Specific and photostable rhodamine-based tracker for 3D video imaging of single acidic organelles

Article abstract of DOI:10.1039/c4ra04091k

Three-dimensional video imaging has emerged as an indispensable tool for real-time monitoring of dynamic acidic organelles. However, the limitation of video imaging is the absence of a specific stain for acidic organelle trackers. The aim of this work was to investigate the applicability of a potential acidic organelle tracker, Lyso-R, in three-dimensional video imaging in live cells. In a close examination of three differently designed rhodamine dyes, Lyso-R outperformed the other two with a suitable pK_a value and higher membrane permeability. The uninterrupted fluorescence of Lyso-R towards macromolecules, e.g. lecithin and proteins, led to higher specificity and signal-to-background ratio than LysoTracker DND 189 and DND 99 for imaging acidic organelles. In addition, Lyso-R was photostable, and MTT assays confirmed its low toxicity towards cells. Inspired by these facts, three-dimensional tracking of a single acidic organelle in a live cell was obtained by staining with Lyso-R under confocal microscopy. The measurement of this organelle demonstrated that the distance change of the organelle centroid on XY plane was sharper than its depth change. The usage of Lyso-R was further extended with two-dimensional video imaging of acidic organelles during various cell metabolisms. All of these results demonstrate the potential applicability of Lyso-R as a three-dimensional imaging tracker of acidic organelles. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra04091k

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Specific and photostable rhodamine-based tracker
for 3D video imaging of single acidic organelles†
Cite this: RSC Adv., 2014, 4, 37547
*
Zhiwei Ye, Yi Xiao, Haiying Guo and Chao Wang
Three-dimensional video imaging has emerged as an indispensable tool for real-time monitoring of
dynamic acidic organelles. However, the limitation of video imaging is the absence of a specific stain for
acidic organelle trackers. The aim of this work was to investigate the applicability of a potential acidic
organelle tracker, Lyso-R, in three-dimensional video imaging in live cells. In a close examination of
three differently designed rhodamine dyes, Lyso-R outperformed the other two with a suitable pKa value
and higher membrane permeability. The uninterrupted fluorescence of Lyso-R towards macromolecules,
e.g. lecithin and proteins, led to higher specificity and signal-to-background ratio than LysoTracker DND
189 and DND 99 for imaging acidic organelles. In addition, Lyso-R was photostable, and MTT assays
confirmed its low toxicity towards cells. Inspired by these facts, three-dimensional tracking of a single
acidic organelle in a live cell was obtained by staining with Lyso-R under confocal microscopy. The
measurement of this organelle demonstrated that the distance change of the organelle centroid on XY
plane was sharper than its depth change. The usage of Lyso-R was further extended with two-
dimensional video imaging of acidic organelles during various cell metabolisms. All of these results
demonstrate the potential applicability of Lyso-R as a three-dimensional imaging tracker of acidic
organelles.
Received 4th May 2014
Accepted 31st July 2014
DOI: 10.1039/c4ra04091k
www.rsc.org/advances
restricted their real-time video imaging ability in three dimen-
sions.13 On the one hand, the non-specic stain of commercial
Introduction
trackers in cells generates the mixed signals of specic uo-
rescence and nonspecic background uorescence noise, which
decreases the resolution and makes 3D video imaging unreli-
able. The rigid polycyclic aromatic structures of LysoTrackers,
like DND 99 and DND 189, generates a strong tendency of
accumulating in the hydrophobic cavities of proteins or lipids,
leading to their poor specicity. Moreover, DND 99 photo-
degrades to another emissive species during imaging,14 which
further reduces its signal-to-background ratio in imaging.
Although conjugating highly hydrophilic groups to some extent
addresses this problem, it results in another serious problem of
membrane impermeability. On the other hand, 3D video
imaging without sacricing lateral resolution requires trackers
to emit more photons before photobleaching than in 2D
imaging or 3D imaging. Although 3D video imaging and 3D
imaging are same in nature: a collection of photons, 3D video
imaging involves a stack of 3D images at different time points;
moreover, 3D video imaging requires a high-photostability
tracker. Poor photostability of trackers, like Neutral Red (NR),
results in fast photobleaching before emitting enough photons
for 3D video imaging. Thus, this paper describes a better tracker
to replace the commercial ones for 3D video imaging of single
acidic organelles, as well as series of video imaging examples of
acidic organelles during various cell metabolisms.
Acidic organelles include lysosomes, late endosomes and other
acidic compartments.1,2 These organelles continuously perform
complex transformations in morphology and cooperate with
other organelles to serve various functions, like the repair of
plasma membranes, the turnover of cellular proteins, and the
down-regulation of surface receptors.3,4 These dynamic changes
are crucial for their functions and also reect their health
status.5,6 The characteristic of these organelles is their acidic
lumens, which is maintained by the proton pump from the
organelle's membrane. In order to visualize the acidic organ-
elles and their cooperation, a series of trackers7–12 attached with
a weak base group have been developed for acidic organelles.
However, these trackers are commonly used in two-dimensional
(2D) imaging, which leads to omission of critical details on Z
dimension. To the best of our knowledge, their usage in three-
dimensional (3D) video imaging (3D video imaging involves
time-lapse imaging) of acidic organelles has rarely been
reported.
The unsatisfactory property of conventional trackers for
acidic organelles, namely, less specicity in staining, has
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong
Road, Dalian 116024, China. E-mail: xiaoyi@dlut.edu.cn
† Electronic supplementary information (ESI) available: The detailed
characterization of compounds and additional gures for the manuscript. See
DOI: 10.1039/c4ra04091k
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intercalating into hydrophobic domains. In fact, even if trace
quantities of trackers are distributed outside those organelles,
their uorescence will be negligible due to the PET processes. In
order to identify the appropriate functionality, we designed
three rhodamine-based trackers, Lyso-R, Lyso-ER and Lyso-PR,
which are shown in Chart 2.
Lyso-R surpasses the other two trackers in cellular staining
Although trackers with basic groups are potentially toxic
towards cells, our designed trackers exhibits low toxicity
towards cells in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) assays (ESI Fig. S1b†). At either a
minimum concentration of 1 mM or a maximum concentration
of 5 mM, up to 85% of the dye-incubated cells are alive aer 24
hours (only Lyso-PR exhibits 70% cell survival). The low toxicity
indicates the potential property of trackers for biological
imaging.
Chart 1 Working principles of Lyso-R, which keeps a good balance
between the cell-membrane permeability and the specificity of
localization in acidic organelles.
Compared with Lyso-ER and Lyso-PR, Lyso-R exhibits far
better applicability for acidic staining (ESI Fig. S1†). As shown in
Table 1, Lyso-R is hydrophobic in neutral aqueous solution with
a positive log P value of 0.85, while the other two trackers are
slightly hydrophilic in the same aqueous solution. This hydro-
phobicity of Lyso-R drives the tracker to diffuse through the
membranes in vivo, which is conrmed by the cell staining
experiment shown in ESI Fig. S1d.† The other two trackers,
hindered due to their hydrophilicity are membrane-imperme-
able as no uorescence is detected in the same experiment. This
result is further conrmed by their higher pKa value (7.82 and
9.95, respectively) than Lyso-R (6.11). This pKa value result
suggests that Lyso-ER and Lyso-PR exist in positively charged
and membrane-impermeable forms in neutral media (pH
around 7), while Lyso-R retains its ring-closed hydrophobic
form to diffuse through the membranes. Furthermore, Lyso-R
owns high quantum yields of 0.729, as well as a high absorption
cross section of 2.9 ꢀ 10ꢁ19 cm2 at 526 nm in aqueous solution
of pH 4.0. Combined with the abovementioned results, Lyso-R
surpasses the other two trackers owing to its better cell
permeability.
Chart 2 Structures of designing trackers, Lyso-ER, Lyso-PR and
Lyso-R.
Results and discussion
Molecular design
The design strategy is exhibited in Chart 1 (Lyso-R has been
published15 before but without any further applicable tests).
Firstly, the uorophore moiety of rhodamine, a well-known
laser dye, has moderate photostability and strong uorescence.
Secondly, owing to the specialty of forming hydrophobic leuco
structure, the tracker freely diffuses through the plasma
membrane and the organelle's membrane. Lastly, but most
importantly, two conjugated alkalescent amino moieties
signicantly contribute to the specic stain of the acidic
organelle. Outside the acidic environment, the free amino
group quenches the uorescence through PET16,17 (photoin-
duced electron transfer) processes. Inside the acidic organelles,
the protonation of the amino moieties converts the hydro-
phobic trackers into highly hydrophilic molecules emitting
strong uorescence; consequently, the hydrophilicity of
trackers stabilizes their retention to prevent them from
Lyso-R's specicity and photostability: co-localization and
comparison with commercial trackers
Firstly, Lyso-R exhibits a good quantum yield and absorption
cross-section compared with commercial trackers (NR, DND
Table 1 Photophysical properties of Lyso-R, Lyso-PR and Lyso-ER
Absorb
Fluorescent
Stokes
shi (nm)
Quantum
yieldsc
Absorption cross
log Pa
pKa
peakb (nm)
peakb (nm)
sectionb (ꢀ10ꢁ19 cm2)
b
Compound
Lyso-R
Lyso-ER
Lyso-PR
0.85
ꢁ0.38
ꢁ0.58
6.11
7.82
9.95
526
533
543
557
555
565
31
22
22
0.73
0.80
0.53
2.9
1.8
3.0
a
log P tests in neutral aqueous solutions; log P (logarithm of octanol–water partition coefficient) value indicates the hydrophobicity or
b
c
hydrophilicity of compound. All labeled data are measured at a concentration of 5 mM in aqueous solution of pH ¼ 4.0. Quantum yields are
measured in aqueous solution of pH ¼ 4.0 using uorescein as a standard (F ¼ 0.92 in 0.1 M NaOH solution).18
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Fig. 1 Applicability of Lyso-R for 3D video imaging of acidic organelles. (a) Colocalization of Lyso-R (0.25 mM, lex ¼ 488 nm, lem ¼ 530 nm–570
nm) with NR (0.25 mM, lex ¼ 559 nm, lem ¼ 570–590 nm) in MCF-7 cells. Sequential images exhibit the two-channel confocal images, the
merged image, intensity profile of ROIs across cells and the intensity correlation profile of Lyso-R and NR (the intensity correlation profile
indicates the correlation between two fluorescent images. When two fluorescent images have perfect correlation, the plots perfectly overlap the
diagonal line22). (b) Colocalization of Lyso-R (0.25 mM, lex ¼ 515 nm, lem ¼ 530 nm–570 nm) with DND 189 (0.25 mM, lex ¼ 458 nm, lem ¼ 470
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Table 2 Co-localization and signal-to-background results of Lyso-R,
DND 99, DND 189 and NR calculated from confocal images
the Z axis in Fig. 1f and ESI movie 1†). Fluorescence signals of
Lyso-R exhibit some globular structures, but DND 189 presents
a cloud of continuous uorescent regions with indenite
outlines.
Pearson's
Manders'
Compound
coefficienta
coefficienta
SBRb
To further understand the factors inuencing the specicity
in the acidic organelle's staining, a comparison study on the
effects of pH, and protein and lipid concentrations on the
uorescence properties of Lyso-R and DND 189 is conducted. As
shown in Fig. 1e, in the pH range from 7.5 to 4.5, Lyso-R shows a
39 times enhancement with a decrease in pH value independent
of the presence or absence of BSA (representing proteins) or
lecithin (representing lipids). In contrast, the plots of DND 189
exhibit high sensitivity towards the presence of BSA or lecithin.
Not only the pKa values vary considerably, but the uorescence
increase tendencies are also different. For example, in the
presence of lecithin, the uorescence intensity of DND 189 at
pH of 4.5 is only 1.5 times higher than at pH of 7.5, which to
some extent explains the non-negligible, non-specic stain of
the dye in the cells. This pKa plot change is possibly caused by
the affinity between the hydrophobic cavities of macromole-
cules and the hydrophobic molecule of DND 189. The above-
mentioned results conrm the origin of the Lyso-R's specic
stain of acidic organelles: the independence of signicant
uorescence enhancement from other macromolecules. More-
over, in a practical cellular environment the pH value of
organelles changes with time. Trackers with a uorescence
intensity inuenced by protein or lipid are unsuitable for
application to pH evaluation of acidic organelles in live cells.
Thus, Lyso-R is capable of the quantitative evaluation of the pH
of acidic organelles as further conrmed in ESI Fig. S7.†
To evaluate the background strength of Lyso-R, we calculate
the signal-to-background ratio of the three dyes (Lyso-R, DND
189 and DND 99) shown in Fig. 2. First, we identify those high-
uorescence regions of interest, representing the acidic organ-
elles, and the same-size region of low uorescence, representing
the background signal. Then, these data are processed through
the algorithm mentioned in the experimental section to obtain
the SBR value. Among all the three dyes, Lyso-R exhibits the
Lyso-R
—
—
12.36 ꢂ 1.8
—
Neutral red
DND 189
DND 99
0.89 ꢂ 0.02
0.82 ꢂ 0.04
0.71 ꢂ 0.11
0.94 ꢂ 0.01
0.87 ꢂ 0.03
0.81 ꢂ 0.06
7.04 ꢂ 1.43
4.57 ꢂ 1.07
a
Colocalization analysis of DND 189, DND 99 or NR with Lyso-R in
MCF-7 cells (n ¼ 3, ꢂ: SEM). Pearson correlation coefficient and
Manders' overlap coefficient are used to quantify the co-localization of
two dyes and a perfect colocalization means a value of 1 for both
b
coefficients.19–21
SBR (signal-to-background ratio) values are
calculated from the average signal divided by the background in
confocal images (n ¼ 18, ꢂ: SEM).
189 and DND 99) listed in ESI Table S1.† Secondly, colocaliza-
tion experiments conrm the specicity of Lyso-R towards
acidic organelles. There is a close synchrony between Lyso-R
and commercial trackers in Fig. 1a–c. This result is also sup-
ported by high Manders' coefficients in Table 2. The higher
Manders' coefficients of NR and Lyso-R suggest an analogous
stain between them. Generally, rhodamine dyes exhibit specic
affinity to mitochondria, but that is not true for Lyso-R. In ESI
Fig. S3,† Lyso-R exhibits a different segregated stain from tet-
ramethyl rhodamine (TMRM),
tracker.
a standard mitochondrial
According to the photostability analysis in Fig. 1d, Lyso-R
owns suitable photostability for long time imaging. In order to
demonstrate the photostability of Lyso-R, the photostable
trackers23–25 DND 189 and DND 99, as well as the photo-unstable
tracker Neutral Red, are chosen for photostability comparison.
Aer successive ten minute imaging under continuous laser
excitation, barely 30% of Lyso-R's origin intensity is quenched,
which is close to the photostable LysoTracker DND 99 and DND
189 and far better than Neutral Red. Thus, photostability will
not restrict Lyso-R's applications on 3D video imaging of acid
organelles.
highest signal-to-background ratio (12.36
ꢂ
1.8), which
suggests that it is the most specic for staining acidic organ-
elles. Consistent with the abovementioned pKa value, the SBR of
DND 189 is the lowest, making DND 189 potentially inappli-
cable for some noisy environments. DND 99 has also been
found to have an unknown mechanism of photodegradation to
another emissive species,14 which possibly reduces its signal-to-
background ratio. With the combination of these facts and the
possible unquenched uorescence in a physiologically neutral
environment, DND 99 exhibits a moderate SBR value, between
Lyso-R and DND 189.
In Fig. 1b and f, there is a sharp contrast between the non-
specic stain of DND 189 and specic stain of Lyso-R. In Fig. 1b,
while DND 189 exhibits diffusive and continuous uorescence
signals in broad intracellular regions, the uorescence of Lyso-R
appears in the separated and punctual areas, consistent with
the characteristics of the acidic organelles. Thus, the colocali-
zation is not so close, supported by the relatively low Manders'
coefficients (0.87 ꢂ 0.03). Because good specicity is necessary
for 3D imaging, a 3D colocalization experiment is conducted to
compare the stain from DND 189 and Lyso-R (rotating around
nm–510 nm) in MCF-7 cells. Sequential images exhibit the two-channel confocal images, the merged image, intensity profile of ROIs across cells
and the intensity correlation profile of Lyso-R and DND 189. (c) Co-localization of Lyso-R (0.25 mM, lex ¼ 488 nm, lem ¼ 530–570 nm) with DND
99 (0.25 mM, lex ¼ 559 nm, lem ¼ 570–630 nm) in MCF-7 cells. Sequential images exhibit the two-channel confocal images, the merged image,
intensity profile of ROIs across cells and the intensity correlation profile of Lyso-R and DND 99. (d) Photostability comparison of four dyes, Lyso-
R, DND 189, DND 99 and NR in live cells under continuous laser excitation (approximately 0.24 mW cmꢁ2) (n ¼ 3, see details in the experimental
section). (e) pKa plot of Lyso-R and DND 189 at 1 mM in aqueous solution, 2% bovine serum albumin (BSA) and 10 mM lecithin, respectively. (f)
Colocalization of Lyso-R (red) and DND 189 (green) in 3D image rotates from 0ꢃ to 90ꢃ, respectively. Scale bar: 10 mm (a and c), 20 mm (b).
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Fig. 2 Quantitative analysis of signal-to-background ratio (SBR) of
Lyso-R, DND 189 and DND 99 for the in vivo acidic organelle's tar-
geting ability. (a) Images acquired from confocal microscopy. Magni-
fication images of the white boxes in the upper images are shown in
the images below. In those below images, the average of signal and
noise were calculated from the fluorescent intensities in the circle
representing the acidic organelles (red circle) and the background area
(yellow circle), respectively. The SBR of a single dye was obtained from
the average signal divided by the background (see Experimental). (b)
SBR value of Lyso-R, DND 189 and DND 99 for the acidic organelle's
targeting ability (n ¼ 18 processes). Scale bar: 5 mm.
Fig. 3 3D tracking of a single acidic organelle in Lyso-R (0.25 mM, lex
¼
515 nm, lem ¼ 530–570 nm)-stained MCF-7 cells. The tracking rate is
approximately 17.5 seconds per image. The resolution of the captured
images is 800 ꢀ 800 pixels. (a) The MCF-7 cell's panorama. (b)
Tracking trajectory of the organelle projects on the XY plane. (c) The XY
distance change and depth of the organelle centroid vs. time. (d) The
calibration bar of color to its depth, respectively. (e) Image sequence of
the organelle. Scale bar: 10 mm (a), 5 mm (e).
The cytotoxicity of Lyso-R is further evaluated in ESI Fig. S8.†
As the data in ESI Fig. S8† shows, no obvious cytotoxicity is
observed for Lyso-R. The cell variability of MCF-7 cells is 93.4%
for 24 h and 92.2% for 48 h at the concentrations of 1 mM. The
concentration experiment on the cell variability of MCF-7 cells
also suggests a good biocompatibility of Lyso-R for cell vari-
ability up to 85% at concentrations as high as 10 mM. Combined
with the abovementioned results, Lyso-R exhibits good
biocompatibility, signicant specicity towards acidic organ-
elles and suitable photostability for 3D video imaging. In order
to further conrm this potential for video imaging of acidic
organelles, the application of Lyso-R for real-time video imaging
of various biological situations is analyzed and discussed.
organelles are analyzed. All data exhibit the sharper change of
the XY position than that of the depth. There may be two factors
contributing to this. Firstly, due to the horizontal extension of
the adherent cells, the length (around 30–40 mm) and width
(around 15–30 mm) of the cell is longer than its depth (around 5–
10 mm). The second factor may be the shape of the cytoskel-
eton.26,27 Furthermore, the recorded trajectory is similar to a
cycle in Fig. 3b. Above all, the successful application of Lyso-R in
3D track of single acidic organelles in live cells under confocal
microscopy conrms the suitable photostability and strong
specicity for the imaging of acidic organelles.
3D tracking of single acidic organelles in MCF-7 cells by using
Lyso-R
Lyso-R tracking by stimulation-induced tubular organelles in
macrophages RAW264.7 cells
By using Lyso-R, a 3D video tracking of single acidic organelles
of MCF-7 cells under confocal microscopy is achieved, as
exhibited in Fig. 3 (and also in ESI movie 2 and 2a†). To prepare
a 3D track of a single acidic organelle, we carefully choose an
organelle with both the highest signal (brighter than
surrounding organelles) and the largest size (nearly 1 mm).
Moreover, in the surrounding area, there are no other acidic
organelles with higher signals and larger size. Thus, despite the
long interval between each image, the tracked organelle in each
image would be the same organelle. The sequential images,
shown in Fig. 3e, present the average depth of the organelle
during sequential time intervals. When these images are
combined with a time resolution of 17.44 s, the 3D confocal
video is generated in ESI movie 2 and 2a.†
Most acidic organelles are known to be small globular struc-
tures. However, upon LPS stimulation or under starvation
condition, partial globular acidic organelles, particularly lyso-
somes, transform into tubular shapes in microphages.28 These
In order to evaluate the depth of a single acidic organelle, we
Fig. 4 Image sequences of tubular organelle formation from Lyso-R
propose a concise algorithm shown in the experimental section. stained macrophages after exposing to 10 mg mLꢁ1 of LPS for 1 h.
These macrophages are incubated with Lyso-R (1.0 mm) for five
Through this algorithm, the depth of the organelle centroid
over time is recorded in Fig. 3c along with the XY distance of the
organelle centroid during the imaging time. The plot shows that
the XY distance of this single organelle centroid changes
minutes. Then, the dyes in the medium are washed out by PBS solu-
tions, and next, the cells are exposed to 10 mg mLꢁ1 of LPS for 1 h. The
cells are imaged under continuous laser excitation for around 1 min.
Scale bar: 1 mm. Color bar indicates the range from low- (black) to
sharper than its depth. In ESI Fig. S9,† six more acidic high-fluorescence intensity (white).
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exhibits a good quantum yield and absorption cross-section
compared with commercial trackers (DND 99, DND 189 and
Neutral Red). Lyso-R also exhibits no obvious cytotoxicity in
MTT experiments. The specicity of acidic organelle staining
from Lyso-R is supported by the coefficients study in colocali-
zation experiments between conventional trackers and Lyso-R.
Moreover, Lyso-R also owns a good photostability close to
conventional DND 99 and DND 189. Furthermore, in pKa plot
studies, the uorescence of Lyso-R exhibits a strong indepen-
dence from the presence of lecithin and proteins, while the
uorescence of DND 189 is strongly affected by them. In addi-
tion, the signal-to-background ratio of Lyso-R is higher than
that in conventional trackers, DND 189 and DND 99. These
results indicate that Lyso-R surpasses the conventional trackers
for its specicity and photostability.
Furthermore, the 3D video imaging of a single acidic
organelle in a MCF-7 cell under confocal microscopy is achieved
by using Lyso-R. The result suggests that the movement of the
organelle is sharper in the plane of focus than in the depth, and
both the photostability and the specicity of Lyso-R are suitable
for the 3D video imaging of acidic organelles. The visualization
of tube phenomenon in Lyso-R-stained macrophages indicates
a higher pH value of the tubular organelle than the original
globular acidic organelle. This video imaging extends the
potential applicability of Lyso-R for the in vivo imaging of acidic
organelles under confocal microscopy. Therefore, it is antici-
pated that Lyso-R would be useful in future acidic organelle-
related research.
Scheme 1 Synthesis route of Lyso-R, Lyso-ER and Lyso-PR.
tubular organelles, which comprise a more motile population
than normal organelles,29 are related to metabolite transport30
and autophagy31 in cell metabolism.
To clearly understand this tube phenomenon, we study the
tubular acidic organelles in macrophages stained with Lyso-R
(Fig. 4, ESI Fig. S5 and ESI movie 3a and b†). In this experiment,
the “bud” formation of a single tubular organelle is captured
unambiguously. Different tubular organelles are stained with
Lyso-R in ESI Fig. S5.† They exhibit low uorescent signals
compared to globular acidic organelles, suggesting their
potentially higher pH value. The formation process of a single
tubular organelle is fully presented in Fig. 4. Single tubular
acidic organelle arises from the globular acidic organelle in less
than 50 seconds, which is consistent with the former ndings.29
Aer maturation, it splits from the original organelle. Thus, the
imaging of tubular organelles extends the application of Lyso-R
in the video imaging of different types of acidic organelles.
Currently, considerable effort is directed toward developing
various molecular probes for ‘static’ 2D imaging of acidic
organelles, including probes for two-photon imaging,7,32–34 for
the sensing of zinc ions,35 hydrogen peroxide,36 hydrogen
sulde,11 nitro oxides,10 iron pool,37 and pH,8,38–40 as well as for
the quantication of the organelle's viscosity.9 As demonstrated
in beginning, these 2D images lose dynamic details in depth,
which could be potentially signicant for the understanding of
acidic organelles. In this case, Lyso-R is a reliable tracker for the
3D co-tracking agent of acidic organelles.
Other applications of Lyso-R
To extend the applicability of Lyso-R, two other different acidic
organelles, involving cellular dynamic processes, have been
real-time imaged and some interesting and/or previously
unavailable phenomena are observed. Firstly, we study the
acidic organelle's behavior during lipid hydrolysis in MCF-7
cells (ESI Fig. S6 and ESI movie 4, 4a and 4b†). The concave
shape of acidic organelles close to lipid droplets indicates the
potential microautophagy regulation during lipid hydrolase. In
addition, the two-channel video imaging suggests the multi-
color video imaging ability of Lyso-R. Lastly, the process of the
clinical medicine chloroquine, inducing pH elevation of the
single acidic organelles of MCF-7 cells, is visualized for the rst
time in ESI Fig. S7 and ESI movie 5.† The result suggests three
potential stages for incubational pharmacology of chloroquine
in the MCF-7 cell. Through this video imaging, Lyso-R also
exhibits potential applicability for pH evaluation of single acidic
organelles in live cells.
Experimental
General information
All chemicals were obtained from commercial suppliers and
used without further purication. Melting points were obtained
with a capillary melting-point apparatus in open-ended capil-
laries and are uncorrected. 1H-NMR and 13C-NMR were
measured in CD3OD with TMS as an internal reference. Multi-
plicities of signals are described in the following manner: s –
singlet, br. s – broad singlet, d – doublet, t – triplet, and m –
multiplet. Coupling constants (J) are given in Hz. Column
chromatography was performed with silica gel (200–300 mesh).
Conclusions
In summary, we design a specic and photostable tracker for
the 3D video imaging of acidic organelles in live cells. Firstly,
three potential trackers based on rhodamine are designed and
tested. Aer careful comparison of their pKa value, log P value,
Quantum yields
cell permeability and cell toxicity, Lyso-R surpasses the other Fluorescence quantum yields were determined using uores-
two dyes with suitable pKa value and cell permeability. Lyso-R cein (in 0.1 M NaOH, F ¼ 0.92) as a reference.18 The given
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quantum yields were calculated using the following equa- (fetal bovine serum) in an atmosphere of 5% CO2 and 95% air at
tion41,42 with the absorption maximum 0.02–0.05.
37 C.
ꢃ
Fsflample ¼ FsfltandardAbsstandardS[Fsample]/AbssampleS[Fstandard] (1)
Effects on cell growth/viability (MTT assay)
MCF-7 cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS) at 37 ꢃC in a 5% CO2/95% air
incubator. The cells in the exponential phase of growth were
seeded on 96-well cell culture plates (1.5 ꢀ 103 cells per well,
100 mL), and maintained for 12 h. Then the probe (1 mM or 5 mM,
dispersed in 100 mL medium) was added to the wells and
incubated for 24 h. MTT (5.0 mg mLꢁ1, 20 mL), a water-soluble
tetrazolium salt, which can be transformed into colored, water-
insoluble formazan crystals by mitochondrial dehydrogenases
in living cells, was then added. Furthermore, aer being
cultured for more than 4 h, the medium was removed and 200
mL DMSO was added to dissolve the formazan crystals. The
optical density (OD) was recorded by an AC100-120 Automated
Microplate Reader (TECAN, Switzerland) at 570 nm (630 nm was
used as the reference wavelength).
F stands for the quantum yields of the tracker (sample) and
the uorescein (standard). Abs stands for the absorption value
recorded at the excited wavelength. F denotes uorescence
intensity at each wavelength, and S[F] was calculated by the
summation of uorescence intensity.42
Synthesis
The synthesis route is shown in Scheme 1.
MR. MR was synthesized according to the literature43
(compound 8 in literature).
General procedure for Lyso-R, Lyso-ER and Lyso-PR. The
procedure for Lyso-R is representative. To a 50 mL Schlenk
ask, MR (300 mg, 0.65 mmol, 1 eq.), Pd(OAc)2 (29 mg, 0.13
mmol, 0.2 eq.), BINAP (125 mg, 0.20 mmol, 0.3 eq.), and Cs2CO3
(847 mg, 2.6 mmol, 4.0 eq.) were added. Then, the ask was
evacuated with nitrogen (3ꢀ). Toluene (20 mL) and N-methyl
piperazine (0.5 mL, 3.81 mꢃmol, 5.9 eq.) were added, and the
reaction was stirred at 110 C for 24 h. It was cooled to room
temperature and washed thrice with saturated Na2CO3 solution.
Then, the organic layer was dried over MgSO4. The solvent was
evaporated, and the residue was puried using silica gel column
chromatography with eluent CH2Cl2–CH3OH–triethylamine (v/
v/v ¼ 40 : 1 : 1) to afford Lyso-R (136 mg, 42%). mp 97–99 ꢃC. 1H
NMR (400 MHz, CD3OD) d 8.01 (d, J ¼ 7.3 Hz, 1H), 7.79–7.61 (m,
2H), 7.17 (d, J ¼ 6.9 Hz, 1H), 6.76 (d, J ¼ 1.6 Hz, 2H), 6.74–6.59
(m, 4H), 3.31 (s, 8H), 2.58 (s, 8H), 2.34 (s, 6H). 13C NMR (100
MHz, CD3OD) d 171.0, 153.8, 135.5, 130.6, 129.6, 126.0, 125.2,
113.0, 110.7, 102.6, 96.9, 55.2, 48.2, 45.9. TOF-MS-ES(+) calcd for
Fluorescent microscopy
MCF-7 cells or macrophages on 35 mm glass-bottom culture
dishes (F 20 mm) were grown for 1–2 days to reach 70–90%
conuency. These cells were further used in the staining
comparison of the four trackers. The cells were washed thrice
wꢁit1h PBS (phosphate buffered solution, 8.0 g Lꢁ1 NaCl, 0.20 g
L
KCl, 0.27 g Lꢁ1 KH2PO4, 1.78 g Lꢁ1 Na2HPO4$2H2O, 1.44 g
Lꢁ1 NaH2PO4, pH 7.4), and then incubated with 0.25 mM solu-
tion of uorescent dyes in an atmosphere of 5% CO2 and 95%
ꢃ
air for 5 min at 37 C. Then, the cells were washed thrice with
PBS. Images were obtained with a confocal microscope with a
100ꢀ objective (NA, 1.40), C.A. 0.105–0.120 mm (pinhole size,
automatically adjusted by the soware) and continuous laser of
2.4 mW, subjected to deconvolution with the manufacturer's
soware and prepared using Adobe Photoshop 6.0 soware
(Adobe Systems Inc.). The detectors were four integrated
confocal PMT detectors. The lters were 405/488 for 405 nm or
488 nm excitation, 405/488/559 for 559 nm excitation, and 458/
515 for 515 nm excitation. Quantication was performed in
individual frames aer deconvolution and thresholding using
ImageJ soware21 (NIH). The details of the imaging were shown
in the following manner: the averaging pixel dwell time was 2 ms
per pixel; PMT voltage was around 550 to 700; the size of the
image was 800 ꢀ 800 pixels with a depth of 12 bit per pixel; and
the scanning rate was 2.18 seconds per frame. The imaging
conditions for each tracker: Lyso-R (lex ¼ 515 nm, lem ¼ 530–
570 nm), Neutral Red (lex ¼ 559 nm, lem ¼ 570–590 nm), DND
189 (lex ¼ 458 nm, lem ¼ 470–510 nm), DND 99 (lex ¼ 559 nm,
lem ¼ 570–630 nm) and TMRM (lex ¼ 559 nm, lem ¼ 570–600
nm). The uorescent image threshold of 3D tracking is 0 to
1500, while for others are 0 to 4095.
C
30H33N4O3 497.2553 found 497.2549.
Lyso-ER (85 mg, 26%). mp 86–89 ꢃC. 1H NMR (400 MHz,
CD3OD) d 8.01 (d, J ¼ 7.3 Hz, 1H), 7.79–7.61 (m, 2H), 7.17 (d, J ¼
6.9 Hz, 1H), 6.76 (d, J ¼ 1.6 Hz, 2H), 6.74–6.59 (m, 4H), 3.31 (s,
8H), 2.58 (s, 8H), 2.34 (s, 6H). 13C NMR (100 MHz, CD3OD) d
161.2, 159.8, 135.3, 135.1, 134.9, 133.9, 133.5, 132.8, 132.6,
117.3, 117.2, 101.0, 57.7, 50.5, 47.4, 42.3. TOF-MS-ES(+) calcd for
C
30H37N4O3 501.2866 found 501.2906.
Lyso-PR (108.00 mg, 31%). mp 118–120 ꢃC. 1H NMR (400
MHz, CD3OD) d 8.17 (d, J ¼ 7.3 Hz, 1H), 7.69 (dt, J ¼ 19.0, 7.3 Hz,
2H), 7.24 (dd, J ¼ 14.5, 8.4 Hz, 3H), 7.00 (d, J ¼ 9.4 Hz, 2H), 6.89
(s, 2H), 3.60 (dd, J ¼ 15.8, 7.6 Hz, 4H), 3.20 (s, 6H), 3.11–3.03 (m,
4H), 2.77 (s, 12H), 2.06–1.91 (m, 4H). 13C NMR (100 MHz,
CD3OD) d 161.5, 160.1, 135.6, 134.3, 133.9, 133.6, 133.0, 117.4,
100.7, 100.0, 58.8, 53.6, 46.5, 42.2, 26.4. TOF-MS-ES(+) calcd for
C
32H41N4O3 529.3179 found 529.3173.
Culture of cells
Photostability
MCF-7 (human breast carcinoma) and RAW 264.7 (macro-
phages cells) were obtained from the Institute of Basic Medical The cells were incubated with 0.25 mM Lyso-R, DND 189, DND
Sciences (IBMS) of the Chinese Academy of Medical Sciences 99 or 10 mM NR (because of the low quantum yields of NR) in an
(CAMS) and cultured in RPMI 1640 supplemented with 10% FBS atmosphere of 5% CO2 and 95% air for 5 min at 37 ꢃC,
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respectively. The cells were washed with PBS three times to
eliminate the non-stain dyes. Then, these cells were exposed to
continuous laser excitation of 0.24 mW cmꢁ2 for approximately
10 min. The uorescence of the cells was recorded with an
interval time of 1 min. The photostability experiment for each
dye was conducted three times.
Real-time microscopy
The real-time video imaging was conducted with the same
microscope and the same method for the aforementioned cell
growth. The frame of the video was captured every 2.18 seconds
with the same aforementioned illustrated imaging conditions
except for the 3D tracking case, which has been separately
described. The video frame rate for ESI movie 2, 2a, 3a, 3b, 4, 4a
and 4b† were 8 frame per second. The video frame rate of ESI
movie 5† was 80 frame per second. Then, all movies were
further merged and compressed to AVI les by ImageJ.
SBR
SBR value is calculated according to the scientic report.44 The
algorithm is as follows:
!,
!
Abbreviations
.
.
X
X
SBR ¼
ISi NS
Ibi Nb
(2)
i˛signal
i˛background
TLC
thin layer chromatography
N and I stand for the number of the pixels in the region of
“signal” or “background” and the intensity of the pixels,
respectively. S represents “signal” and b represents “back-
ground” or “noise.” All acquired images were taken in the
same method as illustrated in the uorescent imaging. In
such images, the strongest signals were taken as the “signal”
region in the red circle as in Fig. 2a, while the relative low
uorescence regions inside the cell were taken as the
“background” region in yellow circle. Using this method, SBR
value of three trackers, Lyso-R, DND 189 and DND 99, were
determined with a total number of 18 (n ¼ 18, six spots in one
image).
TMRM tetramethylrhodamine
SBR
NR
signal to background ratio
Neutral Red
BINAP (ꢂ)-2,20-Bis(diphenylphosphino)-1,10-binaphthalene
SEM
MTT
standard error of the mean
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (no. 21174022), the National Basic Research
Program of China (no. 2013CB733702) and the Specialized
Research Fund for the Doctoral Program of Higher Education
(no. 20110041110009). We also thank Dr Liji Jin at Dalian
University of Technology for his help in cell culture.
Tracking method for a single acidic organelle
The track was performed with the same confocal microscope
(0.3%, 2.4 mW continuous laser excitation) using the same cells
in PBS and maintained at room temperature. A single frame
contains eight depth images of 800 ꢀ 800 pixels, which indi-
vidually needs 2.181 s of photo time. Thus, a frame of the video
was captured every 17.45 s. A single frame was generated from
these images by using “Max Intensity” Z projection in ImageJ.45
These frames were thresholded by ImageJ so that the depth on
individual frames is depicted on a colour scale. Colours run
from hot (white) at high depth through to cold (blue) at low
depth. They were further converted to AVI videos (ESI movie 2
and 2a†) using ImageJ at 8 frame per second using JPEG
compression.
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