Two-photon fluorescent probe for detecting cell membranal liquid-ordered phase by an aggregate fluorescence method

Article abstract of DOI:10.1039/c7tb00979h

The cell membranal liquid-ordered (Lo) phase can control the structure and function of cell membranes. In this study, we have engineered a novel two-photon (TP) fluorescent probe, TP-HVC18, which remarkably displayed two different fluorescence emission profiles in the aggregate and solution states in distinct polar environments. In accordance with its aggregate fluorescence, TP-HVC18 also can emit a red fluorescence signal in Lo phase vesicles. Taking advantage of this unique feature, we have demonstrated that the new TP probe TP-HVC18 is suitable for imaging membranal Lo phase by an aggregate fluorescence method. Furthermore, the robust probe also exhibited uncontinuous red fluorescence distribution in the cell membranal Lo phase. Based on this intriguing character, we also successfully showed that the novel probe can be employed to exhibit uncontinuous distribution of cell membranal Lo phase by a 3D imaging technique. We expect that this aggregation-based fluorescent platform may be extended for the development of a wide variety of TP fluorescent probes for detecting several biological species.

Full text of DOI:10.1039/c7tb00979h

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PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
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DOI: 10.1039/C7TB00979H
Journal Name
ARTICLE
Two-photon fluorescent probe for detecting cell membranal
liquid-ordered phase by an aggregate fluorescence method†
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
c
a
b,
a,
Yong Liu, Fangfang Meng, Jing Nie, Jie Niu Xiaoqiang Yu * and Weiying Lin *
DOI: 10.1039/x0xx00000x
Cell membranal liquid-ordered (Lo) phase controlled structure and function of the cell membranes. In this work, we have
engineered a novel two-photon (TP) fluorescent probe, TP-HVC18, which remarkably displayed two different fluorescence
emission profiles to the aggregate and solution states in the distinct polar environments. In accordance with its aggregate
fluorescence, TP-HVC18 also can emit a red fluorescence signal in Lo phase vesicles. Taking advantage of this unique
feature, we have demonstrated that the new TP probe TP-HVC18 is suitable for imaging membranal Lo phase by an
aggregate fluorescence method. Furthermore, the robust probe also exhibited uncontinuous red fluorescence distribution
in the cell membranal Lo phase. Based on this intriguing character, we also successfully showed that the novel probe can
be employed to exhibit uncontinuous distribution of cell membranal Lo phase by 3D imaging technique. We expect that
this aggregation-based fluorescent platform may be extended for the development of a wide variety of TP fluorescent
probes for detecting many biological species.
www.rsc.org/
10, 11
microfabrication.
To facilitate the applications of TP in
Introduction
Conventional fluorescent dyes have made great contributions to
biomedical research field, constructing a novel TP probe with
excellent TP properties is very important.
1
-
The cell membranes display
a tremendous complexity of
understand luminescence processes of dyes at the molecular level.
3
constituents including various lipids, carbohydrates, and proteins to
The conclusions drawn from the dilute solution data, however,
cannot commonly be extended to the concentrated solutions.
Appearance of aggregation-induced emission dyes expand research
1
2-16
accurately coordinate its biological function.
Cell membranal
liquid-ordered (Lo) phase (also called lipid rafts) was discovered by
1
7
4
Singer and Nicolson in 1972. It plays a critical role in various
scope of fluorescent materials. It is found that fluorescent
materials with aggregate fluorescence property exhibit great
advantages in biological applications, including high fluorescence
quantum yields, good photostability and low signal to noise ratio.
Herein, we have developed a novel fluorescent dye for labeling cell
membranal phase state by an aggregate fluorescence method.
Two-photon (TP) dyes were initially employed to im9 age
intracellular targets by fluorescence microscopy in 1990. TP
technique is an indispensable imaging method in light of its useful
applications in some fields such as localized release of bioactive
species, photodynamic therapy, optical power limiting, and
1
8-20
biological processes.
For instance, Lo phase is associated with
5-7
biological process including the formation of proteins clusters,
signal transduction, apoptosis, cell adhesion and migration, synaptic
transmission, organization of the cytoskeleton, protein sorting
during both exocytosis and endocytosis, and virus invasion. The
structure and function of cell plasma membranes have been
8
2
1
controlled by cell membranal Lo phases. Thus, to understand of
cell membranal function in biology, it is crucial to visualize Lo phase.
In the past few years, fluorescent probes have become powerful
tools for sensing cell membranes and membranal targets in
22-25
biological imaging fields.
Lo phases have been widely
investigated by diverse methods, including dynamic simulations,
detergent resistance experiments, mass spectrometry analysis,
atom force microscopy and fluorescent probes technique.
However, up to present, there have been no reports on fluorescent
probes with unique properties for sensing cell membranal Lo phase
by an aggregate fluorescence method. Thus, developing TP
fluorescent probes for sensing cell membranal Lo phases by an
aggregate fluorescence method is of high interest.
a
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of
Jinan, Shandong 250022, P.R. China. E-mail: weiyinglin2013@163.com.
26
b
Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal
Materials, Shandong University, Jinan, 250100, P. R. China.
E-mail: yuxq@sdu.edu.cn
c
School of Chemical Engineering & Technology, China University of Mining and
As TP fluorescence is more favorable than one-photon (OP)
Technology, Xuzhou, Jiangsu, 221116, P.R. China.
27-29
fluorescence for biological applications,
we selected the 2,7-
†
Electronic Supplementary Information (ESI) available: Experimental details
position substituted carbazole derivative as the TP platform. Based
on this TP platform , we have developed a novel TP fluorescent
probe TP-HVC18 (Fig. 1). It was found that TP dye TP-HVC18
including synthesis, the characterization of the compounds, fluorescence
spectroscopic data, and living cells imaging. See DOI: 10.1039/x0xx00000x.
This journal is © The Royal Society of Chemistry 20xx
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containing long hydrophobic chains could display two different spectrofluorimeter equipped with a 450-W Xe lampV.ieTwwAorti-cplehOontloinne
fluorescence emission profiles to the aggregate and solution states fluorescence spectra were recorded on aDSOpI:e1c0tr.1o0P3r9o/C307T0Bi.00A9ll79oHf
in the distinct polar environments. In accordance with its optical confocal microscopic photos were obtained with Carl Zeiss
properties in the aggregation state, TP-HVC18 also can emit a red Microscopy LSM780. All of TP microscopic photos were obtained
fluorescence signal in Lo phase vesicles. View of this unique with Olympus FV 300 Laser. The total power was provided by laser
property, the novel TP dye TP-HVC18 was capable of imaging source. Dimension Icon scanning electron microscope (SEM) was
membranal Lo phase by an aggregate fluorescence method. produced by Veeco Instruments Inc. Transmission electron
Furthermore, the robust probe also exhibited uncontinuous red microscopy (TEM) images were obtained with a JEOL JEM-2100F
fluorescence distribution in cell membranal Lo phase. By taking transmission electron microscope operating at an acceleration
advantage of this intriguing character, we also successfully showed voltage of 200 kV.
that the novel probe could be employed to image uncontinuous
Cell culture and living cell imaging
distribution of cell membranal Lo phase by a 3D imaging technique
SiHa Cancer cells were cultured in Dulbecco’s modified Eagle’s
for the first time. Compared with the conventional cell membrane
medium supplemented with penicillin/streptomycin and 10%
probes, 1) the TP-HVC18 not only can visualize membranal Lo phase
bovine calf serum in a 5% CO2 incubator at 37°C. For living cells
by an aggregate fluorescence method, 2) but also the probe
imaging experiment of TP-HVC18 (2 μM), cells were incubated in
exhibited uncontinuous red fluorescence distribution in cell
culture medium for 20 min at 37 °C, cells images were carried out
membranal Lo phase. 3) In addition, distribution characteristics of
immediately.
Lo phase were intuitively observed by 3D imaging technique.
One- and two-photon images of TP-HVC18 and DiD: Living cells
were stained with 2 μM TP-HVC18 and 4 μM DiD for 40 min and 15
min at ambient temperature, respectively, and then cell images
were carried out using one- and two-photon fluorescence
microscopy.
Preparation of GUVs
DOPC/CL was used to obtain the giant unilamellar vesicles (GUVs) of
liquid-disordered (Ld) phase, while DPPC/CL was used to construct
the GUVs of Lo phase. Two GUVs were prepared by the following
experimental procedures. Firstly, DOPC (18 mg/mL) and DPPC (18
mg/mL) were dissolved in the mixing solvents of CH Cl and CH OH
3
3
(
V/V, 2:1). Secondly, both solutions were added to flaskets and fully
Fig. 1 Structure and imaging method of the new TP probe TP- shocked. Thirdly, the mixed solutions were carefully laid over the
HVC18
.
inner wall of the flaskets and the solvents were removed under the
protection of N . Fourthly, to further remove residual solvents,
2
flaskets were treated at vacuum conditions. Finally, the flaskets
were filled using 0.1 M sucrose solutions, and further heated at
Experimental
o
60 C for 24 h. The GUVs were formed in the sucrose solutions.
Materials
Before achieving GUVs confocal imaging, some experiments were
All chemical reagents were analytical grade. 4,4’-Dibromobiphenyl
and 1-bromooctadecane were purchased from Sinopharm Chemical
Reagent Co., Ltd (Shanghai, China). DiD, 1,2-dipalmitoyl-sn-glycero-
prepared. First, we prepared 1 mM stock solution of probes in DMF
to stain the GUVs; Second, adding 0.8 mL stock solution to 200 μL
glucose solution (0.1 M) and obtained a diluted solution; Third,
taking out 10 mL GUVs and adding to the above diluted solution
and mixed evenly; Finally, the GUVs confocal imaging were carried
out.
3-phosphocholine(DPPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine
(
DOPC), cholesterol (CL), Palladium (II) acetate and tri-o-
tolylphosphine were purchased from J&K Chemical (Beijing, China).
PBS was purchased from Seikagaku Corporation (Japan). The
solvents used in the spectral measurement are of chromatographic
grade. TLC analyses were performed on silica gel plates and column
chromatography was conducted over silica gel (mesh 200–300),
both of which were obtained from Qingdao Ocean Chemicals.
Real-color images (in situ images) acquirement of GUVs and cells.
The laser scanning microscope imaging was carried out by Zeiss LSM
780 confocal fluorescent microscope. The real-color images were
acquired by the spectra imaging function of the Zeiss LSM 780
confocal microscope. The above function was capable of separately
collecting emission spectra in numerous short wavelength ranges.
For example, the images with green color correspond to
wavelength of 544-552 nm. The images with the wavelength of 578-
Apparatus
1
13
Nuclear magnetic resonance spectra ( H and C) were obtained by
a Bruker Avanace 300/400 spectrometer. The HRMS spectra were
recorded on Agilent Technologies 6510 Q-TOF LC/MS or
ThermoFisher LCQ FLEET. The UV-visible-near-IR absorption spectra
of dilute solutions were recorded on a Cary 50 spectrophotometer
using a quartz cuvette having 1 cm path length. One-photon
5
86 nm exhibited yellow fluorescence, and the images with the
wavelength of 613-621 nm showed red fluorescence, etc. Then the
merge of all these images would give a new image and exhibit its
real emission spectra, called the real-color image. Meanwhile,
management of these images could give the emission intensity at
fluorescence spectra were obtained on
a HITACHI F-2700
2
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different wavelength range and further give the in situ emission yellow solid after the residue was recrystallized from ethanol (yield:
View Article Online
1
DOI: 10.1039/C7TB00979H
50%). H NMR (400 MHz, DMSO-d6) δ (ppm): 8.61 (d, J = 5.44 Hz,
spectra of arbitrary areas.
4
4
H), 8.08 (d, J = 8.08 Hz, 2H), 7.49-7.58 (m, 10H), 7.15-7.26 (m, 2H),
.37 (t, J = 7.20 Hz, 2H), 1.94 (t, J = 7.16 Hz, 4H), 1.23 (s, 28H), 0.87
Preparation of TEM, DLS and SEM
Before achieving TEM experiment, some experiments were (t, J = 6.82 Hz, 3H).
prepared. First, we prepared three TP-HVC18 (4 μM) water/DMF
Synthesis of TP-HVC18
mixtures with different water fraction (f ), including water/DMF (fw
w
The compound 4 (1.25 g, 2 mmol) and excess 1-iodododecane were
dissolved in acetone and stirred for 2 h at room temperature. Then
the mixture was refluxed for another 12 h and a red residue was
obtained. The residue was filtered and then washed with methanol
for 3 times. The title product was obtained as a red solid after the
=
0%), water/DMF (fw = 75%) and water/DMF (fw = 100%); Second,
taking 10 μL liquid from three water/DMF mixtures to bronze for 20
minutes; Third, absorbing residual liquid with sanitary napkin;
Finally, the TEM experiment was carried out. In dynamic light
scattering (DLS) experiment, we prepared water/DMF mixtures (fw =
1
residue was recrystallized from ethanol (Yield: 75%). H NMR (300
MHz, DMSO-d6), δ (ppm): 8.97 (d, J = 6.90 Hz, 4H), 8.22–8.33 (m,
1
00%) of TP-HVC18 (4 μM), and then DLS experiment was carried
out. In this work, we use pure solid TP-HVC18 to achieve SEM
experiment.
SEM was produced by Veeco Instruments Inc. Transmission
electron microscopy. TEM images were obtained with a JEOL JEM-
8
7
H), 8.08 (s, 2H), 7.71 (t, J = 8.80 Hz, 2H), 7.67 (s, 2H), 4.50 (t, J =
.20 Hz, 2H), 1.91 (d, J = 6.00 Hz, 6H),1.15-1.31 (m, 42H), 0.81-0.90
13
(
m, 9H). C NMR (100 MHz, DMSO-d6): δ 152.95, 144.12, 141.99,
1
5
2
41.26 133.53, 123.58, 123.47, 122.78, 121.30, 119.32, 109.75,
9.61, 31.17, 30.48, 30.40, 28.90, 28.87, 28.77, 28.66, 28.57, 28.48,
2
100F transmission electron microscope operating at an
acceleration voltage of 200 kV.
+
6.37, 25.10, 21.96. HRMS (m/z): [M-I] calcd for C H I N ,
56 81 2 3
Synthesis of Probe
397.8200; found, 397.8213.
Chemical synthesis of TP-HVC18 was accomplished in a total of five
steps (Scheme S1). The optimization of 4,4-dibromo-2-nitrobiphenyl
Results and discussion
Optical properties of TP-HVC18
(
1) started from readily available 4,4-dibromobiphenyl and nitryl. 2
was steadily prepared as an isolable intermediate for synthesizing 3.
was obtained by Heck reaction between 3 and 4-vinylpyridine.
4
With TP-HVC18 in hand, we set out to investigate optical properties
of the TP-HVC18 in various solvents. As shown in Fig. S1 and Table
S1 (ESI†), in pure water solution, the compound TP-HVC18 has
maximal absorption and emission at 429 nm and 612 nm,
respectively; In DMF solution, TP-HVC18 has maximal absorption
and emission at 447 nm and 555 nm, respectively. The results
demonstrated that TP-HVC18 exhibited larger Stokes shift in
aqueous solution than organic solution. Moreover, the red solid TP-
HVC18 showed strong red fluorescence signal at wide-field
excitation (Fig. S2, ESI†). Thus, we envisioned that red emission
signal of TP-HVC18 may come from aggregation of compound in
pure water system.
We further investigated TP properties of TP-HVC18 in aqueous
solution. As shown in Table S2 (ESI†), the compound TP-HVC18
possessed large TP action absorption cross-section (δΦ) in pure
water system and buffer solution. The results demonstrated that
the TP-HVC18 should be a novel TP fluorescent material. Thus, we
deduced that the compound TP-HVC18 may be a novel TP probe
with aggregate fluorescence property.
Treatment of iodine hexane with 4 in acetone results in the
formation of material TP-HVC18.
Synthesis of 1 and 2
30
The compound 1 and 2 was synthesized by literature.
Synthesis of 3.
2
0 g KOH was added in DMF (70 mL) and the resulting solution was
stirred for 30 min. 3.23g (10 mmol) of 2 was then added and the
mixture was stirred for another 40 min. Finally, 1-bromooctadecane
(
1
4.98 g, 15 mmol) was added dropwise and the mixture reacted for
2 h at room temperature. White solid was found when the mixture
was poured into water (500 mL). The crude residue was filtered and
washed with ethanol for 3 times. A white solid was obtained for 3
1
after recrystallization from ethanol with a yield of 80%. H NMR
(
400 MHz, DMSO-d6), δ (ppm): 7.89 (d, J = 8.28 Hz, 2H), 7.58 (d, J =
1
4
1
.40 Hz, 2H), 7.35 (dd, J1 = 8.26 Hz, J2 = 1.54 Hz, 2H), 7.26 (s, 2H),
.19 (t, J = 7.34 Hz, 2H), 1.84 (t, J = 7.14 Hz, 2H), 1.25-1.34 (m, 30 H),
.25 (t, J = 6.80 Hz, 3H).
Aggregation of TP-HVC18
Synthesis of 4
The compound 3 (4.60 g, 8.0 mmol) was added into a flask To verify the compound TP-HVC18 could aggregate in aqueous
containing a mixture of palladium(II) acetate (0.18 g, 0.8 mmol), tri- solution, we investigated optical properties of TP-HVC18 in the
o-tolylphosphine (0.72 g, 2.4 mmol) and K CO (8.8 g, 64.0 mmol), distinct polar environments. We have proved that red solid TP-
2
3
and to this mixture N-methyl-2-pyrrolidone (NMP, 40 mL) and 4- HVC18 emitted strong red fluorescence at wide-field excitation.
vinylpyridine (3.4 g, 32.0 mmol) was added. The system was heated Thus, the compound TP-HVC18 may form red nanoribbon in
to 128 °C for 3 days under the protection of argon. A dark-red water/DMF mixtures with a high water fraction (f
suspension was obtained. When the resulting mixture was cooled
To prove the above assumption, we investigated optical
).
w
to room temperature, it was poured into H O (500 mL) and properties of TP-HVC18 in water/DMF mixtures. First, we prepared
2
extracted with CH Cl . Then the organic phases were separated, the eight water/DMF mixtures of TP-HVC18; Under ultraviolet lamp, the
2
2
excess organic solvent was removed by vacuum distillation and a compound TP-HVC18 exhibited bright yellow fluorescence in
dark-red solution was obtained. The title product was obtained as a organic solutions, however, fluorescence signal gradually became
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red with increasing fw values (Fig. 2A). Second, we measured one- 600 nm (Fig. 3E). The results of SEM, TEM and DLS techniques were
View Article Online
DOI: 10.1039/C7TB00979H
and two-photon emission spectra of TP-HVC18 in different consistent with optical properties of TP-HVC18 in different f values.
w
water/DMF mixtures. When fw increased from 0 to 100 %, the one- The above results further demonstrated that TP-HVC18 was a novel
and two-photon fluorescent spectra underwent red shift (Figs. 2B- fluorescent material with aggregate fluorescence feature. We
C), however, fluorescent intensity of TP-HVC18 decreased gradually predicted that TP-HVC18 should possess unique applications by an
(
Figs. 2D-E). Moreover, the fluorescence spectrum of TP-HVC18 in aggregate fluorescence method.
pure water solution was consistent with solid fluorescence of TP-
HVC18 (Fig. 2F). The above results demonstrated that TP-HVC18
displayed two different fluorescence emission profiles to the
aggregate and solution states. Thus, we induced TP-HVC18 should
have unique morphology in definite f values.
w
Fig. 3 (A) SEM picture of the solid TP-HVC18; (B) TEM pictures of
water/DMF (fw = 0%), (C) water/DMF (fw = 75%) and (D) water/DMF
(
fw = 100%) dispersions of TP-HVC18 at 4 μM; (E) DLS of TP-HVC18
dispersions in water/DMF (f = 100 %) mixtures at 4 μM.
w
In situ imaging
We further investigated TP-HVC18 whether possessed unique
applications in vitro and in vivo. According to previous studies on
the physical properties of model membranes, giant unilamellar
31
vesicles (GUVs) were powerful tools to simulate cell membrane.
DOPC/CL can construct cell membranal liquid-disordered (Ld) phase,
32-35
while DPPC/CL can simulate cell membranal Lo phase.
We have
proved that fluorescence spectra of TP-HVC18 exhibited yellow and
red fluorescence emission in solution and aggregation state (Fig.
Fig. 2 (A) Photographs of TP-HVC18 in water/DMF mixtures; (B)
One- and (C) two-photon fluorescence spectra of TP-HVC18 in
water/DMF mixture; Plots of maximum (D) one- and (E) two-photon
emission intensity (I) and wavelength (λem) of TP-HVC18 versus
4A); Next, fluorescence spectra of TP-HVC18 in Lo and Ld phases
solutions were also measured (Fig. 4B). Interestingly, in accordance
with its aggregate properties, TP-HVC18 also can emit red
fluorescence signal in Lo phase solution. Thus, we envisioned that
the probe TP-HVC18 could sense cell membranal Lo phase by an
aggregate fluorescence method.
To prove the above assumptions, we further investigated in situ
imaging of GUVs. Real-color images of GUVs stained with TP-HVC18
were acquired (Figs. 4C and D). The in situ imaging showed that TP-
HVC18 emitted yellow and red fluorescence in Lo and Ld phase,
respectively. In situ imaging results GUVs were consistent with
fluorescence emission of TP-HVC18 in Lo and Ld phases solutions.
Thus, the TP-HVC18 should form aggregation states in Lo phase and
emit red fluorescence signal. We further proved that the probe was
capable of imaging GUV of Lo phase by an aggregate fluorescence
method.
water/DMF (f , vol %) in water/DMF mixtures; (F) Solid
w
fluorescence spectra of TP-HVC18. [TP-HVC18] = 4 μM. λex = 488 nm.
Investigating aggregation of TP-HVC18 by SEM, DLS and TEM
techniques
We further investigated aggregation of TP-HVC18 by scanning
electron microscope (SEM), dynamic light scattering (DLS) and
transmission electron microscopy (TEM). First, we observed
morphology of the solid TP-HVC18 by SEM. As shown in Fig. 3A, the
red solid TP-HVC18 aggregated and formed nanoribbon with
different length and width; Second, as shown in Fig. 3B, it was
found that TP-HVC18 could not occur aggregation in pure orgnic
solvent; We prepared TP-HVC18 water/DMF (fw = 75%) solution,
and then TEM experiment was carried out. Similarly with SEM
experiment, imaging results exhibited nanoribbon with different
length and width (Fig. 3C); Furthermore, the dye TP-HVC18 further
formed irregular aggregation in pure water solution (Fig. 3D). Finally,
we further investigated size of TP-HVC18 in pure water system by
To prove the probe TP-HVC18 can imaging SiHa cell membranal
Lo phase, the cell membranal in situ imaging experiment was
carried out. The toxicity test indicated that the TP-HVC18 had low
toxicity for living cells (Fig. S3, ESI†). The cell membranes imaging
and in situ emission profiles of the TP-HVC18 were displayed in Figs.
4E-G and Fig. S4 (ESI†). The in situ imaging result demonstrated that
DLS technique. Similarly with SEM and TEM, DLS in water/DMF (fw
=
1
00 %) highlighted irregular nanoribbon with an average size of 10-
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TP-HVC18 emitted red fluorescence signal in the cell membranes at Aggregation of TP-HVC18 in cell membranal Lo phaseView Article Online
88 nm excitation (Fig. 4F). This result was similar with fluorescence To further demonstrate TP-HVC18 can labelDmOeIm: 1b0.r1a0n3a9l/CLo7TpBh0a0s9e79bHy
4
characteristics of Lo phase. Furthermore, the in situ emission an aggregate fluorescence method, we further study imaging
spectrum of the cell membrane was consistent with solid characteristics of the TP-HVC18 in the cell membranes. As shown in
fluorescence of TP-HVC18 (Fig. 4G). The results demonstrated that Figs. 6A-C, the TP-HVC18 presented uncontinuous red fluorescence
the TP-HVC18 was suitable for imaging membranal Lo phase by an distribution in the cell membranes at 488 nm excitation. Moreover,
aggregate fluorescence method.
aggregation fluorescence signal was also observed at 800 nm
We have demonstrated that TP-HVC18 displayed red and yellow excitation (Figs. 6D-F). To obtain fluorescence intensity profile of Lo
fluorescence emission profiles to the Lo and Ld phases. However, it phase, we chose a SiHa cell (Fig. 6B) and investigated fluorescence
cannot find yellow fluorescence signal of Ld phase in cell distribution in the cell membranes; The intensity profile of linear
membranes. Thus, we set out to investigate in situ imaging of Ld regions of interest across SiHa cell exhibited uncontinuous
phase by changing experiment condition. Living SiHa cells were fluorescent intensity (Fig. 6G). This result further demonstrated the
incubated with 1 μM TP-HVC18 for a long time (about 1.5 h). As probe TP-HVC18 could label cell membranal Lo phase by an
shown in Fig. 5, the in situ imaging result exhibited red and yellow aggregate fluorescence method. To the best of our knowledge, no
fluorescence signal in the cell membranes (Fig. 5A), in good reports to date have been published on fluorescent probes with
agreement with in site imaging of GUVs. The in situ emission aggregate fluorescence property for visualizing membranal Lo
spectra of the cell membranes were consistant with the in situ phase using one- and two-photon microscope.
emission profiles of both vesicles solutions (Fig. 4B). The results
Conventional membrane probes could not visualize membranal
showed that TP-HVC18 can image cell membranal Lo and Ld phases Lo phase based on aggregate fluorescence method. To prove this
based on two different sets of fluorescence signals.
point, we chose the commercially available membrane tracer DiD to
investigate its one- and two-photon images. Before studying cells
membrane imaging, We firstly study optical properties of DiD in the
distinct polar environments. The results demonstrated that
fluorescence of DiD gradually quenching when fw increased from 0
to 100% (Fig. S5, ESI†). Thus, we envisioned that the commercially
probe DiD could not image cell membanal Lo phase by an aggregate
fluorescence method.
We further investigated cell membranes imaging of DiD. Living
cells were incubated with 2 μM DiD for 15 min at room
temperature, and then one- and two-photon imaging experiment
were carried out. As shown in Figs. 6H-I, the DiD presented
continuous fluorescence distribution in the cell membranes; This
imaging characteristics were intuitively observed by 3D imaging
technique (Fig. 6J). Compared to TP-HVC18, the probe DiD cannot
image cell membranes at 800 nm excitation (Figs. 6K-M). The
intensity profile of linear regions of interest across a SiHa cell
further proved continuous fluorescence distribution of DiD (Fig. 6N).
The above results demonstrated that DiD cannot detect membranal
Lo phase at OP and TP excitation. The confocal image of TP-HVC18
Fig. 4 (A) Fluorescence spectra of TP-HVC18 in organic solution, and
solid fluorescence of TP-HVC18; (B) Fluorescence spectra of TP-
HVC18 (2 μM) in various vesicle solutions; (C) In situ image of Lo did not merge well with that of the DiD. Moreover, the Pearson’s
phase GUVs incubated with TP-HVC18 (2 μM); (D) In situ image of colocalization coefficient is only 0.40, demonstrating that DiD and
Ld phase GUVs incubated with TP-HVC18 (2 μM); (E) Bright-field TP-HVC18 cannot locate in the same region (Fig. S6, ESI†).
images; (F) In situ emission spectra of the cell membranes
incubated with TP-HVC18 (2 μM). λex = 488 nm. Bar = 20 μm.
Compared to commercial membrane probe DiD, the TP-HVC18
not only can visualize membranal Lo phase at one- and two-photon
excitation, but also the probe exhibited uncontinuous red
fluorescence distribution in cell membranal Lo phase. In addition,
the TP-HVC18 showed higher photostability (Fig. S7, ESI†) than DiD
(
Fig. S8, ESI†).
3
D imaging
To further demonstrate the probe TP-HVC18 was capable of
imaging cell membranal Lo phase by an aggregate fluorescence
method, 3D imaging experiment was carried out. Firstly, SiHa cells
were incubated with 2 μM TP-HVC18 for 40 min, and further did
tomoscan experiment. 15 optical sections of cells at different
depths have been recorded (Fig. 7A). The image results exhibited
distribution characteristics of Lo phase at different depths. Secondly,
Fig. 5 (A) Fluorescence images and spectra of the SiHa cells
incubated with TP-HVC18 (1 μM) using the spectra imaging function
of the Zeiss LSM 780 confocal fluorescent microscope. (B)
Fluorescence spectra of the SiHa cells with excitation at 488 nm.
Scale bar = 20 μm.
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Fig. 6 One- and two-photon images of the cell membranes treated with TP-HVC18 (2 μM) and DiD (2 μM). (A) Bright-field image. (B) One-
photon image of the cell membranes treated with TP-HVC18 (2 μM); λex = 488 nm, λem = 675-700 nm. (C) Merged image of A and B. (D)
Bright-field image. (E) Two-photon image of the cell membranes treated with TP-HVC18 (2 μM); λex = 800 nm, λem = 675-700 nm. (F)
Merged image of D and E; (G) Intensity profile of the region of interest (white arrow in B) across the SiHa cells (B). (H) Bright-field image. (I)
One-photon image of the cell membranes treated with DiD (2 μM); λex = 633 nm, λem = 635-691nm. (J) 3D image of I; (K) Bright-field image;
(
L) Two-photon image of the cell membranes treated with DiD (2 μM); λex = 800 nm, λem = 675-700 nm. (M) Merged image of K and L. (N)
Intensity profile of the region of interest (white arrow in I) across the SiHa cells.
Fig. 7 (A) Confocal imaging of TP-HVC18 including 15 optical sections at different depths has been recorded.(B) Confocal 3D imaging of TP-
HVC18 including 15 optical sections ; (C) Changes of fluorescent intensity of TP-HVC18 in cell membrane at different depths. λex = 488 nm.
λem= 650-700 nm.
to intuitive observe aggregation of probe in the cell membranes, 3D profiles to aggregate and solution states. Similarly with its optical
imaging was obtained by Nikon AIMP analysis software (Fig. 7B). It properties of aggregate state, TP-HVC18 remarkably displayed red
successfully showed that the novel probe TP-HVC18 can be fluorescence emission signal in the Lo phase vesicles. View of this
employed to exhibit uncontinuous distribution of cell membranal Lo property, TP-HVC18 can image cell membranal Lo phase by an
phase. In addition, the intensity profile of every pictures aggregate fluorescence method. Furthermore, the robust probe
constituting 3D imaging was acquired. Cell membranal fluorescence also exhibited uncontinuous red fluorescence distribution in cell
intensity enhanced gradually in the range of 0-10 μm; Furthermore, membranal Lo phase. By taking advantage of this intriguing
fluorescence signal further weaken within the range of 10-14 μm character, it was found that the novel TP probe TP-HVC18 was
(
Fig. 7C). Thus, by taking advantage of the above character, it was capable of visualizing membranal Lo phase by a 3D imaging
firstly found that TP-HVC18 can be employed to image technique. We expect that this fluorescent platform might be
uncontinuous distribution of cell membranal Lo phase by 3D extended for the development of a wide variety of functional
imaging technique for the first time.
probes for detecting intracellular phase states.
Conclusions
Acknowledgements
In summary, we have developed a novel TP fluorescent probe TP-
HVC18 based on the 2,7-position substituted carbazole derivative. This work was financially supported by NSFC (21472067, 21672083,
The new probe displayed two different fluorescence emission 51503077), Taishan Scholar Foundation (TS 201511041), and the
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startup fund of the University of Jinan (309-10004). Doctor start up 29 F. Helmchen and W. Denk, Nat. Methods., 2005, 2, 932.
View Article Online
DOI: 10.1039/C7TB00979H
fund of University of Jinnan (160082102) and NSFSP (ZR2015PE001). 30 Y. Liu, F. Meng, L. He, X. Yu and W. Lin, Chem. Commun., 2016,
52, 8838.
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Journal of Materials Chemistry B
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In this work, we have engineered a novel two-photon fluorescent probe, TP-HVC18, which
remarkably displayed two different fluorescence emission profiles to the aggregate and solution
states in the distinct polar environments. Taking advantage of this unique feature, we have
demonstrated that the new TP probe TP-HVC18 was suitable for imaging membranal Lo phase by
an aggregate fluorescence method.