
3b and a high overlap coefficient of 0.88 was observed with
LysoTracker Red (Fig. S23). These results further verified the
organelleꢀspecific targeting ability and high photoactivation
For the in situ generated compound 3b, a very fast lightꢀup
fluorescence within 20 s and a more than 6ꢀfold lightꢀup ratio
was observed for HeLa cells under light irradiation at 405 nm
(Fig. 4AꢀG). The coꢀstaining experiment of photoactivated 3b
with LysoTracker Red showed a high overlap coefficient of
0.86 and an excellent signal synchrony for the ROI line across
the cells (Fig. 4IꢀL, Fig. S21B). These results suggest that the in
efficiency of the in situ generated compound 3.
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To reduce the photoꢀdamage effect of oneꢀphoton UV CNERC14SC01); Guangdong Innovative Research Team
irradiation, the photoactivatable imaging experiment was then Program (201101C0105067115).
investigated under twoꢀphoton irradiation at 780 nm. The timeꢀ
lapse fluorescence images under twoꢀphoton irradiation showed
Notes and references
a very fast lightꢀup fluorescence within 45 s for both 3a and 3b
(Fig. 5A, C, Fig. S24). Based on the high spatial imaging
resolution under twoꢀphoton irradiation, the photoactivation of
LDs and lysosomes for selected cells was conducted in a multiꢀ
cellular environment. To our satisfaction, the selected cells can
be sequentially photoactivated with a high spatiotemporal
resolution (Fig. 5B, D), which suggests that the
1
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photoactivatable probes
3 can be used for organelle study in a
complex biological environment.
3
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compounds
3 from chemical oxidants. For both compounds 3a
and 3b in aqueous solution, almost no fluorescence increase
was observed even in the presence of H2O2 and NaClO (100
µM) for 2 h (Fig. S25). Moreover, no fluorescence changes
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3 and further treated
with H2O2 or NaClO (100 µM) for 1 h (Fig. S26). In contrary, a
fast lightꢀup fluorescence within 30 s to 5 min was observed
under light irradiation at 405 nm, which could thus efficiently
preclude the interference of chemical oxidants for the
photoactivation process.14
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Conclusions
In conclusion, we have developed photoactivatable AIE probes
for organelleꢀspecific imaging through in situ quantitative
generation from easily available disulfide and thiol substrates.
The in situ generated AIE probes could be directly used for
photoactivatable bioꢀimaging without tedious purification
procedures. Based on the adjustable organelleꢀtargeting ability
and excellent photoactivation efficiency, a high spatiotemporal
resolution for photoactivatable imaging of LDs and lysosomes
were respectively achieved under both oneꢀ and twoꢀphoton
irradiation. Through avoiding the complicated separation steps,
the photoactivatable AIE probe could act as an easyꢀtoꢀuse
imaging tool and is expected to have broad applications in
biological study.
Conflict of interest
9
(a) J. Qian and B. Z. Tang, Chem, 2017, 3, 56ꢀ91; (b) J. Liang,
B. Z. Tang and B. Liu, Chem. Soc. Rev., 2015, 44, 2798ꢀ2811;
(c) Y. Yuan, S. Xu, X. Cheng, X. Cai and B. Liu, Angew.
Chem. Int. Ed., 2016, 55, 6457ꢀ6461; (d) C. Y. Y. Yu, H. Xu,
S. Ji, R. T. K. Kwok, J. W. Y. Lam, X. Li, S. Krishnan, D.
Ding and B. Z. Tang, Adv. Mater., 2017, 29, 1606167.
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Science
Foundation of China (21788102, 51620105009 and 21602063);
the Science and Technology Planning Project of Guangzhou
(201607020015 and 201704030069); Pearl River S&T Nova
Program of Guangzhou (201806010152); Natural Science
Foundation of Guangdong Province (2013A030313852 and
2013A030312002); the Fundamental Research Funds for the
Central Universities (2015ZY013 and 2015ZZ104); the
Innovation and Technology Commission of Hong Kong (ITCꢀ
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