Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging

Article abstract of DOI:10.1021/acs.bioconjchem.0c00023

The plasma membrane (PM) plays a major role in many biological processes; therefore, its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535, were found to display enhanced photophysical properties (reduced cross-talk, higher brightness, improved photostability) and, unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell types including neurons (primary culture and tissue imaging). Taking advantage of these features, we successfully used SP-468 in STED super resolution neuronal imaging. Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we showed that the new probes kept the ability to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Full text of DOI:10.1021/acs.bioconjchem.0c00023

Subscriber access provided by Access provided by University of Liverpool Library
Article
Molecular Tuning of Styryl Dyes Leads to Versatile and
Efficient Plasma Membrane Probes for Cell and Tissue Imaging
Mayeul Collot, Emmanuel Boutant, Kyong Tkhe Fam, Lydia DANGLOT, and Andrey S. Klymchenko
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.0c00023 • Publication Date (Web): 13 Feb 2020
Downloaded from pubs.acs.org on February 14, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 10
Bioconjugate Chemistry
Molecular Tuning of Styryl Dyes Leads to Versatile and
Efficient Plasma Membrane Probes for Cell and Tissue
Imaging
1
2
3
4
5
6
7
8
Mayeul Collot,*1 Emmanuel Boutant,1 Kyong Tkhe Fam,1 Lydia Danglot,2 Andrey S. Klymchenko*1
1 Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, University of Strasbourg, France.
2Université de Paris, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, " Membrane Traffic in
Healthy and Diseased Brain ", F-75014 Paris, France
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords: Fluorescent probes, Plasma membrane probes, Bioimaging, Neuron, Brain slice, Plant imaging
ABSTRACT: The plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence
staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely
used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The
newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher
brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian
cell types including neurons (primary culture and tissue imaging). Tacking advantage of these features we successfully used SP-468
in STED super resolution neuronal imaging. Additionally, we showed that the new probes displayed differences in their
internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did
not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM
imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.
the phospholipids through opposite charge attraction.25 Unlike
Introduction
The plasma membrane (PM), as a barrier between the
hydrophobic carbocyanines, such as DiI, DiD and DiR,
bearing two C-18 hydrocarbon chains,20 FM dyes display a
extracellular and intracellular environments, maintains the
lower hydrophobicity and thus do not precipitate in aqueous
integrity of various organisms at the cellular level and plays
media. Therefore, FM probes are successfully and widely used
key roles in biology.1,
Consequently, the proper and
2
3
for imaging PM or vesicle trafficking in plants.26,
In
27
selective staining of the PM is important in bioimaging to
delimit the cells and to continue deciphering its role in various
cellular mechanisms. Nowadays, fluorescence microscopy
remains the most widely used bioimaging technique as it is
relatively affordable and it rapidly provides important
information with good resolution. From these two
observations, numerous efforts have been made to develop
fluorescent PM probes,
Noteworthy that efficient targeting of the PM also led to
probes compatible with super resolution imaging
mammalian cells they were applied to study vesiculation28, 29
and to image recycling synaptic vesicles in neuroscience.30, 31,
32 Unfortunately, the reduced hydrophobicity of FM1-43 leads
to a lack of affinity towards the PM and inefficient PM
staining in fixed cells.9 Moreover, FM probes tend to
internalize quickly by endocytosis33 and their broad absorption
and emission spectra limit their use in multicolor imaging.
Nevertheless, attractive optical properties of FM dyes, such as
their photostability, can constitute an advantage for long-term
imaging and their large Stokes shifts are appealing for
4,
5
7, 8
including from our group.6,
9, 10, 11, 12
or
probes able to locally sense biologically active molecules,13
viscosity14 or polarity15, 16, 17, 18 at the level of the PM. Among
PM probes, FM dyes (named after Fei Mao19), which are styryl
dyes introduced by Betz and colleagues in the early 90s’ as
synaptic vesicle markers,19 rapidly became among the most
Stimulated Emission Depletion (STED) super resolution
35
imaging34,
as STED requires the use of a high power
depletion laser that can photobleach the probe when its
excitation spectrum is not sufficiently separated from the
depletion wavelength. We recently introduced a family of PM
probes with balanced hydrophobicity that provided efficient
PM staining for cells and tissue, namely MemBright.6, 10 These
PM probes were obtained by decorating BODIPY6 and
cyanines10 with a clickable and selective PM targeting moiety:
CAZ7 (Scheme 1, Figure 1).
famous and used commercially available PM probes.20,
21
These amphiphilic dyes were further developed by the group
of Loew to give rise to PM voltage sensitive probes22, 23 and
membrane lipid domains probes.24 These probes are composed
of an aniline or naphthylamine donor linked to a pyridinium
acceptor moiety through a double bond or a polymethine
chain, which can tune their color.22, 23 However, efforts were
mainly focused on designing probes bearing polar groups on
one end and hydrophobic groups on the other end, which thus
imposed vertical orientation of styryl fluorophores in the
membrane. In the case of FM1-43 (Figure 1), one of the most
commonly used FM probes, it was claimed that the dibutyl
chains bore by the aniline moiety interact with the
hydrocarbon chains of the lipid bilayer via hydrophobic
interactions whereas the triethylammonium cationic moiety
bore by the pyridinium interacts with the negative charges of
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 10
Synthesis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Structures of FM1-43 and the newly developed styryl
probes SP-468 (Styryl Pyridinium - Ex 468) and SQ-535
(Styryl Quinolinium - Ex max 535) and their orientation within the
plasma membrane.
Scheme 1. Synthesis of SP-468 and SQ-535. a) Ethyl 4-
bromobutyrate, NaI, NaHCO3, DMSO, 80°C. b) POCl3, DMF,
55°C. c) NaOH (1M), MeOH. d) Dipropargylamine, DIEA,
HATU, DMF, RT. e) Picolinium propane-1-sulfonate or
lepidinium propane-1-sulfonate, piperidine (cat.), MeOH, 110°C.
f) CAZ, CuSO4∙5H2O, AscNa, water, DMF, 60°C.
max
The synthesis started with the N-alkylation of N-methylaniline
to introduce an ester group (Scheme 1). The aniline 1 was then
formylated using the Vilsmeier reaction to obtain 2. The ester
was saponified and the obtained carboxylic acid was directly
coupled to dipropargylamine using HATU as a coupling agent
to give compound 3. This aldehyde was then involved in
Knoevenagel reaction with picolinium propane-1-sulfonate
and lepidinium propane-1-sulfonate to obtain SP-dialkyne and
SQ-dialkyne respectively. These styryl dyes were clicked to
CAZ7 to finally lead to the probes SP-468 and SQ-535
(Scheme 1).
Results and discussion
Design of the probes
FM1-43 is a red emitting plasma membrane probe from the
FM family. It is a styryl dye composed of an N,N-dialkylated
aniline donor moiety linked through an ethylene connector to a
4-pyridinium acceptor moiety (Figure 1). It exhibits some
affinity to PM due to two synergic interactions. First, the butyl
groups bring hydrophobicity to enhance the affinity towards
the hydrophobic tails of the lipids, while the cationic
pyridinium and the ammonium moieties interact with the polar
head of the lipids.25 In order to improve the PM imaging
properties of FM1-43, we performed three different
modifications. 1) We first chose to annihilate the cationic
nature of FM dyes since cationic and hydrophobic molecules
tend to accumulate inside the cells.36 Therefore, the
ammonium group of FM was replaced by a sulfonate group to
give rise to a neutral zwiterionic dye (Figure 1). 2) Then, one
of the two butyl groups borne by the aniline moiety of FM1-43
was replaced by a methyl group in order to reduce the
inductive donor effect that electronically enriches the nitrogen.
This modification should provoke a hypsochromic shift in the
absorption spectrum and thus should reduce the cross talk
phenomenon due to the relatively high extinction coefficient
of FM1-43 at 530 nm and 560 nm that are generally dedicated
to excitation sources for the red channel in fluorescence
microscopy. 3) Finally, two amphiphilic moieties were
introduced by CuAAc click chemistry to ensure an efficient
PM targeting.6 All these modifications led to a probe called
SP-468 (Styryl Pyridinium). As an additional modification, the
pyridinium moiety of the fluorophore has been replaced by a
quinolinium in order to extend the -conjugation of the
fluorophore.37 This extension led to SQ-535 and is expected to
provoke a bathochromic shift in both absorbance and emission
spectra, which would ensure its use in the red channel with
limited crosstalk in the green one.
Spectroscopic studies
To evaluate their photophysical properties, the absorption and
emission spectra of the three styryl dyes were measured in
various conditions. Methanol and DMSO were chosen as polar
and solubilizing solvents, whereas PBS was chosen as a
typical aqueous medium for cell imaging microscopy. In order
to mimic the presence of plasma membrane, liposomes
composed
of
DOPC
(1,2-Dioleoyl-sn-glycero-3-
phosphocholine) were used in the presence of the dyes. Their
photophysical properties are reported in table 1.
Table 1. Photophysical properties of the probes.
Abs max Em max FWHM FWHM Stockes Shift
d
Probe
Solvent
QY
F/F0
(Abs)
(Em)
(nm)
(nm)
497
509
499
(nm)
635
630
637
N/Ab
N/Ab
N/Ab
99
PBS
95
84
86
138
121
138
0.01
0.02
0.05
1
2
8
MeOH
DMSO
FM1-43
Liposomesa
PBS
486
602
90
116
0.15
31
472
485
483
613
615
625
97
85
86
85
81
81
141
130
142
0.03
0.03
0.07
1
1
2
MeOH
SP-468
SQ-535
DMSO
Liposomesa
PBS
468
600
80
91
132
0.26
8
N/Ac
N/Ab
N/Ab
99
N/Ac
109
N/Ac
0.01
525
555
557
N/A
664
704
114
104
108
1
4
8
MeOH
DMSO
147
0.002
Liposomesa
535
665
103
130
0.10
53
a Liposomes (size 100 nm) are composed of DOPC (200 M final
concentration in phosphate buffer 20 mM, pH 7.4).
b
The low fluorescence intensity did not allow to determine the
FWHM from the spectrum
ACS Paragon Plus Environment

Page 3 of 10
Bioconjugate Chemistry
both absorption (up to 25 nm) and emission (up to 22 nm). As
c No proper fluorescence spectrum was obtained.
1
2
3
4
5
6
7
8
expected and discussed above, the replacement of a butyl
chain for a methyl group decreased the electron density of the
nitrogen donor, thus provoking the blue shift. In the presence
of liposomes, the new probes showed significant blue shift in
the absorption and emission spectra compared to water and
polar organic solvents, similarly to that of FM1-43. However,
their Stokes shift was much larger, reaching 130 nm. This
larger Stokes shift could be explained by a different insertion
of the probes, where the fluorophore of new probes will adopt
a parallel orientation to the membrane surface.
d Fluorescence enhancement: F= Fluorescence intensity at Em max
in the presence of liposomes, F0= fluorescence intensity at the
same wavelength in PBS.
Similarly to the parent probe FM1-43, the new probes showed
hypochromism in water compared to organic solvents (Figure
S1), and quantum yields in water and methanol were low
(Table 1). However, the newly designed SP-468 displayed
significant differences compared to FM1-43 (Figure 2A).
First, a hypsochromic shift was observed in all solvents, in
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FM1-43
SP-468
SQ-535
FM1-43
SP-468
SQ-535
PBS
DOPC
Abs
Em
A
B
C
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.9
0.8
0.7
0.6
0.5
FM1-43
SP-468
SQ-535
400
500
600
700
800
500 550 600 650 700 750 800
0
10
20
30
40
50
60
Wavelength (nm)
Wavelength (nm)
Time (min)
Figure 2. (A) Normalized absorption and emission spectra of the styryl probes in the presence of DOPC vesicles (200 M). The 3 vertical
lines indicate the most common laser lines used in imaging namely: 488, 530 and 560 nm. Absorption spectra in presence of liposomes
were corrected from scattering. (B) Normalized fluorescence spectra depicting the fluorescence enhancement of the probes from PBS to
insertion into DOPC vesicles (200 M). (C) Photostability: evolution of the fluorescence intensity over the time under continuous
irradiation. The probes were excited at wavelengths corresponding to the same optical density values (0.03). Concentration of the probes
was
2
M
It was noteworthy that SP-468 displayed a sharper absorption
band (FWHM= 80 nm) compared to FM1-43 (90 nm) (Figure
2A), whereas both SP-468 and FM1-43 are efficiently excited
by the commonly used 488-nm laser (84 and 99%
respectively). Advantageously, SP-468 with a blue shifted
excitation wavelength combined to a sharper peak is less
prone to cross-talk compared to FM1-43. Indeed, while FM1-
43 is also well excitable at 530 and 560 nm (52% and 13%
respectively), SP-468 is only excited at 14 and 3% at these
laser wavelengths (see laser lines on Figure 2A). The presence
of liposomes resulted in significant enhancement of
fluorescence intensity for all three probes, which according to
the earlier studies indicates that the probe is located in highly
organized environment of lipid membranes that minimize
rotation-induced quenching in the styryl dyes. However, in
case of SP-468, the quantum yield in PBS was slightly higher
compared to FM1-43, which decreased fluorescence
enhancement from PBS to liposomes (8-fold vs 31 fold,
respectively, Figure 2B). The monitoring of the fluorescence
signal over the time and in the presence of lipid vesicles
indicated that FM1-43 and SP-468 immediately incorporated
the lipid bilayer, whereas SQ-535 showed binding within less
than a minute (Figure S2). We can conclude that both FM1-43
and SP-468 are present in PBS in molecular form, showing
very fast binding kinetics. On the other hand, a more
hydrophobic SQ-535 is probably partially aggregated in PBS,
which leads to slower binding kinetics, similarly to previously
reported MemBright probes.10 The aggregation of SQ-535 in
PBS is in line with the strong blue shift in absorbance
compared to organic solvents, which are much less
pronounced in case of FM1-43 and SP-468. Extending the -
system from SP-468 to SQ-535 led to a 65-67 nm red shift in
both absorption and emission spectra of dyes in DOPC
liposomes (Figure 2A). SQ-535 constitutes a suitable PM
probe with high fluorescence enhancement 53-fold, figure
2B). Although it is well excitable at 488 nm (57%), SQ-535
starts emitting at only 560 nm and thus can be used in
combination with green emitting dye like BODIPY,
fluorescein or eGFP (vide infra). We then assessed the
photostability of the probes in the presence of DOPC
liposomes and it was noteworthy that FM1-43 photobleached
in an exponential manner (30% decrease of the fluorescence
intensity after 15 min irradiation), while after 1 h SP-468 and
SQ-535 lost only 10% and 8% of their fluorescence intensity
respectively (Figure 2C). This difference might be assigned to
the design of the probes that impose fluorophore of SP-468
and SQ-535 to locate parallel to the membrane plane, in
contrast to FM1-43 where the fluorophore itself insert
perpendicular to the lipid bilayer.25
Cellular imaging
Prior to imaging studies, the effect of the probes on the cell
viability was assessed by MTT assay. As expected, whereas
SP-468 and SQ-535 did not show significant signs of
cytotoxicity up to 5 M, the cationic nature of the FM1-43
provoked a notable decrease of the cell viability after 24h at
various concentrations (Figure S3). As a first imaging
experiment, the ability of FM1-43 and SP-468 to stain the PM
of live cells was evaluated on 4 different eukaryotic cell lines
at a concentration of 1 M in no-wash conditions. Since FM1-
43 and SP-468 possess similar photophysical properties, the
same image acquisition and processing settings were applied
for both. The results showed that in the same conditions,
whereas FM1-43 failed at providing any signal, SP-468
immediately displayed a selective and intense staining of the
PM regardless the cell line (Figure 3). When the laser power
was increased, the images obtained with FM1-43 were of poor
quality with a weak signal/background ratio even after 20
minutes whereas SP-468 maintained a high quality staining
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 10
over that same period (Figure S4). SQ-535, when excited at imaged under continuous laser scanning illumination for 10
1
2
3
4
5
6
7
8
560 nm, showed similar good PM staining properties (Figure
S5) and thus constitutes a red shifted alternative to SP-468. At
this point, SP-468 and SQ-535 were compared to
commercially available PM probes (Figure S6). SP-468 was
used in combination with a far-red PM probe, MemBright®
MB-Cy5. Both probes showed clean and selective PM staining
with a high colocalization. SQ-535 was compared to WGA-
488, a fluorescently labeled lectin. Unlike WGA-488, SQ-535
provided a homogeneous staining of the PM. Indeed WGA
failed at efficiently staining the PM involved in cell-cell
contacts, which is consistent with our previous work.10 In a
second time, KB cells stained with SP-468 and SQ-535 were
minutes with negligible loss of intensity and with conserved
PM staining thus demonstrating their photostability (movie 1
and 2). The PM staining efficiency of SP-468 was then
assessed in fixed cells. We established that cell fixation
followed by addition of the probe was preferable over the
other way around and provided excellent results with no sign
of internalization (Figure S7).
Additionally, the use of SQ-535 at only 500 nM on fixed
primary culture of hippocampal neurons and astrocytes in no
wash conditions led to a homogeneous and bright staining of
their PM (Figure 4A and B).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Laser scanning confocal imaging of live cells from four different cell lines stained with 1 M of FM1-43 (A, B, C, D) and SP-
468 (E, F, G, H). Images were acquired 5 minutes after addition of the dyes in the medium. Excitation wavelength was 488 nm and the
fluorescence signal was collected between 520 and 700 nm. Scale bar is 15 m. On the right is displayed the color lookup table.
The photostability and quality of staining allowed to obtain
high quality 3D images that revealed the dense neuronal
network (Figure 4C). After incubation on fixed brain slices,
SQ-535 provided a homogeneous staining of the whole tissue
(Figure 4D). When zoomed on the cortical layer, some
dendritic processes were finely revealed in a multicolor 3D
rendering (Figure 4D).
Figure 4. Laser scanning confocal imaging (with 20× objective)
of fixed hippocampal neurons (A) and fixed astrocytic glial cells
(B) incubated with SQ-535 (500 nM). (C) 3D rendering of the
complex neuronal network (10 m thick 3D volume, 93×
objective). (D) Fixed brain slice images using SQ-535 (in red) and
ACS Paragon Plus Environment

Page 5 of 10
Bioconjugate Chemistry
DAPI (cell nuclei in green). Some dendritic processes within the
conditions (Figure S9), a time laps imaging was performed for
1
2
3
4
5
6
cortical layer (top right inset, scale bar 500 m) are revealed in
the 3D rendering of a 12 m thick Z stack (scale bar: 20 m).
10 minutes (Movie 3). The results first showed that FM1-43 at
high concentration and after several minutes, provided a weak
PM staining accompanied with non-specific staining of
intracellular membranes (Figure 6A). Conversely, SQ-535
provided a fast and bright PM staining that kept its selectivity
along the imaging period (Figure 6B, C and Movie 3).
Encouraged by these results and taking advantages of the large
Stokes shifts of these new probes, we assessed their ability to
provide live super resolution imaging using Stimulated
35
Emission Depletion (STED).34,
To this end, live
In order to compare the fate of the probes over incubation,
FM1-43 and SP-468 were incubated for 1h at 37°C to provoke
endocytosis. Imaging revealed that FM1-43 rapidly stained
intracellular membranes along with endosomes vesicles
(Figure S10A, movie 4). SP-468 kept a PM staining after
incubation and showed punctiform staining in the cytoplasm
with characteristic movement when imaged over the time
(Movie 5) depicting endosomes (Figure S10B). To assess the
difference in the internalization within the same cellular
experiment, FM1-43 and SQ-535 were co-incubated in KB
cells for 1h at 37°C before being fixed. Analysis of images
revealed that FM1-43 was actually distributed between
endosomes and intracellular membranes (Figure 6D), whereas
SQ-535 was distributed between the PM and endosomes
(Figure 6E). Interestingly the merge images revealed that
FM1-43 and SQ-535 do not fully follow the same
internalization pathway as green and red signals poorly
colocalized in the endosomes with a measured Pearson
correlation coefficient of 0.32 (Figure 6F).
7
8
9
hippocampal neurons were stained with SP-468 in no-wash
conditions and were imaged by laser confocal microscopy
directly followed by STED (Figure 5). Overall, STED imaging
led to a clear gain of resolution. Indeed, unlike conventional
confocal imaging, STED allowed to reveal the lumen of the
dendritic processes by discrimination of the plasma
membranes from either side of the latter (Figure 5B).
Additionally, plot profiles on different regions of interest
(dendrite and vesicles) displayed a quantifiable gain of
resolution (Figure S8) thus illustrating the compatibility of SP-
468 with STED super resolution imaging.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Specificity and Cellular uptake
We then aimed at comparing the specificity of the probes
towards the PM. To this end, KB cells were co-stained with
SQ-535 (1 M) and FM1-43 at a higher concentration (5 M)
in order to increase its signal in imaging. After checking that
crosstalk did not occur between the 2 channels in these
Figure 5. Laser scanning confocal (A) and STED (B) imaging of live hippocampal neurons stained with 1 M of SP-468 (no-wash
conditions). Scale bar is 5 m.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 10
crosstalk was reduced and the brightness as well as the
1
2
3
4
5
6
7
8
photostability were significantly enhanced. The newly
developed probes displayed fast, efficient and selective
staining of PM in 5 different mammalian cell lines, including
primary neurons, in contrast to parent FM1-43 that showed
weak signal and significant intracellular staining. Remarkably,
the new probes showed very good PM staining of fixed cells
and brain slices. Taking advantages of its enhanced
photostability and its large Stokes shifts, SP-468 was
successfully used in live neuronal super resolution imaging
using STED microscopy. Additionally the ability to stain PM
of plant cells was overall conserved making these new dyes
adapted for both mammalian and plant cells experiments. The
present work demonstrated the importance in the design of
molecular fluorescent probes to fit with the bioimaging
requirements. SP-468 and SQ-535 constitute reliable PM
probes for users who want to benefit from large Stokes shift of
PM markers with high photostability and to work with both
live and fixed samples of cells and tissues.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Laser scanning confocal imaging of KB cells stained
with Hoechst (5 g/mL) and 5 M of FM1-43 (A, D, G) and 1
M SQ-535 (B, E, H). (A, B, C) Images of live cells after 10 min
of continuous imaging. (D, E, F) Images of fixed cells after 1h
incubation in Opti-MEM. Merged channels (C, F) depict the
different staining pattern and internalization pathway of the
probes. G and H are zoomed images in the region of interest
displayed in F, highlighting endosomes using spots detection
plugin. (I) The resulting merged binary images of the detected
endosomes depicted a weak colocalization. Scale bars are 15 m.
PM staining of plant cells
FM dyes are widely used in cellular plant imaging as it
efficiently stains the PM of the latter and thus is useful for
segmentation and for tracking trafficking vesicles.26, 27 We thus
investigated on the ability of our modified FM probes to
conserve efficient PM staining in plant cells. For this two
different experiments were conducted. On one hand, a solution
of probe was injected in the leaf of 6 weeks old Nicotiana
Benthamiana plant (Figure 7A-D); on the other hand, whole
seedlings were incubated in a solution of probe to stain the
roots (Figure 7E-F). The results showed that using the same
imaging and processing conditions, FM1-43 and SP-468 both
stained the PM and allowed segmentation of the cells thus
showing that the newly developed PM probes can be used with
both mammalian and plant cells. Noteworthy, although FM1-
43 and SP-468 displayed similar staining properties in the
roots, FM1-43 displayed slightly more homogeneous staining
in leaf cells compared to SP-468 that displayed enhanced
signal in stomata (Figure 7D).
Figure 7. Laser scanning confocal imaging of plant cells
(Nicotiana Benthamiana) stained with FM1-43 (A, C, E) and SP-
468 (B, D, F). Images at the level of leafs without stomata (A, B)
and with stomata (C, D). Images at the level of roots (E, F). Scale
bar is 15 m. On the right is the color lookup table.
Conclusion
Although FM dyes are known as plasma membrane probes,
FM1-43 displayed rather weak PM staining properties in
eukaryotic cells. We showed that by conserving the same core
fluorophore and by means of rational chemical modifications
using adapted targeting moieties and neutralizing the cationic
nature of FM1-43, styryl dyes could be efficiently converted
into PM probes with interesting properties for PM bioimaging.
Notable improvements were obtained as the tendency for
Materials and methods
ACS Paragon Plus Environment

Page 7 of 10
Bioconjugate Chemistry
Synthesis. All starting materials for synthesis were purchased HTB-14™) were grown in Minimum Essential Medium
1
2
3
4
5
6
7
8
from Alfa Aesar, Sigma Aldrich or TCI Europe and used as
received unless stated otherwise. FM1-43 was purchased from
Biotium.17 The solvents were not dried unless mentioned in the
protocol. DOPC Liposomes of ∼100 nm were obtained
following a described protocol after several rounds of
extrusion.38 NMR spectra were recorded on a Bruker Avance
III 400 MHz spectrometer. Mass spectra were obtained using
an Agilent Q-TOF 6520 mass spectrometer. Synthesis of all
new compounds is described in the supporting information.
(MEM, Gibco-Invitrogen) supplemented with 10% FBS, 1%
Ultra-Glutamine (Gibco-Invitrogen) and 1% antibiotic
solution. KB cells (ATCC® CCL-17) were grown DMEM
(Gibco-Invitrogen) supplemented with 10% FBS (Lonza), 1%
non-essential amino acids (Gibco-Invitrogen), 1% MEM
vitamin solution (Gibco-Invitrogen), 1% L-Glutamine (Sigma
Aldrich) and 0.1% antibiotic solution (gentamicin, Sigma-
Aldrich) at 37C in humidified atmosphere containing 5%
CO2. Cells were seeded onto a chambered coverglass (IBiDi®)
at a density of 5104 cells/well 24 h before the microscopy
measurement. For imaging, the culture medium was removed
and the attached cells were washed with Opti-MEM (Gibco–
Invitrogen). Next, the cells were incubated in Opti-MEM with
Hoechst (5 µg/mL) to stain the nuclei for 1h00. The cells were
then washed with Opti-MEM and a freshly prepared solution
of Styryl probe (2 M in Opti-MEM) was added to the cells
prior to imaging. Confocal fluorescence images of the cells
were taken on a Leica TSC SPE confocal microscope using a
63 × oil immersion objective. The images were processed with
Icy software.42 For colocalization between FM1-43 and SQ-
535 the following settings were used: FM1-43 (ex: 488 nm,
Em: 515-600 nm) and SQ-535 (ex: 561 nm, Em: 650-750 nm).
Colocalization study was performed with Icy software:
Endosomes were localized by detecting intense spots using
“spot detector” plugin. The obtained binary images of
endosomes were merged and the “colocalization Studio”
plugin43 was used to determine the colocalization Pearson’s
colocalization coefficient. Neuronal imaging: All animal
experiments were performed in accordance with European
Community guidelines legislation and reviewed by the local
ethical committee of the Paris Diderot University.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Spectroscopy. For spectroscopy studies the concentration of
the probes was 2 M and solvents were of spectroscopic
grade. The water was miliQ water. Absorption and emission
spectra were recorded on a Cary 400 Scan ultraviolet–visible
spectrophotometer
(Varian)
and
a
FluoroMax-4
spectrofluorometer (Horiba Jobin Yvon) equipped with a
thermostated cell compartment, respectively. For standard
recording of fluorescence spectra, the emission was collected
10 nm after the excitation wavelength. All the spectra were
corrected from wavelength-dependent response of the detector.
The quantum yields were determined by comparison with
fluorescein39 (in NaOH 0.1 M, = 0.95) for FM1-43 and SP-
468 (excitation at 460 nm), and rhodamine B40 (in water, =
0.31) for SQ-535 (excitation at 520 nm), following equation
(1):
where QY is the quantum yield, I is the integrated
fluorescence intensity, n is the refractive index, and OD is the
optical density at the excitation wavelength. R represents the
reference. The concentrations were adjusted by absorption
considering the following extinction coefficients in MeOH:
FM1-43, 52,000 M-1.cm-1 (according to the litterature41); SP-
468, 50,000 M-1.cm-1 (determined from measured precursor
SP-dialkyne) and SQ-535, 49,800 M-1.cm-1 (determined from
measured precursor SQ-dialkyne).
Hippocampal cultures from 18-day-old Sprague-Dawley rat
45
embryos were prepared as described.44,
Cells were
dissociated by treatment with 0.25% trypsin for 15 min at
37°C and plated on poly-Ornithine (1 mg/mL, Sigma) coated
glass coverslips in MEM supplemented with 10% horse
serum, 0.6% glucose, 0.2% sodium bicarbonate, 2 mM
glutamine, and 10 IU/mL penicillin- streptomycin. After
attachment neurons were grown in neurobasal medium
supplemented with B27 (Thermo Fisher) conditioned on
astroglia. Neurons were then imaged on Leica SP8 confocal
microscope. For imaging, the medium was removed and the
attached cells were washed with warm HBSS (Gibco-
Invitrogen) three times. Neurons were fixed with 4% PFA,
0.1% glutaraldehyde during 10 min at 37°C. Samples were
washed 3 times with HBSS and incubated with 500 nM SQ-
535 and processed directly for confocal imaging in open
chamber. Mice tissue: C57BL6/J mice were maintained on a
12ꢀh light-dark cycle with ad libitum access to food and water.
All animal work was conducted following protocol approved
by ethical committee. Adult C57BL6 mice were euthanized
and fixed with 4% paraformaldehyde by transcardial
perfusion, post-fixed for 30 min in the same fixative, rinsed in
PBS and kept in PBS-30% sucrose at 4°C until use. Brains
were sectioned on a cryotome at 40  and floating sections
were processed for labeling as described for cultured neurons
with slight modifications: slices were washed 3 times in PBS-
NH4Cl 50 mM and incubated 3 h with DAPI (10 g/ L) and
SQ-535 (8 µM) at room temperature under agitation. The
microscope settings were: 405 nm laser for excitation of
DAPI, emission was collected between 420 and 470 nm. SQ-
535 was excited with 561 nm laser line and fluorescence was
collected with 650-750 nm detection range. The images were
processed with LAsX and Icy software.
Photostability assay. The dyes (2 µM) in the presence of
vesicles (100 eq of DOPC) in phosphate buffer (pH 7.4) were
all excited at the same optical density value based on their
absorption spectra, namely @ OD= 0.03. FM1-43 was excited
at 450 nm, SP-468 at 430 nm and SQ-535 at 478 nm. Optical
slits were set at 16 mm for excitation and 1 mm for emission.
The emission was monitored at 600 nm for FM1-43 and SP-
468 and at 660 for SQ-535.
Membrane binding kinetics. A solution of DOPC (200 µM)
vesicles in PB at pH 7.4 were stirred under excitation for 1
min, then 5 µL of probe (5 µL of stock at 400 µM in DMSO)
was added. Excitation was at 460 nm for FM 1-43 and SP-468
and the fluorescence was monitored at 600 nm. For SQ-535 it
was 520 nm and 660 nm respectively.
Cellular imaging. HEK293 (ATCC® CRL-3216™) and
HeLa (ATCC® CCL-2™) cells were grown in Dulbecco's
Modified Eagle Medium without phenol red (DMEM, Gibco-
Invitrogen) supplemented with 10% fetal bovine serum (FBS,
Lonza), 1% L-Glutamine (Sigma Aldrich) and 1% antibiotic
solution (Penicillin-Streptomycin, Sigma-Aldrich) at 37°C in
humidified atmosphere containing 5% CO2. U87 (ATCC®
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 10
STED imaging. Neuronal membranes were labeled on live in system purchase. We are also grateful to IBMP’s gardener and
1
2
3
4
5
6
7
8
vitro hippocampal neurons using SP-468 in incubation
chamber at 1µM. Neurons were imaged with a 93× glycerol
objective with automatic correction collar on a Leica SP8
STED microscope. Thanks to the white laser technology,
STED was performed in two different modalities. 2D STED
was tested with laser line at 490 nm for excitation and the 592
nm depletion laser (Figure 5), detection was set between 502
and 585 nm. 3D STED was realized using 514 nm laser line
for excitation and the 775 nm pulsed depletion laser (Figure
S8) with detection between 520 and 663 nm. Acquisitions
were done so that the pixel size was 29 nm in XY.
Elodie Klein for providing the plants and Dmytro Danylchuk
for the preparation of liposomes.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website. Data on synthesis protocol, 1H and 13
C
NMR and mass spectra, spectroscopy, additional cellular imaging,
and cytotoxicity assays.
9
Movie 1. Photostability SP-468 (AVI)
Movie 2. Photostability SQ-535 (AVI)
Movie 3. FM1-43 & SQ-535 time laps (AVI)
Movie 4. Incubation of FM-1-43 (AVI)
Movie 5. Incubation of SP-468 (AVI)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cytotoxicity assay. Cytotoxicity assay was quantified by the
MTT
assay
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide). A total of 1x104 KB cells/well
were seeded in a 96-well plate 24 h prior to the cytotoxicity
assay in Dulbecco’s Modified Eagle Medium (Gibco Life
Technologies -DMEM) complemented with 10% fetal bovine
serum, Gentamicin (100 µg/mL), L-Glutamine (2 mM), non-
essential amino acids (1 mM), MEM vitamin solution (1%)
and were incubated in a 5% CO2 incubator at 37°C. After
medium removal, an amount of 100 µL DMEM containing 5
µM, 1 µM or 0.2 µM of SMCy (SMCy3, SMCy3.5, SMCy5,
SMCy5.5, SMCy7 and SMCy7.5) was added to the KB cell
and incubated for 3 h at 37°C (5% CO2). As control, for each
96-well plate, the cells were incubated with DMEM
containing the same percentage of DMSO (0,5% v/v) as the
solution with the tested dyes or with Triton 1% as a positive
control of cytotoxicity. After 24h of dye incubation, the
medium was replaced by 100 µL of a mix containing DMEM
+ MTT solution (diluted in PBS beforehand) and the cells
were incubated for 4 h at 37°C. Then, 75 µL of the mix was
replaced by 50 µL of DMSO (100%) and gently shaken for 15
min at room temperature in order to dissolve the insoluble
purple formazan reduced in living cells. The absorbance at 540
nm was measured (absorbances of the dyes at 540 nm were
taken into account). Each concentration of dye was tested in
sextuplicate in 3 independent assays. For each concentration,
we calculated the percentage of cell viability in reference of
the control DMEM+ 0.5% DMSO.
Conflict of interest
The authors declare no conflict of interest.
References
(1)
Lingwood, D.; Simons, K. Lipid Rafts As a Membrane-
Organizing Principle. Science 2010, 327 (5961), 46–50.
Sezgin, E.; Levental, I.; Mayor, S.; Eggeling, C. The
Mystery of Membrane Organization: Composition,
Regulation and Roles of Lipid Rafts. Nat. Rev. Mol. Cell
Biol. 2017, 18 (6), 361–374.
(2)
(3)
(4)
(5)
(6)
(7)
Rello, S.; Stockert, J. C.; Moreno, V.; Gámez, A.; Pacheco,
M.; Juarranz, A.; Cañete, M.; Villanueva, A. Morphological
Criteria to Distinguish Cell Death Induced by Apoptotic and
Necrotic Treatments. Apoptosis 2005, 10 (1), 201–208.
Zhang, X.; Wang, C.; Jin, L.; Han, Z.; Xiao, Y. Photostable
Bipolar Fluorescent Probe for Video Tracking Plasma
Membranes Related Cellular Processes. ACS Appl. Mater.
Interfaces 2014, 6 (15), 12372–12379.
Heek, T.; Nikolaus, J.; Schwarzer, R.; Fasting, C.; Welker,
P.; Licha, K.; Herrmann, A.; Haag, R. An Amphiphilic
Perylene Imido Diester for Selective Cellular Imaging.
Bioconjug. Chem. 2013, 24 (2), 153–158.
Collot, M.; Boutant, E.; Lehmann, M.; Klymchenko, A. S.
BODIPY with Tuned Amphiphilicity as a Fluorogenic
Plasma Membrane Probe. Bioconjug. Chem. 2019, 30 (1),
192–199.
Collot, M.; Kreder, R.; Tatarets, A. L.; Patsenker, L. D.;
Mely, Y.; Klymchenko, A. S. Bright Fluorogenic Squaraines
with Tuned Cell Entry for Selective Imaging of Plasma
Membrane vs. Endoplasmic Reticulum. Chem. Commun.
2015, 51 (96), 17136–17139.
Plant imaging. Nicotiana Benthamiana were used as plant
model and were obtained from the Institut de biologie
moléculaire des plantes (IBMP) greenhouse. Staining of leaf
cells: leaves from 5-6 weeks old plants were infiltrated with
the help of a syringe containing a solution of probes (5 M in
water). After 30 min, disk of leafs were taken off using a hole
punch of 0.7 cm in diameter. The leaf samples were
maintained in water between cover slips and were directly
imaged. Staining of root cells: young plants (3 to 5 days after
germination) were placed in 1 mL solution of probe (5 M in
water) for 1 hour at RT. The roots were washed with water
and placed under cover slips before being imaged.
(8)
(9)
Kreder, R.; Oncul, S.; Kucherak, O. A.; Pyrshev, K. A.;
Real, E.; Mély, Y.; Klymchenko, A. S. Blue Fluorogenic
Probes for Cell Plasma Membranes Fill the Gap in
Multicolour Imaging. RSC Adv. 2015, 5 (29), 22899–22905.
Revelo, N. H.; Kamin, D.; Truckenbrodt, S.; Wong, A. B.;
Reuter-Jessen, K.; Reisinger, E.; Moser, T.; Rizzoli, S. O. A
New Probe for Super-Resolution Imaging of Membranes
Elucidates Trafficking Pathways. J. Cell Biol. 2014, 205 (4),
591–606.
(10)
Collot, M.; Ashokkumar, P.; Anton, H.; Boutant, E.;
Faklaris, O.; Galli, T.; Mély, Y.; Danglot, L.; Klymchenko,
A. S. MemBright: A Family of Fluorescent Membrane
Probes for Advanced Cellular Imaging and Neuroscience.
Cell Chem. Biol. 2019, 26 (4), 600–614.e7.
Acknowledgements
(11)
(12)
Danylchuk, D. I.; Moon, S.; Xu, K.; Klymchenko, A. S.
Switchable Solvatochromic Probes for Live-Cell Super-
Resolution Imaging of Plasma Membrane Organization.
Angew. Chem. Int. Ed. 2019, 58 (42), 14920–14924.
Ye, Z.; Yang, W.; Wang, C.; Zheng, Y.; Chi, W.; Liu, X.;
Huang, Z.; Li, X.; Xiao, Y. Quaternary Piperazine-
Substituted Rhodamines with Enhanced Brightness for
Super-Resolution Imaging. J. Am. Chem. Soc. 2019, 141
(37), 14491–14495.
This work was supported by the European Research Council
ERC Consolidator grant BrightSens 648528, ANR
BrightRiboProbes
(ANR-16-CE11-0010)
and
HumanBrainProject Partnering project “Sensei” (LD). The
authors would like to thank Neurimag (Paris) and Romain
Vauchelles for his assistance at the PIQ platform as well as the
PACSI analysis platform. We are grateful to Leducq
foundation for its support concerning NeurImag SP8 confocal
ACS Paragon Plus Environment

Page 9 of 10
Bioconjugate Chemistry
(13)
Zhang, X.; Wang, B.; Xiao, Y.; Wang, C.; He, L. Targetable,
(29)
Wiederkehr, A.; Avaro, S.; Prescianotto-Baschong, C.;
Two-Photon Fluorescent Probes for Local Nitric Oxide
Capture in the Plasma Membranes of Live Cells and Brain
Tissues. Analyst 2018, 143 (17), 4180–4188.
López-Duarte, I.; Vu, T. T.; Izquierdo, M. A.; Bull, J. A.;
Kuimova, M. K. A Molecular Rotor for Measuring Viscosity
in Plasma Membranes of Live Cells. Chem. Commun. 2014,
50 (40), 5282–5284.
Kwiatek, J. M.; Owen, D. M.; Abu-Siniyeh, A.; Yan, P.;
Loew, L. M.; Gaus, K. Characterization of a New Series of
Fluorescent Probes for Imaging Membrane Order. Plos One
2013, 8 (2), e52960.
Shaya, J.; Collot, M.; Bénailly, F.; Mahmoud, N.; Mély, Y.;
Michel, B. Y.; Klymchenko, A. S.; Burger, A. Turn-on
Fluorene Push–Pull Probes with High Brightness and
Photostability for Visualizing Lipid Order in Biomembranes.
ACS Chem. Biol. 2017, 12 (12), 3022–3030.
Kucherak, O. A.; Oncul, S.; Darwich, Z.; Yushchenko, D.
A.; Arntz, Y.; Didier, P.; Mély, Y.; Klymchenko, A. S.
Switchable Nile Red-Based Probe for Cholesterol and Lipid
Order at the Outer Leaflet of Biomembranes. J. Am. Chem.
Soc. 2010, 132 (13), 4907–4916.
Klymchenko, A. S.; Kreder, R. Fluorescent Probes for Lipid
Rafts: From Model Membranes to Living Cells. Chem. Biol.
2014, 21 (1), 97–113.
Betz, W. J.; Mao, F.; Bewick, G. S. Activity-Dependent
Fluorescent Staining and Destaining of Living Vertebrate
Motor Nerve Terminals. J. Neurosci. 1992, 12 (2), 363–375.
Life Technologies https://www.thermofisher.com/.
Biotium Https://Biotium.Com/.
Wuskell, J. P.; Boudreau, D.; Wei, M.; Jin, L.; Engl, R.;
Chebolu, R.; Bullen, A.; Hoffacker, K. D.; Kerimo, J.;
Cohen, L. B.; et al. Synthesis, Spectra, Delivery and
Potentiometric Responses of New Styryl Dyes with
Extended Spectral Ranges. J. Neurosci. Methods 2006, 151
(2), 200–215.
Yan, P.; Acker, C. D.; Zhou, W.-L.; Lee, P.; Bollensdorff,
C.; Negrean, A.; Lotti, J.; Sacconi, L.; Antic, S. D.; Kohl, P.;
et al. Palette of Fluorinated Voltage-Sensitive Hemicyanine
Dyes. Proc. Natl. Acad. Sci. 2012, 109 (50), 20443–20448.
Jin, L.; Millard, A. C.; Wuskell, J. P.; Dong, X.; Wu, D.;
Clark, H. A.; Loew, L. M. Characterization and Application
of a New Optical Probe for Membrane Lipid Domains.
Biophys. J. 2006, 90 (7), 2563.
Schote, U.; Seelig, J. Interaction of the Neuronal Marker
Dye FM1-43 with Lipid Membranes: Thermodynamics and
Lipid Ordering. Biochim. Biophys. Acta BBA - Biomembr.
1998, 1415 (1), 135–146.
Bolte, S.; Talbot, C.; Boutte, Y.; Catrice, O.; Read, N. D.;
Satiat‐Jeunemaitre, B. FM-Dyes as Experimental Probes for
Dissecting Vesicle Trafficking in Living Plant Cells. J.
Microsc. 2004, 214 (2), 159–173.
Jelínková, A.; Malínská, K.; Simon, S.; Kleine‐Vehn, J.;
Pařezová, M.; Pejchar, P.; Kubeš, M.; Martinec, J.; Friml, J.;
Zažímalová, E.; et al. Probing Plant Membranes with FM
Dyes: Tracking, Dragging or Blocking? Plant J. 2010, 61
(5), 883–892.
Haguenauer-Tsapis, R.; Riezman, H. The F-Box Protein
Rcy1p Is Involved in Endocytic Membrane Traffic and
Recycling out of an Early Endosome in Saccharomyces
Cerevisiae. J. Cell Biol. 2000, 149 (2), 397–410.
Murthy, V. N.; Stevens, C. F. Synaptic Vesicles Retain Their
Identity through the Endocytic Cycle. Nature 1998, 392
(6675), 497–501.
Betz, W. J.; Bewick, G. S. Optical Analysis of Synaptic
Vesicle Recycling at the Frog Neuromuscular Junction.
Science 1992, 255 (5041), 200–203.
Rea, R.; Li, J.; Dharia, A.; Levitan, E. S.; Sterling, P.;
Kramer, R. H. Streamlined Synaptic Vesicle Cycle in Cone
Photoreceptor Terminals. Neuron 2004, 41 (5), 755–766.
Darwich, Z.; Klymchenko, A. S.; Dujardin, D.; Mély, Y.
Imaging Lipid Order Changes in Endosome Membranes of
Live Cells by Using a Nile Red-Based Membrane Probe.
RSC Adv. 2014, 4 (17), 8481–8488.
Sednev, M. V.; Belov, V. N.; Hell, S. W. Fluorescent Dyes
with Large Stokes Shifts for Super-Resolution Optical
Microscopy of Biological Objects: A Review. Methods Appl.
Fluoresc. 2015, 3 (4), 042004.
Sezgin, E.; Schneider, F.; Zilles, V.; Urbančič, I.; Garcia, E.;
Waithe, D.; Klymchenko, A. S.; Eggeling, C. Polarity-
Sensitive Probes for Superresolution Stimulated Emission
Depletion Microscopy. Biophys. J. 2017, 113 (6), 1321–
1330.
Lin, J.; Alexander-Katz, A. Cell Membranes Open “Doors”
for Cationic Nanoparticles/Biomolecules: Insights into
Uptake Kinetics. ACS Nano 2013, 7 (12), 10799–10808.
Jędrzejewska, B.; Pietrzak, M.; Pączkowski, J. Solvent
Effects on the Spectroscopic Properties of Styrylquinolinium
Dyes Series. J. Fluoresc. 2010, 20 (1), 73–86.
Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R.
Production of Large Unilamellar Vesicles by a Rapid
Extrusion Procedure: Characterization of Size Distribution,
Trapped Volume and Ability to Maintain a Membrane
Potential. Biochim. Biophys. Acta 1985, 812 (1), 55–65.
Lakowicz, J. R. Principles of Fluorescence Spectroscopy;
Springer Science & Business Media, 2007.
Magde, D.; Rojas, G. E.; Seybold, P. G. Solvent Dependence
of the Fluorescence Lifetimes of Xanthene Dyes.
Photochem. Photobiol. 1999, 70 (5), 737–744.
Schote, U.; Seelig, J. Interaction of the Neuronal Marker
Dye FM1-43 with Lipid Membranes: Thermodynamics and
Lipid Ordering. Biochim. Biophys. Acta BBA - Biomembr.
1998, 1415 (1), 135–146.
Chaumont, F. de; Dallongeville, S.; Chenouard, N.; Hervé,
N.; Pop, S.; Provoost, T.; Meas-Yedid, V.; Pankajakshan, P.;
Lecomte, T.; Montagner, Y. L.; et al. Icy: An Open
Bioimage Informatics Platform for Extended Reproducible
Research. Nat. Methods 2012, 9 (7), nmeth.2075.
Lagache, T.; Sauvonnet, N.; Danglot, L.; Olivo-Marin, J.-C.
Statistical Analysis of Molecule Colocalization in
Bioimaging. Cytom. Part J. Int. Soc. Anal. Cytol. 2015, 87
(6), 568–579.
1
2
3
4
5
6
7
8
(14)
(15)
(16)
(30)
(31)
(32)
(33)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17)
(34)
(35)
(18)
(19)
(36)
(37)
(38)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
Banker, G. A.; Cowan, W. M. Rat Hippocampal Neurons in
Dispersed Cell Culture. Brain Res. 1977, 126 (3), 397–342.
Danglot, L.; Triller, A.; Bessis, A. Association of Gephyrin
with Synaptic and Extrasynaptic GABAA Receptors Varies
during Development in Cultured Hippocampal Neurons.
Mol. Cell. Neurosci. 2003, 23 (2), 264–278.
(28)
Jennings, P.; Bertocchi, C.; Frick, M.; Haller, T.; Pfaller, W.;
Dietl, P. Ca2+ Induced Surfactant Secretion in Alveolar
Type II Cultures Isolated from the H-2Kb-TsA58 Transgenic
Mouse. Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol.
Biochem. Pharmacol. 2005, 15 (1–4), 159–166.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 10
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table of content graphic (TOC)
10
ACS Paragon Plus Environment