Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging

Article abstract of DOI:10.1021/jacs.0c00451

Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.

Full text of DOI:10.1021/jacs.0c00451

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Article
Spontaneously blinking fluorophores based on nucleophilic addition/
dissociation of intracellular glutathione for live-cell super-resolution imaging
Akihiko Morozumi, Mako Kamiya, Shin-nosuke Uno, Keitaro Umezawa,
Ryosuke Kojima, Toshitada Yoshihara, Seiji Tobita, and Yasuteru Urano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00451 • Publication Date (Web): 28 Apr 2020
Downloaded from pubs.acs.org on April 28, 2020
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Spontaneously blinking fluorophores based on nucleophilic addi-
tion/dissociation of intracellular glutathione for live-cell super-
resolution imaging
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Akihiko Morozumi†, Mako Kamiya‡,§,*, Shin-nosuke Uno†, Keitaro Umezawa†, Ryosuke Koji-
⊥
,*
ma‡,§, Toshitada Yoshihara¶, Seiji Tobita¶, Yasuteru Urano†,‡,
†Graduate School of Pharmaceutical Sciences and ‡Graduate School of Medicine, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
§PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
¶Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, Gunma 376-8515, Japan
⊥AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-
0004, Japan
ABSTRACT: Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution
images, but generally requires prior intense laser irradiation and in some cases additives to induce blinking of
conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on
an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a
novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack
of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine
fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form
and the non-fluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules in living
cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green
ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.
Introduction
to induce appropriate blinking states of plural fluoro-
phores2, 10-11
.
Super-resolution fluorescence imaging provides micro-
scopic images with a resolution well below the diffraction
limit, and is a powerful tool for detailed investigation of
cellular structures and processes1-5. Single-molecule local-
ization microscopy (SMLM) is one of the most frequently
used methods, and reconstructs super-resolution images
through detection and high-precision localization of indi-
vidual fluorescent molecules attached to the observation
target2, 6. In order to perform SMLM with conventional
organic fluorophores, approaches known as direct sto-
chastic optical reconstruction microscopy (dSTORM)7 or
ground-state depletion microscopy followed by individual
molecule return (GSDIM)8 have been developed. These
methods require the fluorophores to be converted into a
dark state via the excited state and then to revert stochas-
tically to the fluorescent state under intense laser irradia-
We previously reported a first-in-class spontaneously
blinking fluorophore, HMSiR, based on an intramolecular
spirocyclization reaction in the ground state. This fluoro-
phore exists mostly in a colorless and non-fluorescent
form and spontaneously blinks with fluorescence emis-
sion in the near-infrared (NIR) range. In contrast to con-
ventional fluorophores, HMSiR does not require intense
laser irradiation or any additive for blinking, so it is suita-
ble for live-cell SMLM under physiological conditions12. In
order to expand the color range of spontaneously blinking
fluorophores, we also developed a spontaneously blinking
fluorophore with green-light emission, HEtetTFER, and
by using the two fluorophores, we succeeded in dual-
color SMLM of fixed cells in additive-free buffer solution
without optimization10. Unfortunately, however, we found
that HEtetTFER could not be used for live-cell SMLM,
probably due to an unfavorable subcellular localization,
poor cell-membrane permeability or other reasons. These
results clearly demonstrate that spontaneously blinking
tion in the presence of reducing agents such as thiols2, 9
.
However, intense laser irradiation can be cytotoxic and
may cause photobleaching of the fluorophores; also, espe-
cially in the context of multi-color SMLM, it can be diffi-
cult to find a suitable composition of the imaging buffer
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fluorophores that work in vitro do not always work in live
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cells, and therefore we need to prepare a variety of
candidate blinking fluorophores and select those which
can work in live cells in order to achieve dual-color live-
cell SMLM.
5
Here, in order to expand the molecular design strategy
of spontaneously blinking fluorophores, we develop a
novel principle of spontaneous blinking in living cells,
which builds on our previous finding that some xanthene
fluorophores convert between a colored, fluorescent form
and a colorless, non-fluorescent form as a result of a re-
versible ground-state nucleophilic attack of intracellular
glutathione (GSH) upon the 9th carbon atom of the xan-
thene ring (Figure 1). We previously utilized this thermal
equilibrium of intermolecular nucleophilic addi-
tion/dissociation to develop a reversible fluorescent probe
for monitoring intracellular GSH concentration. The
probe has a suitable dissociation constant toward GSH
(Kd,GSH) of millimolar level and a sub-second response rate
for real-time monitoring of intracellular GSH dynamics13.
In particular, we thought that the lack of background flu-
orescence of the GSH adduct in this thermal equilibrium
would be advantageous for achieving high localization
precision in SMLM. In order to utilize this intermolecular
reaction for SMLM, it is essential to ensure that only a
small subset of the fluorophores is switched on, followed
by stochastic reversion to the non-fluorescent state with
appropriate kinetics12. Therefore, we started by derivatiz-
ing xanthene fluorophores to look for candidate fluoro-
phores with appropriate Kd,GSH values and blinking kinet-
ics. As a result, we found two candidate fluorophores,
SiP650 and CP550, working in the NIR and green wave-
length ranges, respectively, with which we were able to
perform SMLM of microtubules in living cells by utilizing
the endogenous intracellular GSH. Furthermore, we
achieved dual-color live-cell SMLM by using CP550 to-
gether with our previously developed NIR spontaneously
blinking fluorophore, HMSiR, and succeeded in observ-
ing two targets in mammalian cells and in bacterial cells
without the need for optimization of the imaging medium.
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Figure 1. Spontaneously blinking fluorophores for SMLM
based on the ground state nucleophilic addition and dis-
sociation of intracellular GSH. (a) Fluorescence switching
based
on
intermolecular
nucleophilic
addi-
tion/dissociation of GSH to/from xanthene derivatives as
a novel mechanism of fluorescence blinking for SMLM.
Xanthene derivatives can convert between the fluorescent
dissociated form and the non-fluorescent GSH adduct
form. Kd,GSH is the dissociation constant toward GSH and 
is the lifetime of the dissociated form. (b) Preparation of
new xanthene derivatives with sufficiently low Kd,GSH val-
ues. Chemical structures of candidate fluorophores based
on silicon pyronine (SiP) and carbopyronine (CP) scaffolds
are shown with the measured Kd,GSH values and fluores-
cence quantum yields (fl). (c) Dose-response curves of
the candidate fluorophores versus GSH. The normalized
absorbance at the indicated wavelengths (absorption max-
ima of the dissociated forms) is plotted against GSH con-
centration to evaluate Kd,GSH for each fluorophore. Ab-
sorption spectra were measured in 200 mM sodium phos-
phate buffer (pH 7.4) containing various concentrations of
GSH and 1% dimethyl sulfoxide (DMSO) as a cosolvent,
and normalized with respect to those in the absence of
GSH. The Kd,GSH of SiP600 was not determined because
the majority of the molecules underwent nucleophilic
addition of hydroxide ion in 200 mM sodium phosphate
buffer (pH 7.4) in the absence of GSH.
Results and discussion
Design and synthesis of novel blinking fluoro-
phores: evaluation of thermal equilibrium of addi-
tion and dissociation of GSH. In order to develop a new
class of spontaneously blinking fluorophores for SMLM
based
on
intermolecular
nucleophilic
addi-
tion/dissociation of intracellular GSH (Figure 1a), we con-
sidered that it would be essential to optimize two param-
eters: (1) the dissociation constant toward GSH (Kd,GSH), so
that a small subset of fluorophores would exist in the flu-
orescent state in the physiological GSH concentration
range, in order to avoid overlapping signals; (2) the life-
time of the fluorescent form (τ, the duration until the
fluorescent dissociated form reverts to the non-
fluorescent GSH adduct form) in order to match the ex-
posure time of microscope cameras so that sufficient pho-
tons can be detected for precise localization12.
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Table 1. Properties of SiP650 and CP550 derivativesa
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τ (ms) c
Absorbance
maximum (nm)
Emission
maximum (nm)
Fluorescence quan-
tum yieldb
Kd,GSH (μM)
5 mM GSH
1.0
633
636
639
550
567
576
654
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663
570
586
593
0.39
0.46
0.52
0.70
0.69
0.65
1.0
2.9
25
SiP650
SiP650-BA
SiP650-HaloTag
CP550
2.3
9.9
0.46
1.7
3.1
CP550-BA
35
CP550-HaloTag
210
7.6
a Measured in 200 mM sodium phosphate buffer (pH 7.4), except for τ values measured in 10 mM sodium phosphate buff-
er (pH 7.4); Absolute quantum yield; c The lifetime of the fluorescent dissociated form. Note: this parameter is not the
b
fluorescence lifetime.
We first tried to optimize the Kd,GSH values of xanthene
Kd,GSH values of 3.1 μM and 96 μM, respectively, probably
due to the increased LUMO energy levels of the fluoro-
phores (Figure 1c and S1 and Table S1). These results
strongly suggested that the LUMO energy level of the
core xanthene ring and the steric hindrance around the
9th carbon of a xanthene fluorophore are indeed im-
portant determinants of the Kd,GSH value. Through this
derivatization, we obtained four candidates with accepta-
ble Kd,GSH values in the range of 1-100 μM (9Phe SiP600,
SiP650, CP550, CP600). Among these derivatives, SiP650,
CP550, and CP600 showed sufficiently high quantum
yields (Φfl = 0.39, 0.70, and 0.49, respectively), while 9Phe
SiP600 exhibited a low fluorescence quantum yield (Φfl =
0.09), probably due to the rotation of its pendant phenyl
ring13, 23. Low fluorescence quantum yield would be disad-
vantageous considering that the localization precision of
a single molecule essentially depends on the number of
photons emitted from the molecule24-25, so we excluded
9Phe SiP600 as a candidate. Among the remaining three
candidates, we selected two pyronines, SiP650 and CP550
as different-colored candidate scaffolds, since these fluor-
ophores can be efficiently excited by commonly used laser
derivatives to control the percentage of the fluorescent
dissociated form at physiological GSH concentrations of 1-
10 mM14-15. The Kd,GSH value can be determined from the
dose-response curve of the fluorophore, as the GSH con-
centration at which the absorbance of the dissociated
form is reduced to half of the maximum (Figure 1c). When
the Kd,GSH value of a fluorophore is 1 mM, as is the case
with silicon-substituted rhodamine 2’Me SiR600 (Figure
1b)13, more than 10% of the fluorophore exists in the fluo-
rescent dissociated form in the presence of 1-10 mM GSH.
In contrast, a Kd,GSH value of less than 10 μM implies that
less than 1% of the fluorophore would be in the fluores-
cent form and most of it would be in the non-fluorescent
GSH adduct form at physiological GSH concentrations.
Considering recent advances in multi-emitter localization
algorithms16-21, we set a criterion Kd,GSH value of roughly 1-
100 μM so that the percentage of the fluorescent form
would be less than 10%. Since it has been suggested that
the affinity of a xanthene fluorophore for GSH or other
nucleophiles is correlated with the lowest unoccupied
molecular orbital (LUMO) level of the fluorophore and
the steric hindrance around the 9th carbon atom of the
xanthene ring12-13, 22, we focused on modifying 2’Me
SiR600 and 9Phe SiP650, both of which showed low
Kd,GSH values in our previous study (1.0 mM and 1.1 mM,
respectively)13, aiming to prepare silicon-substituted xan-
thene fluorophores with decreased Kd,GSH values by reduc-
ing the steric hindrance around the 9th carbon atom
(Figure 1b). We prepared 9Phe SiP600, SiP600, and
SiP650 by removing the methyl group from the pendant
phenyl group of 2’Me SiR600 or removing the pendant
phenyl group itself from 2’Me SiR600 and 9Phe SiP650.
9Phe SiP600 and SiP650 exhibited Kd,GSH values of 15 μM
and 1.0 μM, respectively; however, it was difficult to de-
termine the Kd,GSH value of SiP600, since nucleophilic
attack of hydroxide ion in the buffer solution readily oc-
curred due to the high electrophilicity of the fluorophore
(Figures 1c and S1 and Table S1). Further, in order to ex-
pand the color range, we also prepared carbopyronine
derivatives CP550 and CP600 by replacing the silicon
atom at the 10th position of SiP600 and SiP650 with a
carbon atom. CP550 and CP600 exhibited increased
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1
lines. In addition, H NMR analyses provided evidence of
nucleophilic attack by thiol at the 9th carbon of the xan-
thene units of SiP650 and CP550 (Figure S2).
Evaluation of duration of the dissociated form by
laser photolysis. The lifetime of the fluorescent dissoci-
ated form (τ) is another critical parameter for spontane-
ously blinking fluorophores (note: this parameter is not
the fluorescence lifetime). We supposed that an appropri-
ate τ value would be one that matches the exposure time
of the camera in order to achieve detection of sufficient
photons for precise localization12, and we set a criterion τ
value of millisecond order, taking account of the exposure
time of cameras recently used for SMLM2, 26
.
The τ values of the candidate fluorophores, SiP650 and
CP550, were determined by laser photolysis as the time
constant of conversion from the fluorescent dissociated
form to the non-fluorescent GSH adduct form (Figure S3).
In sodium phosphate buffer (pH 7.4) containing physio-
logical concentrations of GSH (1-10 mM), most of the
fluorophores exist in colorless, non-fluorescent GSH ad-
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Figure 2. Evaluation of switching properties of the HaloTag protein-fluorophore conjugates by single-molecule fluores-
cence imaging. (a) Chemical structures of SiP650- and CP550-based HaloTag ligands, and labeling of purified HaloTag
proteins to prepare fluorophore-protein conjugates (SiP650-HaloTag, CP550-HaloTag). (b) Single-molecule fluorescence
time traces of SiP650-HaloTag (left) and CP550-HaloTag (right). (c,d) Excitation intensity dependence of photon number
per switching event (c) and lateral localization precision (d) of SiP650-HaloTag (red) and CP550-HaloTag (green) (mean
± s.e., N = 416-12802). Single-molecule imaging was performed in 10 mM sodium phosphate buffer (pH 7.4) containing 5
mM GSH. Excitation: 647 nm (100 W/cm2 for (b)) for SiP650 and 561 nm (100 W/cm2 for (b)) for CP550. Exposure: 8.8
ms/frame.
duct form. Upon pulsed irradiation with an ultraviolet
laser (308 nm), transient absorption decay was observed
at a wavelength characteristic of each xanthene fluoro-
phore (Figure S4). As the shape of the transient absorp-
tion spectrum corresponded well to that of the dissociat-
ed form of the fluorophore (Figure S4), the observed tran-
sient absorption and its decay were attributed to the tran-
sient dissociation of GSH from the xanthene fluorophore,
followed by the re-addition of GSH. By fitting the ob-
tained decay curve to a pseudo-first-order equation, we
could calculate the τ values of the candidate fluorophores.
Although the τ values varied depending on GSH concen-
tration, SiP650 and CP550 exhibited appropriate τ values
in the millisecond range in the presence of 1-10 mM GSH
(Table 1 and S2). In the case of 9Phe SiP600, which we
excluded from our candidates due to its low fluorescence
quantum yield, we observed relatively long τ values, par-
ticularly at lower concentrations of GSH, which would
probably due to the steric hindrance around the 9th car-
bon. These results confirm that SiP650 and CP550 are
promising candidates as different-colored scaffolds.
Evaluation of the fluorescence properties of SiP650
and CP550 by single-molecule fluorescence imaging.
We next examined the fluorescence properties of SiP650
and CP550 by means of single-molecule imaging using
total internal reflection fluorescence (TIRF) microscopy.
We introduced a ligand unit of HaloTag27, one of the most
widely used protein tags, into SiP650 and CP550 to pre-
pare SiP650-Halo and CP550-Halo, which were then
coupled with a purified HaloTag protein to afford SiP650-
HaloTag and CP550-HaloTag as fluorophore-protein
conjugates (Figure 2a). These protein conjugates showed
larger Kd,GSH and τ values than those of their parental
small molecules SiP650 and CP550 (Table 1 and Figures
S5 and S6), probably because of the increased electron
density of the xanthene ring due to the N-alkylation or N-
alkyl extension and the increased steric hindrance around
the 9th carbon of the xanthene ring due to the protein
labeling. Nevertheless, both of the τ values and the Kd,GSH
values remained almost within the original target range
(τ: 9.9 ms for SiP650-HaloTag and 7.6 ms for CP550-
HaloTag in the presence of 5 mM GSH, Kd,GSH: 25 μM for
SiP650-HaloTag and 210 μM for CP550-HaloTag). The
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percentage of the fluorescent form in the presence of 5 HaloTag (647 nm excitation), and 1600 photons and 6.1
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mM GSH was estimated to be 0.5% for SiP650-HaloTag
and 4% for CP550-HaloTag, which would still be within
the acceptable range when using multi-emitter localiza-
tion algorithms16-21. However, the Kd,GSH values of these
conjugates increased further under more acidic pH condi-
tion (Figures S7), indicating that our fluorophores would
be less suitable for SMLM at lower pH. We further con-
firmed that SiP650-HaloTag and CP550-HaloTag exhibit
sufficient fluorescence quantum yields (Φfl = 0.52 and 0.65,
respectively) (Table 1), which are comparable to those of
the parental small molecules, SiP650 and CP550.
nm precision for CP550-HaloTag (561 nm excitation)
(Figures 2c,d, S10 and S11). These values are comparable to
those of conventional fluorophores used for SMLM
(Alexa6477, 28-30 and TMR28-31) at the highest laser intensity
(1.5 kW/cm2) in an optimized buffer solution containing
β-mercaptoethylamine (MEA) and an enzymatic oxygen
scavenging system (GLOX): 1100 photons and 7.3 nm pre-
cision for Alexa647-HaloTag conjugate (647 nm excita-
tion) and 300 photons and 15 nm precision for TMR-
HaloTag conjugate (561 nm excitation) (Figure S12). These
results demonstrated that SiP650-HaloTag and CP550-
HaloTag, working in different color regions, both exhibit
appropriate blinking for SMLM at physiological GSH con-
centrations.
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In order to examine the fluorescence properties of
SiP650-HaloTag and CP550-HaloTag at the single-
molecule level, these fluorophore-protein conjugates were
adsorbed onto a coverslip, and their single-molecule fluo-
rescence behaviors were observed with a TIRF microscope
in sodium phosphate buffer (pH 7.4) containing 1-10 mM
GSH. We confirmed that the majority of SiP650 and
CP550 fluorophores existed in the non-fluorescent GSH
adduct form, and showed reversible fluorescence blinking
even under low-intensity excitation (Figure 2b and Movie
S1). The average durations of the fluorescent states of
SiP650-HaloTag and CP550-HaloTag were calculated to
be 7.4-10 ms and 6.5-12 ms, respectively, in the presence of
1-10 mM GSH, showing clear GSH concentration depend-
ence (Figures S8 and S9). As well as being consistent with
the results of the bulk transient absorption measurements,
these values meet the criterion for use in SMLM. The
number of emitted photons detected per switching event
and the localization precision of single molecules varied
depending on the laser intensity (40 W/cm2-1.5 kW/cm2),
and with the highest intensity, we obtained the best val-
ues: 1300 photons and 8.0 nm precision for SiP650-
Interfering effects of other intracellular nucleo-
philes. In order to examine the effects of other intracellu-
lar nucleophiles on the optical properties of SiP650 and
CP550, we measured the in-vitro absorption spectra of
SiP650-HaloTag and CP550-HaloTag in the presence of
thiol-containing species other than GSH, such as cysteine,
homocysteine, and H2S at the maximum concentrations
possible in a live-cell environment32-36 (Figures S13 and
S14). These thiol-containing species also induced consid-
erable decreases in the absorbance, suggesting that these
nucleophiles are reactive with the fluorophores in vitro.
Interestingly, the intermolecular reactions with cysteine
and homocysteine were reversible, whereas the reaction
with H2S was irreversible for some reason (Figure S15).
But, considering that the physiological concentration of
H2S in living cells is in the sub-micromolar range37, this
irreversibility should not be an impediment to practical
use for live-cell imaging (we were able to obtain images
without difficulty), but should be taken into account
Figure 3. Live-cell SMLM with SiP650-Halo and CP550-Halo. -Tubulin-Halo fusion proteins were transiently expressed in
Vero cells and labeled with SiP650-Halo (a) or CP550-Halo (c) for 30 min. Imaging was performed in cell culture medium
(DMEM) after washing. (a,c) Conventional images (averaged projection images, left) and SMLM images (right). Excitation:
647 nm (200 W/cm2) for (a) and 561 nm (200 W/cm2) for (c). Acquisition: 8.8 ms/frame, 2000 frames (= 17.6 s) for (a) and
1000 frames (= 8.8 s) for (c). (b,d) Transverse profiles of fluorescence intensity in the conventional images (black) and local-
izations in the SMLM images (red) corresponding to the regions outlined by the solid yellow lines (left) and by the dotted
yellow lines (right). (b) and (d) correspond to (a) and (c), respectively. FWHM = 347.4 ± 25.5 nm (conventional) and 109.2
± 8.4 nm (SMLM) (mean ± s.e., N = 6) for (b); 339.3 ± 11.1 nm (conventional) and 100.4 ± 5.1 nm (SMLM) (mean ± s.e.,
N = 6) for (d). Scale bars: 3 m (a) and 2 m (c).
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Conventional
SMLM
SMLM (CP550-mitochondrion, HMSiR-microtubule)
CP550-FtsZ
HMSiR-
cell membrane
0 min
3 min
6 min
Figure 4. Time-lapse/dual-color live-cell SMLM. (a,b) Time-lapse live-cell SMLM of mitochondria with CP550-BnClPy.
Mitochondria-localizable SNAP-tag protein molecules were transiently expressed in living Vero cells and labeled with
CP550-BnClPy for 60 min. Imaging was performed in cell culture medium (DMEM). Excitation: 561 nm (400 W/cm2).
Acquisition: 8.8 ms/frame, 5000 frames (= 44 s) in total. (a) Sub-diffraction localizations collected from the whole dataset
of 5000 frames. Each localization is color-coded according to the time at which it was detected. (b) Fast time-lapse
SMLM image sequence of the region boxed in (a). Each SMLM image was reconstructed from 500 consecutive frames
(4.4 s) starting at the indicated time point. Scale bars: 2 m. (c,d) Dual-color live-cell SMLM with CP550 and HMSiR. (c)
Mitochondria-localizable SNAP-tag (green) and -tubulin-Halo (microtubules, red) were transiently expressed in living
Vero cells and labeled with CP550-BnClPy and HMSiR-Halo, respectively, for 60 min. Time-lapse imaging was performed
at 0, 3, and 6 min in cell culture medium (DMEM). Significant changes in the mitochondrial structure and arrangement
can be seen in the boxed region (dotted boxes), with the magnified views shown in the insets. Laser illumination (561
nm, 300 W/cm2 for CP550 and 647 nm, 150 W/cm2 for HMSiR) and image acquisition were performed at 8.8 ms/frame in
an alternate manner for the two colors with an 8-frame duration for each turn. Each SMLM image was reconstructed
from 960 frames for each color (16.9 s in total). Scale bars: 3 m (main images) and 500 nm (insets). (d) Halo-Tagged
FtsZ (cell-division-related protein, green) and outer membrane (red) were labeled with CP550-Halo and HMSiR-NHS,
respectively, in living C. crescentus cells. Imaging was performed on 1.5% agar. Laser illumination (561 nm, 200 W/cm2 for
CP550 and 647 nm, 200 W/cm2 for HMSiR) and image acquisition were performed at 15 ms/frame in an alternate manner
for the two colors with a 7-frame duration for each turn. Each SMLM image (right for each cell) was reconstructed from
7000 frames for each color (210 s in total). Conventional images (averaged projection images, left) were generated from
the corresponding raw images. Scale bars: 1 m.
when the probes are applied to cells with high levels of
intracellular H2S. Further, considering the fact that GSH
exists at a much higher concentration than the other nu-
cleophiles in cells, the fluorescence behavior of the fluor-
ophores should be predominantly determined by GSH in
live-cell environments. We also confirmed that a physio-
logical concentration of taurine (a representative amine-
containing molecule) or a basic pH of 9.0 had little effect
on the performance of the fluorophores.
living cells, we applied the HaloTag ligands SiP650-Halo
and CP550-Halo to Vero cells expressing β-tubulin fused
with HaloTag (Figure S16a). SiP650-Halo and CP550-
Halo specifically labeled microtubules, indicating that
both ligands are cell-permeable and react specifically with
the HaloTag proteins (Figure S17a,b). Most importantly,
these fluorophores exhibited spontaneous blinking based
on the reversible reaction with the endogenous GSH in-
side the living cells without prior intense laser irradiation
and in the absence of any chemical additive (Movies S2
and S3).
Validation of SiP650 and CP550 for live-cell SMLM.
Encouraged by the above results, we next attempted to
apply our spontaneously blinking fluorophores to live-cell
SMLM. First, in order to examine whether the fluoro-
phores could be used to label specific target proteins in
We next examined whether SMLM images can be re-
constructed from the blinking fluorescence signals of our
fluorophores. Because intense laser illumination is not
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required to induce blinking with our fluorophores, the
position10. Therefore, we next focused on performing du-
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illumination power can be reduced to minimize photo-
bleaching and potential photodamage. Thousands of con-
secutive images were recorded at a laser intensity of 200
W/cm2, which is lower than the illumination power typi-
cally required for dSTORM7, 30 or GSDIM8, and analyzed
to reconstruct a super-resolution image (see Supporting
Information for localization analysis employing a multi-
emitter fitting algorithm). As a result, the microtubules
were visualized more sharply and with better separation
than a conventional image (Figure 3), while no obvious
cellular structures were constructed when these fluoro-
phores were applied to cells not expressing tag protein
(Figure S18a,b). These results indicate that our spontane-
ously blinking fluorophores enable SMLM with minimal
phototoxicity.
al-color SMLM by utilizing our spontaneously blinking
fluorophores with different colors. Specifically, we labeled
mitochondria-localized SNAP-tag with CP550-BnClPy
and β-tubulin-HaloTag with HMSiR-Halo, a HaloTag
ligand of HMSiR, which is our previously reported NIR-
emitting, spontaneously blinking fluorophore based on
intramolecular spirocyclization (Figure S17c)12. Irradiation
with 561 nm and 647 nm lasers was applied alternately to
excite CP550 and HMSiR, which allowed us to obtain
dual-color SMLM images of the mitochondria and the
microtubules, respectively, and thus to visualize the rela-
tive arrangement of the two targets. Further, as we used a
low power level to minimize photobleaching and photo-
damage, we were able to repeat dual-color SMLM imaging,
for example, at 3-min intervals to allow time-lapse exper-
iments (Figure 4d).
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Time-lapse SMLM in live cells. In order to expand the
utility of our newly developed spontaneously blinking
fluorophores, we further prepared SiP650-BnClPy and
CP550-BnClPy by introducing a benzylchloropyrimidine
(BnClPy) unit, a known substrate for SNAP-tag38-40, into
SiP650 and CP550, respectively (Figure 4a). When we
applied these substrates to live Vero cells expressing
SNAP-tag proteins in their mitochondria (Figure S16b),
we observed efficient labeling of the mitochondria with
CP550-BnClPy (Figure S17d), but only subtle labeling was
observed with SiP650-BnClPy, probably due to its poor
cellular permeability (data not shown). Therefore, we
focused on investigating whether super-resolution imag-
ing of mitochondria in living cells can be performed with
CP550-BnClPy. Considering the mobility (movement,
fusion, or fission) of mitochondria on the time scale of
seconds to minutes41-42, we recorded thousands of consec-
utive images at an exposure time of 8.8 ms/frame to
match the blinking kinetics of CP550, and then recon-
structed sequential SMLM images from partial (500-
frame) subsets with 400-frame overlaps (frames 1-500; 101-
600; 201-700; 301–800; and so on)12, 43-44 (Figure 4b,c and
Movie S4). The resultant SMLM image sequence success-
fully visualized the dynamics of the mitochondria at a
temporal resolution of a few seconds per reconstructed
image. We also confirmed that no obvious cellular struc-
tures were constructed when CP550-BnClPy was applied
to cells not expressing tag protein (Figure S18c). These
results suggest that its appropriately fast spontaneous
blinking rate makes CP550 an excellent choice for track-
ing mobile targets with minimal damage, perturbation, or
artifacts.
Next, we examined whether dual-color SMLM imaging
with our spontaneously blinking fluorophores would be
applicable to live bacterial cells, which contain structures
too tiny to be analyzed by conventional fluorescence mi-
croscopy. HaloTag fusion protein of FtsZ45, a bacterial
cytoskeletal protein forming a part of the cell division
machinery (Figure S16a)46-49, in live Caulobacter crescen-
tus bacterial cells was labeled with CP550-Halo, and the
outer membrane of the cells was labeled with HMSiR-
NHS, an N-hydroxysuccinimidyl ester of HMSiR12. Then,
the bacterial cells were immobilized on an agarose pad for
imaging. CP550 showed appropriate blinking inside the
bacterial cells, as expected, since the concentration of
GSH in bacteria is reported to be in the millimolar
range50-54. HMSiR also showed spontaneous blinking even
on the cell surface, since it blinks on the basis of intramo-
lecular spirocyclization. Reconstruction of dual-color
SMLM images enabled detailed observations of the locali-
zations and structures of protein complexes formed by
FtsZ, together with fine profiling of the bacterial mor-
phology by imaging the cell membrane. As previously
reported46, FtsZ exhibits a continuous band-like pattern
(Figure 4e, Cell 1) or a spot-like pattern (Figure 4e, Cell 2),
which cannot be distinguished by conventional microsco-
py. Further investigations, e.g., by three-dimensional46, 55
and/or time-lapse SMLM,46 should lead to a better under-
standing of the distribution and behavior of FtsZ.
Conclusion
We have established a novel principle of spontaneous
blinking for live-cell SMLM on the basis of nucleophilic
addition/dissociation of endogenous intracellular GSH
to/from xanthene fluorophores. By structural optimiza-
tion of the fluorophore, we have developed novel sponta-
neously blinking fluorophores, SiP650 and CP550, with
appropriate equilibrium constants between the fluores-
cent dissociated form and the non-fluorescent GSH ad-
duct form, and with adequately fast blinking kinetics. We
confirmed that HaloTag ligands of SiP650 and CP550 can
specifically label target proteins and show spontaneous
blinking in live cells as a result of reversible nucleophilic
Dual-color SMLM in live cells. Multi-color super-
resolution imaging has been a powerful tool for detailed
studies of biological processes involving multiple cellular
components. However, when conventional organic fluor
ophores are used for multi-color SMLM, it is particularly
troublesome to optimize the experimental conditions for
inducing appropriate blinking of plural fluorophores2, 10-11
.
Spontaneously blinking fluorophores with different colors
would circumvent this difficulty, since their blinking oc-
curs spontaneously under intracellular conditions, with-
out the need for optimization of the imaging buffer com-
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(7) Heilemann, M.; van de Linde, S.; Schuttpelz, M.; Kasper,
R.; Seefeldt, B.; Mukherjee, A.; Tinnefeld, P.; Sauer, M.
Subdiffraction-resolution fluorescence imaging with
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attack of intracellular GSH, enabling SMLM images to be
obtained. Further, by using CP550 in combination with
our NIR-emitting HMSiR, we achieved dual-color live-
cell SMLM in mammalian cells and in bacterial cells. Our
dual-color approach should be useful for investigating the
relationships between two biological targets in live cells
with minimum photodamage and without the need to
optimize the imaging buffer. We anticipate that the mo-
lecular design strategy described in this work can be fur-
ther applied to develop new fluorophores with different
optical properties and blinking kinetics, thereby offering a
wider range of tools for super-resolution studies.
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ASSOCIATED CONTENT
Supporting Information. Synthesis and characterization of
compounds, evaluation of optical properties and response to
GSH, fluorescence imaging (movies and data analysis), and
experimental details. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*mkamiya@m.u-tokyo.ac.jp
*uranokun@m.u-tokyo.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported in part by AMED under grant
number JP19gm0710008 (to Y.U.), by JST/PRESTO grant
JPMJPR14F8 (to M.K.), by MEXT/JSPS KAKENHI grants
JP16H02606, JP26111012, and JP19H05632 (to Y.U.) and
JP15H05951 “Resonance Bio”, JP19H02826, and JP19K22242 (to
M.K.), by JSPS Core-to-Core Program (grant number
JPJSCCA20170007), A. Advanced Research Networks, by Ja-
pan Foundation for Applied Enzymology (to M.K.), and The
Naito Foundation (to M.K.), as well as a stipend from the
Graduate Program for Leaders in Life Innovation (GPLLI)
and a JSPS stipend (to A.M.). The authors thank S. Manley
for sharing the plasmid encoding mitochondria-localizable
SNAP-tag and C. crescentus bacterial strain (EG603) and for
advice on bacterial imaging.
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