Ratiometric emission NIR-fluorescent probe for the detection of lysosomal pH in living cells and: In vivo

Article abstract of DOI:10.1039/c9nj02771h

The lysosome is known to be an acidic organelle that is ubiquitous in cells. Acidic pH is a characteristic feature of lysosomes, and aberrant lysosomal pH values have been manifested to be associated with diverse diseases. In order to monitor the lysosoma

Full text of DOI:10.1039/c9nj02771h

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Ratiometric emission NIR-fluorescent probe for
the detection of lysosomal pH in living cells and
in vivo†
Cite this:DOI: 10.1039/c9nj02771h
Weifen Niu, *‡ Juan Jia,‡ Junkai Li, Chao Zhang and Keming Yun*
The lysosome is known to be an acidic organelle that is ubiquitous in cells. Acidic pH is a characteristic
feature of lysosomes, and aberrant lysosomal pH values have been manifested to be associated with
diverse diseases. In order to monitor the lysosomal pH changes, we developed a ratiometric emission
NIR-fluorescent probe (CzQl) for the quantitative analysis of the lysosomal pH in living cells and in vivo.
CzQl was constructed via the ethylene bridging of carbazole derivatives and quinoline. The protonation
of the probe resulted in a marked green-to-red emission color change. The probe exhibited an ideal pK
a
Received 28th May 2019,
Accepted 15th July 2019
value of 4.60 with a linear relationship in the pH range of 3.9–5.3, which covered most of the pH ranges
of lysosomes, and was useful for the quantitative detection and imaging of lysosomal pH changes.
Moreover, CzQl exhibited easy loading capability, high selectivity, a large Stokes shift, specific lysosome-
targeting ability and low cytotoxicity; thereby, it was successfully used for monitoring the lysosomal pH
changes at a cellular level as well as the LPS-mediated inflammation in vivo.
DOI: 10.1039/c9nj02771h
rsc.li/njc
selective pH probes have been developed for biological
applications. Some recent studies on ratiometric fluorescent
Introduction
1
1
Lysosomes, single-membrane organelles defined by a uniquely probes for lysosomal pH sensing published in the last three
1
2–21
strong acidic lumenal pH (4.5–5.5) and high content of acid years have been summarized in Table 1.
hydrolases, are the shared degradative compartments of the the single emission detection, the emission ratio simultaneously
Compared with
1
,2
endocytic and autophagic pathways. A variety of hydrolases reflects the population of the protonated and deprotonated forms
are responsible for substrate degradation. Besides, lysosomes of the fluorescent probe, which can cancel out the most possible
are the points of particular vulnerability in many neurodegen- effects of environmental variations, probe distribution, and
1
,3
1
,4,5
erative diseases.
Beyond their role in substrate degradation, instrumental performance, thus offering more accurate analysis
new findings have ascribed to lysosomes a leading role in sensing because of its self-referencing capability by the calculation of the
2
2–25
and responding to cellular nutrients, growth factors and cellular two emission intensity ratio.
In addition, as seen in Table 1,
1
stress. However, the acidic pH environment is the crucial bio- some probes have short emission wavelengths, which can be
marker for lysosomes; abnormal fluctuations in the lysosomal pH due to the interference from cellular autofluorescence. NIR-
value can cause lysosomal dysfunction, which is associated with fluorescence emissions can largely eliminate the interference of
5–10
several diseases.
pH value in living cells and in vivo is vital for investigating to living cells and organisms. Moreover, some probes have
lysosome-associated physiological and pathological processes. complex structures and synthesis processes. Thus, the design and
Consequently, the detection of the lysosomal cellular autofluorescence and also reduce light-induced damage
26
Organelle-specific fluorescent probes are robust molecular development of ratiometric emission NIR-fluorescence probes
tools for sensing and monitoring trace amounts of biological with simple structures and easy synthesis for sensing the lysosomal
molecules and the processes of individual subcellular organelles pH are of great concern and significantly challenging.
in living cells and in vivo by virtue of their excellent sensitivity and
In this study, a ratiometric emission NIR-fluorescent probe
high spatiotemporal resolution. To date, numerous lysosome- (CzQl) (Scheme 1) was achieved via the chemical conjugation of
methylcarbitol-substituted carbazole and quinoline; the methyl-
carbitol substituent served as the targeting unit for lysosomes, and
the quinoline moiety acted as a proton reaction site. Upon proto-
nation, CzQl could exhibit a pronounced pH-dependent red shift
in both absorption and emission spectra, which was attributed to
the significant enhancement of the electron-withdrawing ability
School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001,
P. R. China. E-mail: niuweifen_2000@163.com
1
†
Electronic supplementary information (ESI) available: H NMR titration, rever-
sibility, photostability cytotoxicity assay, characterization of fluorescent probe.
See DOI: 10.1039/c9nj02771h
‡
These authors contributed equally to this work.
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Table 1 Comparison of the properties of recently reported probes for lysosomal pH sensing with the ratiometric method
Probe
l
ex/nm
l
em/nm
pK
a
Linearity pH range
Ref.
12
4
73
522/557
662/460
5.96
5.35
3.5–6.0
a
3
4
75
05
—
13
14
641/483
4.8
—
470
633/518
3.4
—
15
3
4
60
70
514/454
515/665
4.46
2.00
3.82–5.00
16
17
—
4
4
95
70
697/618
613/560
4.5
3.5–5.2
4.0–6.0
18
19
5.00
385
540/425
5.02
3.0–5.0
20
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Table 1 (continued)
Probe
l
ex/nm
80
l
em/nm
pK
a
Linearity pH range
3.8–6.0
Ref.
21
3
511/439
—
a
Not reported in the corresponding paper.
Scheme 1 Synthetic scheme and the acid–base form equilibrium of CzQl.
1
of the protonated quinoline moiety. Remarkably, this probe
H NMR titration experiment (Fig. S1, ESI†) showed that the
exhibited an expected pK value of 4.60 with a linear relationship chemical shift values of the quinoline protons H1 and H2
a
in the pH range of 3.9–5.3 and could quantitatively detect and shifted down-field, which confirmed the above conclusion.
image the lysosomal pH changes.
Furthermore, an easy-to-discern solution color change from
nearly colorless to orange was observed on decreasing the pH
value (Fig. 1A, inset), which suggested that CzQl could serve as
a colorimetric probe in acidic pH conditions.
The fluorescence spectral changes of CzQl at various pH
values are shown in Fig. 1B. When the pH was basic-neutral, the
Results and discussion
Optical response to pH
Spectroscopic studies of CzQl in solutions of different pH probe exhibited a fluorescence emission maximum at 530 nm
values were performed using UV-vis absorption spectroscopy (lex = 415 nm, F = 0.38) with a large Stokes shift of 115 nm,
and fluorescence spectroscopy. Fig. 1A shows the UV-vis which could greatly reduce the excitation interference. As the
absorption spectral changes of CzQl at various pH values. pH decreased from 7.0 to 1.9, the fluorescence intensity at
Under basic-neutral pH conditions, CzQl displayed an absorp- 530 nm was reduced gradually concomitant with the red shift of
4
À1
À1
tion maximum (labs) at 383 nm (emax = 6.36 Â 10 M cm ). an emissive band to 637 nm (D = 107 nm, F = 0.21), leading to
When the pH was changed from neutral (7.0) to acidic (2.5), the an isoemissive point at B595 nm. The large red shift in the
absorption maximum shifted to a longer wavelength at 464 nm emission signals was caused by the enhanced ICT effect.
4
À1
À1
(emax = 7.84 Â 10
M
cm ) with a well-defined isosbestic Importantly, the marked red shift of the emission spectra
point at 415 nm. The red shift in the absorption spectra was offered the possibility to carry out ratiometric detection. With
attributed to the protonation of the quinoline moiety of CzQl, the decrease in pH, the emission ratio of the fluorescence
which enhanced the intramolecular charge transfer (ICT) effect intensities (F530nm/F637nm) also changed dramatically. As seen
+
on decreasing pH because of the H binding-induced enhance- in Fig. 1C, F530nm/F637nm is reduced by more than 260-fold
ment of the electron-withdrawing ability of quinoline. The (from 7.733 to 0.029) over the pH range of 7.0–1.9. The pK
a
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Fig. 2 The fluorescence emission ratios (F530nm/F637nm) of CzQl (10 mM) at
pH 7.0 (black bars) and 4.5 (red bars) in the presence of various metal ions
+
+
and bioactive species. (1) blank; (2) 20 mM K ; (3) 20 mM Na ; (4) 20 mM
2+
2+
2+
3+
2+
Ca ; (5) 20 mM Mg ; (6) 15 mM Zn ; (7) 0.15 mM Fe ; (8) 1 mM Pb
;
2+
(
9) 2 mM Cd ; (10) 5 mM glucose; (11) 5 mM GSH; (12) 2 mM vitamin C;
13) 1 mM leucine; (14) 1 mM valine; (15) 1 mM L-threonine; (16) 1 mM cysteine;
17) 1 mM arginine; (18) 1 mM serine; (19) 1 mM histidine. lex = 415 nm. Data
(
(
are expressed as mean values with the standard error of the mean of three
independent experiments.
significant effect on the fluorescence emission ratio (F530nm
/
F
637nm) of the probe at pH 7.0 and 4.5, respectively. These results
+
confirm that CzQl exhibits an excellent selective response to H
ions in the presence of metal ions and bioactive molecules.
Moreover, the emission ratio (F530nm/F637nm) of CzQl was
recorded by robustly cycling back and forth between pH 7.0 and
2.2 using concentrated hydrochloric acid and aqueous sodium
hydroxide (Fig. S2, ESI†), indicating the reversible sensing ability
of CzQl. Also, the response and recovery times in different pH
solutions were rapid, i.e., within seconds. Thus, the probe can be
used to monitor pH variations in real time. Likewise, the
experimental results indicated that the probe solution possessed
good photostability (Fig. S3, ESI†). Therefore, this probe shows a
promising potential for the quantitative detection of pH changes
by means of the ratiometric fluorescence method.
Fig. 1 UV-vis absorption (A) and fluorescence (B) pH titration curves of
CzQl (10 mM) with decreasing pH values. (C) Sigmoidal fitting of the pH-
dependent emission intensity ratio (F530nm/F637nm). Insets: Photographs of
the solution color change (A) and fluorescence change (B) at different pH
conditions. (C) The linear relationship over the pH range from 3.9 to 5.3.
Confocal fluorescence imaging in cells
Because lysosomes are the typical acidic organelles in cells and
CzQl has the appropriate pH range for lysosomes, it is practical
for the probe to sense this acidity if it can exhibit subcellular
localization of lysosomes. To confirm the lysosome-targeting
value calculated from the titration curve of the emission ratios ability of CzQl, colocalization experiments were carried out.
F530nm/F637nm) was 4.60. Interestingly, the plot of F530nm/F637nm B16-F10 cells were stained with CzQl and LysoTracker Red
versus pH values shows a linear relationship in the pH range of DND-99 (a well-known commercially available lysosome-specific
.9–5.3, which covers most of the pH ranges of lysosomes. The red-emissive probe). In Fig. 3, excellent overlapping images with
(
3
linear regression equation was F530nm/F637nm = À16.10 + 4.34 pH, a high Pearson’s co-localization coefficient (A) of 0.93 can be
with a linear coefficient of 0.9987. Consequently, CzQl showed a observed (Fig. 3D), clearly demonstrating that CzQl predomi-
marked green-to-red emission color change in response to the nantly resided in the lysosomes. Additionally, CzQl exhibited
change in pH from neutral to acidic (Fig. 1B, inset) and an minimal cytotoxicity, which was confirmed by an MTT viability
appropriate pK value for assessing the change in the lysosomal pH. assay (Fig. S4, ESI†).
a
It is necessary that CzQl shows a highly selective response
towards H ions over other potentially competitive species such as
+
Lysosomal pH imaging
various metal ions and some representative bioactive molecules. Based on the confirmed intrinsic lysosome-targeting ability, we
As shown in Fig. 2, high concentrations (over their physiological next tested if CzQl could quantitatively detect the lysosomal pH
concentrations) of metal ions and bioactive molecules cause no changes. pH-dependent ratiometric fluorescence imaging of
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Fig. 3 Colocalization imaging of CzQl and LysoTracker Red DND-99 in
B16-F10 cells. (A) CzQl imaging (10 mM, lex= 405 nm; lem= 510–550 nm).
(
B) LysoTracker Red DND-99 imaging (1.0 mM, lex = 543 nm; lem
70–610 nm). (C) Overlay of (A) and (B). (D) Intensity correlation plot of
CzQl and LysoTracker Red DND-99.
=
5
the probe in living cells at different pH conditions was performed;
the results are shown in Fig. 4. The excitation wavelength was Fig. 5 In vivo fluorescence images of CzQl in mice with stimulation
À1
by LPS. (A) LPS (1.0 mg mL in saline, 200 mL) was injected, followed
at 405 nm, and the fluorescence images were recorded at 510–
by injection with CzQl (40 mM, 200 mL). (B) Only CzQl (40 mM, 200 mL)
treated. (C) Only LPS (1.0 mg mL in saline, 200 mL) treated. (E
550 nm (green channel) and 620–660 nm (red channel). It was
À1
x
= 420 nm,
obvious that the cells displayed bright green fluorescence and
very weak red fluorescence at pH 7.4. When the extracellular pH
decreased to 5.1, the fluorescence in the red channel slightly
E
m
= 535 nm).
enhanced. As the pH continued to decrease to 4.0, the green For comparison, mouse B was subcutaneously injected only
fluorescence intensity was significantly reduced accompanied by with CzQl. It was obvious that mouse A (Fig. 5A) treated with
the enhancement in red fluorescence. When pH dropped to 3.2, both LPS and CzQl exhibited lower fluorescence intensity
there was faint green fluorescence and strong red fluorescence. (pseudo-color) than mouse B treated with only CzQl (Fig. 5B),
2
9–31
The results matched well with those of the fluorescence titra- indicating decrease in the pH value in the inflamed tissues.
tions using the solutions of different pH values. Hence, these As a control, mouse C treated with LPS but not with CzQl
outcomes clearly demonstrated that CzQl is capable of monitor- showed no fluorescence (Fig. 5C). Therefore, these results
ing the changes in the lysosome pH values in living cells.
demonstrate that CzQl is useful for in vivo imaging and detecting
the pH changes in small animals.
In vivo fluorescence imaging
Experimental
Material and apparatus
We next investigated the application of CzQl to visualize the pH
changes in vivo induced by lipopolysaccharide (LPS)-triggered
2
7,28
inflammation.
As shown in Fig. 5, mouse A is subcuta-
All chemicals and solvents were of analytical grade and used
without further purification. 2-(2-Methoxyethoxy) ethanol was pur-
chased from Aladdin Industrial Inc. Lepidine and p-toluenesulfonyl
chloride were purchased from TCI chemical industrial develop-
ment Co., Ltd. Carbazole was provided by Sigma-Aldrich. Co.,
neously injected with LPS in the peritoneal cavity to cause an
acute inflammatory response and then is injected with CzQl.
Ltd. Trimethyl chlorosilane (TMSCl) and POCl were purchased
3
from Alfa Aesar Chemical Co., Ltd. All other commercially
available chemicals and solvents were obtained from Beijing
Chemical Reagent Co.
1
13
3 6
H and C NMR spectra were collected in CDCl or DMSO-d
as the solvent with tetramethylsilane (TMS) as the internal
standard on a Bruker 400 MHz NMR spectrometer (Bruker
biospin, Switzerland). High-resolution mass spectrometry (HRMS)
analyses were carried out using a Bruker Autoflex II matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectrometer (Bruker Daltonics, Germany). Absorption
spectra were recorded on a Varian Cary 300 UV-vis spectro-
photometer (Varian Technology Co., Ltd, USA). Fluorescence
spectra were recorded using a PTI Luminescence spectrophoto-
meter. Standard quartz cuvettes with 1 cm light path were used
for all optical measurements. Cell imaging was carried out using
a confocal laser scanning microscope (Zeiss, LSM880) with a
Fig. 4 pH-dependent ratiometric fluorescence imaging of CzQl (10 mM)
in B16-F10 cells. The excitation wavelength was at 405 nm. The fluores-
cence images were collected at 510–550 nm (green channel, second
column) and 620–660 nm (red channel, third column).
6
3Â oil-immersion objective lens. In vivo images were obtained
using a Bruker small animal in vivo imaging system. Ultrapure
water was obtained from a Millipore-Q ultrapurification system.
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pH measurements were obtained with a Beckman F 50 pH meter using n-hexane/ethyl acetate (3/1) to afford compound 3 (0.82 g) in
(
Shanghai, China).
69% yield as a yellow solid.
1
H NMR (CDCl , 400 MHz), d (ppm): 3.276 (s, 3H), 3.375–
3
Synthesis and characterization of fluorescent probe
3.397 (m, 2H), 3.481–3.503 (m, 2H), 3.850–3.879 (t, J = 5.8 Hz,
Synthesis of 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate 2H), 4.481–4.510 (t, J = 5.8 Hz, 2H), 7.260–7.329 (m, 1H), 7.468–
(1). A mixture of 2-(2-methoxyethoxy) ethanol (1.20 g, 10 mmol), 7.534 (m, 3H), 7.958–7.983 (dd, J = 10.0 Hz, 1.6 Hz, 1H), 8.099–
sodium hydroxide (0.80 g, 20 mmol), THF (20 mL), and water 8.118 (d, J = 7.6 Hz, 1H), 8.546–8.549 (d, J = 1.2 Hz, 1H), 10.061
1
3
(
3
20 mL) was added into a three-necked flask. The solution (s, 1H). C NMR (CDCl , 100 MHz), d (ppm): 43.77, 59.28,
mixture was stirred at 0 1C. To the solution mixture, a THF 69.49, 71.12, 72.17, 109.64, 109.77, 120.72, 120.90, 123.28,
solution (50 mL) of p-toluenesulfonyl chloride (2.28 g, 12 mmol) 123.39, 124.05, 126.96, 127.41, 128.95, 141.52, 144.66, 192.05.
was added dropwise, and the reaction mixture was stirred for
Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-3-((E)-2-(quinolin-
12 h at room temperature. The reaction mixture was poured into 4-yl)vinyl)-9H-carbazole (CzQl). To the solution of lepidine
aqueous hydrochloric acid, and the product was extracted with (0.1 mL, 0.75 mmol) and compound 3 (0.15 g, 0.5 mmol) in
dichloromethane three times. The organic layer was dried over DMF (5 mL) in a sealed tube, TMSCl (0.64 mL, 5 mmol) was
anhydrous magnesium sulfate and filtered and concentrated added, and the resulting mixture was heated to 100 1C for 24 h.
by evaporation under reduced pressure. The crude product After cooling down to room temperature, water was added,
was purified by column chromatography using n-hexane/ethyl followed by aqueous Na
acetate (5/1, V/V) as the eluent to give a colorless oil. Yield: aqueous solution was extracted with dichloromethane three times.
.38 g (87%). The combined organic phase was washed with brine and dried
H NMR (CDCl , 400 MHz), d (ppm): 2.450 (s, 3H), 3.355 over anhydrous sodium sulfate. After removing the solvent, the
2 3
CO solution to adjust the pH to 8. The
2
1
3
(
(
(
(
s, 3H), 3.474–3.496 (m, 2H), 3.573–3.596 (m, 2H), 3.682–3.706 crude product was purified by silica gel column chromatography
t, J = 4.8 Hz, 2H), 4.160–4.184 (t, J = 4.8 Hz, 2H), 7.332–7.352 using a dichloromethane and ethyl acetate (5/1) mixture as the
1
3
d, J = 8.0 Hz, 2H), 7.793–7.814 (d, J = 8.4 Hz, 2H). C NMR eluent to afford CzQl (0.112 g) in 53% yield as an orange solid.
1
CDCl
14.61, 129.72, 131.60, 163.23, 190.41.
Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (2). (t, J = 5.4 Hz, 2H), 4.577–4.604 (t, J = 5.4 Hz, 2H), 7.240–7.277
To a solution of carbazole (0.84 g, 5 mmol) in THF (30 mL) at (t, J = 7.4 Hz, 1H), 7.460–7.495 (t, J = 7.0 Hz, 1H), 7.643–7.701
1C, NaH (0.18 g, 7.5 mmol) was added. The solution was (m, 3H), 7.780-7.821 (t, J = 8.2 Hz, 2H), 7.890–7.902 (d, J = 4.8 Hz,
3
, 100 MHz), d (ppm): 58.91, 67.18, 69.34, 70.59, 71.73,
H NMR (DMSO-d
6
, 400 MHz), d (ppm): 3.119 (s, 3H), 3.299–
1
3.323 (t, J = 4.8 Hz, 2H), 3.456–3.480 (t, J = 4.8 Hz, 2H), 3.807–3.834
0
stirred for 30 min, and compound 1 (2.06 g, 7.5 mmol) was 1H), 7.948–7.966 (d, J = 7.2 Hz, 1H), 8.032–8.055 (d, J = 9.2 Hz, 1H),
added with constant stirring. The resulting mixture was stirred 8.111–8.152 (d, J = 16.4 Hz, 1H), 8.225-8.243 (d, J = 7.2 Hz, 1H),
overnight at room temperature. The solution was poured into 8.614–8.634 (d, J = 8.0 Hz, 1H), 8.670–8.673 (d, J = 1.2 Hz, 1H),
13
6
ice water, and the product was extracted with ethyl acetate three 8.876–8.887 (d, J = 4.4 Hz, 1H). C NMR (DMSO-d , 100 MHz),
times. The organic layer was washed with water and brine and d (ppm): 54.91, 58.12, 68.88, 69.82, 71.29, 109.45, 109.81, 109.98,
then dried over anhydrous sodium sulfate to remove the 115.96, 118.91, 119.28, 119.62, 120.36, 120.76, 122.60, 122.75,
solvent. The crude product was separated by silica gel column 124.20, 125.02, 125.85, 126.35, 127.68, 129.35, 134.75, 136.15,
chromatography with n-hexane and ethyl acetate (3/1) to give 140.83, 142.69, 148.47, 150.21. MS (MALDI-TOF) m/z: calcd for
+
the desired product 2 (1.07 g) in 80% yield as a brown oil.
[M + H] , 423.2067; found, 423.2079.
1
H NMR (CDCl
3
, 400 MHz), d (ppm): 3.310 (s, 3H), 3.405–
UV-vis and fluorescence pH titrations
3
2
7
1
1
.429 (m, 2H), 3.514–3.536 (m, 2H), 3.844–3.876 (t, J = 6.4 Hz,
H), 4.494–4.526 (t, J = 6.4 Hz, 2H), 7.210–7.262 (m, 2H), 7.420– A stock solution of CzQl (1 mM) was prepared in DMSO and was
1
3
.492 (m, 4H), 8.081–8.097 (d, J = 6.4 Hz, 2H). C NMR (CDCl
00 MHz), d (ppm): 43.02, 59.01, 69.14, 70.72, 71.79, 108.71, an analytical concentration of 10 mM for absorption and emis-
18.87, 120.20, 122.82, 125.64, 140.51. sion measurements in quartz cuvettes of 1 cm optical path
Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3- length (3 mL volume). Slight pH variations in the solution were
carbaldehyde (3). A 100 mL three-necked flask containing achieved by adding minimal volumes of HCl (0.01 M). The
3
,
2
further diluted with a mixed solution of DMSO/H O (1/1, v/v) to
2
6
0 mL dry DMF was cooled to 0 1C and then, POCl
mmol) was added dropwise. The solution mixture was bandwidths were both set at 1.5 nm. The fluorescence quantum
3
(0.6 mL, excitation wavelength was 415 nm. The excitation and emission
warmed to room temperature and stirred for 1 h. To this yield was determined using quinine sulfate solution (F = 0.577
reaction mixture, compound 2 (1.07 g, 4 mmol) in dichloro- in 0.1 M H SO ) at pH 7.0 and fluorescein solution (F = 0.95 in
methane (5 mL) was added slowly. The reaction temperature 0.1 M NaOH) at pH 1.9 as the reference according to a method
2
4
3
2
was raised to 75 1C and maintained for 8 h. After cooling to described in the literature. All spectroscopic experiments
room temperature, the solution was poured into ice water and were carried out at room temperature.
extracted with dichloromethane. The organic phase was
Colocalization-imaging experiments in B16-F10 cells
washed with water and brine. Then, the organic layer was dried
over anhydrous sodium sulfate, and the solvent was removed. The B16-F10 cells were cultured in DMEM medium supplemented
crude product was purified by silica gel column chromatography with 10% FBS and incubated at 37 1C in a 5% CO atmosphere
2
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and passed and dispersed one day before imaging. Then, the were found for CzQl, including a large Stokes shift (4100 nm),
cells were incubated with LysoTracker Red DND-99 (1.0 mM) good selectivity, high photostability, low cytotoxicity, and excel-
and CzQl (10 mM) for 30 min. The medium was removed, and lent cell permeability. All these features enable the probe to
the cells were washed with PBS (pH 7.4) three times. Cell successfully achieve the real-time imaging of lysosomal pH in
imaging was performed using a confocal laser scanning micro- living cells and in vivo.
scope (Zeiss, LSM880) with a 63Â oil-immersion objective lens.
Confocal images of the cell fluorescence of LysoTracker Red
DND-99 were captured using a 543 nm laser, and the collection
window was 570-610 nm. The excitation wavelength for CzQl
Conflicts of interest
was 405 nm, and the collection window was 510–550 nm.
There are no conflicts to declare.
Intracellular pH imaging
B16-F10 cells were prepared and incubated with 10 mM final Acknowledgements
concentration of CzQl for 15 min. Then, excess CzQl solution
This work was supported by the National Natural Science
was removed by washing with PBS (pH 7.4) three times, and the
À1
+
Foundation of China (No. 21705105). The project was funded by
the China Postdoctoral Science Foundation (No. 2019M651087),
Scientific and Technologial Innovation Programs of Higher
Education Institutions in Shanxi (No. 201802063), and Doctor
Fund of Shanxi Province (No. SD1818).
cells were incubated with 10 mg mL nigericin in high K
buffer (including 120 mM KCl, 30 mM NaCl, 0.5 mM MgSO
4
,
1
5
mM CaCl
2 2 4
, 20 mM HEPES, 20 mM NaOAc, 1 mM NaH PO and
mM glucose) at different pH values (7.40, 5.1, 4.0 and 3.2) for
another 15 min. Cell imaging was performed using a confocal
laser scanning microscope (Zeiss, LSM880) with a 63Â oil-
immersion objective lens (l = 405 nm; l = 510–550 nm).
ex
em
Notes and references
In vivo fluorescence imaging
1
2
P. Y. Lie and A. Nixon, Neurobiol. Dis., 2019, 122, 94.
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BALB/c mice (20–25 g) were obtained from the Laboratory
Animals Center of Shanxi Medical University (Taiyuan, China)
for in vivo imaging. All animal experiments were performed in
compliance with the Animal Management Rules of the Ministry
of Health of the People’s Republic of China (Document no. 55,
3
4
2001) and approved by the ethics committee of Shanxi Medical
University. We have taken great efforts to reduce the number of
animals used in these studies; we have also taken efforts to
reduce the pain and discomfort experienced by the animals.
5
6
3
3,34
According to previous works,
BALB/c mice were given an
À1
intraperitoneal injection of LPS (1.0 mg mL in saline, 200 mL).
F. M. Platt, B. Boland and A. C. Spoel, J. Cell Biol., 2012,
1
After 4 h, the mice were intraperitoneally injected with CzQl
99, 723.
7 L. Zhang, R. Sheng and Z. Qin, Acta Biochim. Biophys. Sin.,
009, 41, 437.
(
40 mM, 200 mL) for another 15 min. For comparison, mice with
À1
only LPS (1.0 mg mL in saline, 200 mL) treatment for 4 h but
no CzQl and with only CzQl (40 mM, 200 mL) treatment for
2
8
A. Nemchenko, M. Chiong, A. Turer, S. Lavandero and
J. A. Hill, J. Mol. Cell. Cardiol., 2011, 51, 584.
W. Ge, D. Li, Y. Gao and X. Cao, Int. Rev. Immunol., 2015,
15 min without stimulation by LPS were prepared. These mice
were anesthetized by an intraperitoneal injection of 4% chloral
hydrate (300 mL) for 10 min before imaging. In vivo images were
then obtained using a Bruker small animal in vivo imaging
system with an excitation filter of 420 nm and an emission filter
of 535 nm.
9
34, 415.
1
1
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In summary, to detect the pH changes in lysosomes, we 14 J. Wang, S. Xia, J. Bi, Y. Zhang, M. Fang, R. L. Luck, Y. Zeng,
developed a ratiometric emission NIR-fluorescent probe (CzQl)
via an ethylene bridge electron-donating carbazole group
T.-H. Chen, H.-M. Lee and H. Liu, J. Mater. Chem. B, 2019,
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