Strategy to Lengthen the On-Time of Photochromic Rhodamine Spirolactam for Super-resolution Photoactivated Localization Microscopy

Article abstract of DOI:10.1021/jacs.8b11369

Rhodamine derivatives and analogues have been widely used for different super-resolution imaging techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit great superiority for PALM imaging due to a de

Full text of DOI:10.1021/jacs.8b11369

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Article
A Strategy to Lengthen the On-time of Photochromic Rhodamine
Spirolactam for Super-Resolution Photoactivated Localization Microscopy
Zhiwei Ye, Haibo Yu, Wei Yang, Ying Zheng, Ning Li, Hui Bian,
Zechen Wang, Qiang Liu, Youtao Song, Mingyan Zhang, and Yi Xiao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11369 • Publication Date (Web): 02 Apr 2019
Downloaded from http://pubs.acs.org on April 2, 2019
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Journal of the American Chemical Society
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A Strategy to Lengthen the On-time of Photochromic Rhodamine
Spirolactam for Super-Resolution Photoactivated Localization
Microscopy
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a,b‡
a,*‡
b‡
b‡
b
b
a
a
Zhiwei Ye, Haibo Yu, Wei Yang, Ying Zheng, Ning Li, Hui Bian, Zechen Wang, Qiang Liu, Youtao
a
c
b*
Song, Mingyan Zhang and Yi Xiao
a. College of Environmental Sciences, Liaoning University, Shenyang 110036, P.R.China.
b. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P.R.China.
c. Liaoning Center of Disease Prevention and Control, Shenyang 110001, P.R. China.
‡.
These authors contributed equally to this work.
ABSTRACT: Rhodamine derivatives and analogues have been widely used for different super-resolution imaging
techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit
great superiority for PALM imaging due to a desirable bright-dark contrast during the photochromic switching process.
Although considerable attention has been paid to the chemical modifications on rhodamine spirolactams, the on-time of
photochromic switching, one of the key characteristics for PALM imaging, has never been optimized in previous
developments. In this study, we proposed that simply installing a carboxyl group close to the lactam site could impose an
intramolecular acidic environment, stabilize the photoactivated zwitterionic structure and thus effectively increase the
on-time. Based on this idea, we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably
longer on-time than the other tested analogues, as well as an enhancement of single molecule brightness, an
improvement on signal-to-noise ratio and an enlargement of total collected photons of single molecule before
photobleach. Finally, super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a
good temporal resolution of 10 s, as well as a satisfactory localization precision of ~25 nm. Through self-labeling protein
tags, Rh-Gly modified with HaloTag ligand enabled super-resolution imaging of histone H2B proteins in live HeLa cells;
through immunostaining antibodies labeled with an isothiocyanate-substituted Rh-Gly, super-resolution imaging of
microtubules was achieved in fixed cells. Therefore, our simple and effective strategy provides novel insight for
developing further enhanced rhodamine spirolactams recommendable for PALM imaging.
poisonous species and, thus, represents an important
advantage of the rhodamine spirolactam over the caged
rhodamine.
Introduction
Single molecule localization microscopy (SMLM)
techniques,
microscopy
reconstruction microscopy (STORM) , call for high
performance fluorescent dyes.
including
(PALM)
photoactivated
and
localization
optical
In 2007, for the first time, a photoactivatable
rhodamine spirolactam (Rh-Pht, in Scheme 1a) was
1–3
stochastic
4–6
introduced into localization-based super-resolution
7–12
18
Recently, rhodamine
imaging by Bossi, Hell and their coworkers. Since then, a
derivatives and analogues have been widely applied for
PALM imaging. Generally, there are two kinds of
variety of rhodamine spirolactams have been applied in
subcellular super-resolution imaging to study the
1
3–16
18,19
20
19
rhodamines for PALM imaging, i.e., caged rhodamines
architecture of tubulin,
clathrin
and keratin,
1
7,18
9
and rhodamine spirolactams.
The first subclass
dynamics of actin filaments, the 3D structure of the
1
7
requires a caging group (usually an o-nitrobenzyl moiety)
that quenches the fluorescence and undergoes
irreversible photocleavage to recover fluorescence upon
UV irradiation. Unfortunately, the applications of caged
rhodamines in live cell imaging are limited because the
potential toxicity of the photoreleased fragments remains
unclarified. The second subclass, i.e., rhodamine
bacterial cell membrane. By rational design of an amide
1
7
substituent for rhodamine spirolactam, Moerner et al.
shifted the activation wavelength to the visible region,
which greatly enhanced the biocompatibility of PALM
1
9
imaging. Recently, Belov and Bossi et al. extended the
spirolactam design with enhanced hydrophilicity, feasible
for super-resolution imaging in live and fixed cells.
spirolactam,
undergoes
a
reversible
structural
However, in PALM imaging, there always exists a trade-
off between spatial and temporal resolutions.
investigate fine architectures of organelles in fixed cells,
earlier PALM imaging required an acquisition time of
transformation from nonfluorescent leuco form to highly
fluorescent zwitterion under UV light irradiation. Such
reversible off-on switching avoids the additional release of
1,21
To
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several minutes to hours. To monitor the dynamic
spirolactam (Scheme 1a), the key to prolong on-time, in
fact, is to stabilize the transitional bright zwitterionic
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changes inside live cells, such a time requirement is
impractical. Therefore, various mathematical or
instrumental methods, e.g., algorithms for solving the
30,31
structure.
In the fields of chemosensors where
rhodamine derivatives are studied intensively, it is widely
accepted that an acidic environment (e.g., lower pH
solution or coordination with transition metal ions,
Scheme 1b) is a stabilization factor for the zwitterionic
22–24
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multi-emitter problems
and faster speed cameras,
have been developed to improve the temporal resolution
of PALM imaging. Chemists also seek to solve this
dilemma by rational design of fluorophores with
3
2,33
structures.
Based on this principle, a massive number
26,27
enhanced brightness. Remarkably, Lavis et al.
doubled
of rhodamine-spirolactam-based fluorescent sensors has
29,34,43,44,35–42
the quantum yield of rhodamine by replacing N,N-dialkyl
units with four-membered azetidine rings, and have
generalized this strategy to other fluorophores. Later, Liu,
been reported.
This knowledge inspires us that
if we place the rhodamine spirolactam molecules into
such an acidic environment, then the on-time -- i.e., the
lifetime of the photogenerated zwitterionic structures --
will be extended. However, for super-resolution imaging
of live cells, it might be impractical to lower pH or to add
highly toxic metal ions safely, precisely and selectively at
the intracellular sites where the rhodamine spirolactam
molecules are located.
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Xu et al. further introduced a three-membered azetidine
replacement to achieve enhanced brightness in highly
polar fluorophores. However, as every acquired single
molecule signal consists of hundreds to thousands of
photon emission events, the sustainability of emission,
reflected by the duration of on-time, constitutes another
major impact on the brightness besides ensemble
fluorescence performance. Thus, a lengthened on-time
and a stabilization of bright state can enhance the single
molecule brightness. This enhancement further generates
blink events with high signal-to-noise ratio (SNR), and
ultimately improves the spatial resolution of obtained
Here, we report a simple and effective strategy to
design rhodamine spirolactams with prolonged on-time
of photogenerated zwitterions. Our idea is the installation
of a carboxyl group close to the lactam site, imposing an
intramolecular acidic environment upon rhodamine
spirolactams. The new dye, Rh-Gly, outperforms its
esterified counterpart Rh-MGly as well as a pioneering
model Rh-Pht in terms of on-time and single molecule
brightness. Finally, through PALM imaging of live cells
stained with Rh-Gly, we obtain high-quality super-
resolution images of live cell mitochondria with relatively
good spatial and temporal resolutions. Through HaloTag
technique and immunostaining method, we further
achieved super-resolution images of histone H2B proteins
and microtubules with derivatives of Rh-Gly in HeLa cells.
PALM images during
a shortened time of image
acquisition. Hence, the design of photoswitchable
rhodamines with prolonged on-time leads to possibilities
of achieving high quality PALM imaging.
Results and Discussion
Molecular design and synthesis
Our idea in the design of a photoswitchable rhodamine
spirolactam with prolonged on-time is to add an acidic
carboxyl group in close proximity to the lactam (Scheme
1c). Photoactivation of Rh-Gly will first generate an
unstable and short-lived amide anion. Simultaneously,
the carboxylic acid could immediately protonate the
amide nitrogen in an intramolecular fashion. Since the
nucleophilicity of carboxylic anion is much lower than
amide anion, the final zwitterionic structure of Rh-Gly
should be more stable and long-lived. Therefore, we
expect Rh-Gly to possess a longer on-time of the bright
zwitterionic state and superior potential for high-quality
PALM imaging in live cells.
Scheme 1. (a) Photochromic mechanism of common
rhodamine spirolactam, e.g., Rh-Pht and Rh-MGly. (b)
Photochromic mechanism of
a
rhodamine spirolactam
Rh-Gly and Rh-MGly were readily prepared from
commercial starting materials, and detailed procedures
were provided in the supporting information. The
esterified intermediate Rh-MGly, lacking acidity, is a
good control compound for Rh-Gly to estimate the
intramolecular acidic effect on the on-time of the
zwitterion.
29
stabilized by zinc ion.
(c) Proposed hypothesis on
photochromic reaction of Rh-Gly with light-induced
activation (hν) of fluorescence (zwitterion) and thermal
relaxation (∆) to the deactivated state (spirolactam).
The optimization of photochromic on-time has never
been attempted in previous designs of PALM-applicable
rhodamine spirolactams. Considering the rapid relaxation
from photogenerated rhodamine zwitterion back to dark
Ensemble photochromic and pK study
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Figure 2. (a) Spontaneous spirocyclization equilibrium of
rhodamine spirolactam. (b) Integrated emission intensity vs
pH of both Rh-MGly and Rh-Gly in C OH/H O (v:v=3:7).
(c) Emission spectral changes of Rh-Gly vs pH in
OH/H O (v:v=3:7).
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H
2 5
2
C H
2 5
2
To better understand the acidity effect on the existence
in terms of spirolactams or zwitterions, we further
measured the pK values of both dyes under identical
conditions. The spirocyclization equilibrium of
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Figure 1. (a) Emission spectral changes of Rh-Gly and Rh-
MGly (5×10 M) in glycerol (containing 30% CH OH, mixed
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45–48
rhodamine spirolactam
corresponding to the pK
a
is
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shown in Figure 2a. As shown in Figure 2b, the pK
value
a
well) before and after UV irradiation (Ac stands for after
activation, λuv= 365 nm). Inset: (left) magnified spectrum of
the boxed region in (a) and (right) photographs of Rh-Gly
of Rh-Gly is 5.86, which is ca. 1 pH unit higher than Rh-
MGly. The result reveals that transformation of Rh-Gly
into the corresponding zwitterion does not require a
strong acidity as in the case of Rh-MGly. This decreased
demand for acidity also proves that zwitterion
transformed from Rh-Gly can be stabilized to some
extent through a “self-stabilized” structure (Scheme 1c),
while there is no such stabilization effect for Rh-MGly.
This stabilization extends the lifetime of the zwitterion
and contributes to the improved photoactivation
efficiency of Rh-Gly (Figure 1). In addition, the
corresponding fluorescence spectra of Rh-Gly during pH
titration are shown in Figure 2c. The dye shows a
significantly enhanced fluorescence (186-fold) at 585 nm,
which results from lowering pH values from 10.45 to 3.87.
At neutral pH, a small peak in the fluorescence spectrum
(red curve in Figure 2c) is detected, indicating that a
small subset of Rh-Gly molecules exist in zwitterion
forms. The existence of open-ring zwitterions is also
evidenced by the small visible band of the absorption
spectrum in PBS in Figure S1b. As rhodamine spirolactams
continuously undergo spontaneous ring-opening in
-4
3
solutions (5×10 M) in glycerol (containing 30% CH OH,
mixed well) before and after UV irradiation. (b) Emission
spectral changes of Rh-Gly and Rh-MGly in thin PVA film
before and after UV irradiation.
Rh-Gly exhibits excellent photoactivation performance
upon irradiation with UV light both in solution and in
film (Figure 1). The absorption band of Rh-Gly and Rh-
MGly tails beyond 350 nm (Figure S1), which is sufficient
20
for sparse activation required for PALM. A majority of
Rh-Gly molecules remain nonfluorescent in glycerol
(
containing 30% methanol); however, the color of
solution changes to pink (Figure 1a inset) upon exposure
to UV light, with an appearance of the characteristic
emission peak of rhodamine fluorophore at 585 nm
(
Figure 1a). In addition, both absorption and emission
spectra of Rh-Gly in glycerol upon UV irradiation match
those measured in acidic aqueous solution (Figure S2),
suggesting the ring-opening transformation from
spirolactam leuco to zwitterionic chromophore upon
photoactivation. In contrast to the 6.1-fold photoactivated
fluorescence enhancement of Rh-Gly, its esterified
counterpart Rh-MGly exhibits essentially no change in
glycerol solution. The photoactivation phenomenon was
further studied in thin PVA film. As recorded in Figure 1b,
fluorescence of Rh-Gly in PVA film shows a 7-fold
increment (λfl = 570 nm) after 375 nm irradiation for 5 s,
while the enhancement of Rh-MGly is just 3.5-fold under
identical conditions. The above results demonstrate that
Rh-Gly possesses an enhanced photoactivation capability
versus Rh-MGly.
47
neutral medium, the slightly acidic pKa of Rh-Gly
guarantees that the majority of its fluorophores exist in
the nonfluorescent spirolactam form under the
physiological condition, thus avoiding signal overlap
during live cell PALM imaging.
Photoactivation and single molecule fluorescence
on-time measurements of Rh-Gly
To investigate the repeatable activation performance of
Rh-Gly, we performed a photoactivation experiment in
PVA film. Rh-Gly molecules dispersed were continuously
2
excited with a strong 532 nm laser (~2 kW/cm ) and
periodically activated by UV irradiation of 375 nm. Upon
exposure to UV irradiation, newly formed zwitterions
sharply improve the total detected number of activation
2
events to approximately 6.0 × 10 , as shown in Figure 3a.
The activation cycle is repeated 46 times, and the process
lasts for over 10 min until the majority of Rh-Gly
fluorophores are bleached. The photoactivation images of
the investigated region are further shown in Figure S3b.
The fraction of fluorescent signals observed before UV
photoactivation is due to the proton-controlled ring-
opening equilibrium (Figure 2a). Thus, Rh-Gly
demonstrates a repeatable photoactivation performance.
This light-controlled characteristic between dark
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spirolactam and bright zwitterion of Rh-Gly satisfies the information section 4.4). These results further allowed us
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photoswitching nature of PALM imaging.
to propose a structural transformation model to explain
the blink behaviors of three dyes. As shown in Figure 3c,
the activation of spirolactam (dark state) is a double-
recovery (kr1 and kr2) process under UV irradiation. The
slow recovery rate kr1 possibly corresponds to the UV
photoactivated transfer to non-protonated zwitterion (A1),
containing an unstable amide ion, while the fast recovery
rate kr2 probably corresponds to the transformation into
The photoswitching behaviors were further identically
investigated at the single molecule level upon continuous
UV photoactivation for Rh-Gly, Rh-Pht and Rh-MGly.
All studied PVA films show sparsely distributed single
molecule signals (Figure 3b inset) with distances
significantly greater than the full width of the single
molecular spot of approximately 330 nm, thus avoiding
the overlap between close signals. The blink behavior is
protonated
zwitterion
(A2)
through
either
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photoactivation or spontaneous ring-opening during
spirocyclization equilibrium. However, once the
fluorophore is in active state, the two states (A1 and A2)
are undistinguishable as they represent the same bright
state of ring-opened zwitterion. After a short residence
time in the active (bright) state, the zwitterion either
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described by a kinetic model following Bustamante et al.
A fluorophore could be activated from dark state to active
(bright) state. Upon continuous excitation, the active
state either transforms to dark state or bleach state. Based
on the model, photoswitching rates between different
states are estimated through the fittings of the probability
distribution of four characteristic parameters: the on-time
of bright state (ton), the dark time of nonfluorescent state
transforms to the ring-closed dark state with rate k
d
or
bleaches with rate To compare the rates of
b
k .
photoswitching transformations of spirolactams, we
performed the single molecule experiment for 5 times
independently (details of obtained results are shown in
Table S1 and discussed in supporting information section
(
(
t
n
dark), the number of blinks after fluorophore activation
blinks) and the photobleach time (tbleach). Figure 3b
exhibits an example of an integrated intensity trace from
a single Rh-Gly fluorophore along with its measurement
of blink parameters. The theoretical kinetic model
provided good fittings and predictions on the probability
4
.4); since the results exhibit a similar tendency, one
result close to median is listed in Figure 3d (from n > 1000
single molecule intensity traces for each fluorophore).
2
distributions of the experimental blink parameters (R >
0
.90, Figure S4, detailed discussion in supporting
Figure 3. Single-molecule characterization of Rh-Gly, Rh-MGly and Rh-Pht embedded in PVA film in conventional (wide-field
imaging) mode. (a) Time trace of numbers of activation events of Rh-Gly upon periodically controlled 375 nm laser and
continuous irradiation of 532 nm laser. (b) One single molecule intensity trace of Rh-Gly and the measurement of its blink
parameters. Inset presents two fluorescence images of single molecule signals recorded in control and Rh-Gly-dispersed PVA
film, respectively. Control shows almost no noise signals. Scale bar: 10 μm. (c) Proposed mechanism explains the blink behavior
of rhodamine spirolactam. Upon self-ring-opening or photoactivation, the dark spirolactam transforms to its non-protonated
(
A1, kr1) or protonated (A2, kr2) zwitterionic structure (active state). The zwitterion is stabilized by an intramolecular proton
transform (Rh-Gly) or an attraction on an environmental proton (Rh-Pht and Rh-MGly). After some dwelling time at active
state, the ring-opened structure either transforms to dark state (k ) or bleaches (k ). (d) Summary of kinetic rates and photon
statistics. α is the fraction of kr1 recovery; ‘SNR’ is the signal-to-noise ratio. (e) Probability density distribution of on-times from
d
b
Rh-Gly fits well to a single exponential decay. Ton was calculated as the inverse of summed rates (k
structures of Rh-Asp, Rh-dMAsp, Rh-Ser and Rh-MSer.
d b
+ k ). (f) Molecular
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To avoid bias introduced by large values, Ton, i.e., the
fewer, its overall photostability is improved as indicated
by the significant enlargement of total collected photons
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average on-time of bright state, was properly calculated
from the probability distribution fit of on-times during
photoswitching quantification analysis (as shown in
Figure 3e; other fits are provided in Figure S4), providing
an accurate estimation of the mean lifetime of active
states. Among three dyes, Rh-Gly exhibits a prolonged
on-time of 68.5 ms, which is apparently longer than the
other two analogs (37.7 ms for Rh-MGly and 29.9 ms for
Rh-Pht, respectively). The prolonged on-time of Rh-Gly
demonstrates the existence of a stabilization effect on the
zwitterion, consistent with the one described in the
ensemble study. As the average on-time (Ton) was
5
of single molecule before photobleach (1.90 × 10 ),
5
compared to the others (Rh-Pht: 0.79 × 10 ; Rh-MGly:
5
1.34 × 10 ). The improvement is attributed to the same
rigidification of zwitterion through hydrogen bonding,
consistent to the on-time extension. Moreover, the
enhanced single molecule signals further benefit the
localization reconstruction. The average localization
uncertainty of Rh-Gly is 22.1 (Table S2), which is lower
than its analogs (Rh-Pht: 26.1; Rh-Pht: 24.6). Therefore,
owing to the carboxylic group, the single molecule
fluorescence of Rh-Gly is synergistically strengthened by
both the intramolecular proton transfer and the hydrogen
bonding, making the fluorophore recommendable for
single molecule localization-based super-resolution
imaging.
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calculated as the inverse of the sum of rates (k
extension can be further explained by the analysis of
photoswitching rates (k and k ) for three compared
d b
+ k ), its
d
b
spirolactams. For Rh-Gly, the short-lived negative
nitrogen atom of the active state is stabilized by a
following intramolecular proton transfer, which induces a
To further understand the relevance between the on-
time extension and the acid substituents, four additional
analogues, i.e., Rh-Asp, Rh-Ser, Rh-dMAsp and Rh-
MSer, were prepared (synthesis procedures are provided
in supporting information) and investigated at single
molecule level under identical conditions. According to
their substituents, the added four fluorophores (Figure 3f)
are classified into two groups i.e., acid installed
spirolactams and esterified analogues. As listed in Figure
-1
d
lower k of 12.7 s , whereas the others rely on the small
possibility of catching nearby protons from hydroxyl
groups of the PVA molecule, resulting in faster
-
1
transformations to dark states (Rh-MGly k
d
= 23.8 s and
-1
Rh-Pht k
d
= 29.9 s ). In other words, the ring-close
reaction for Rh-Gly is slowed by the intramolecular
proton transfer. In addition, Rh-Gly exhibits the lowest
-1
bleach rate (k
b
= 1.9 s ), supporting the enhanced
3
d, the acid installed spirolactams (Rh-Asp, Ton = 67.1 ms;
stabilization effect in an intramolecular fashion. As all
Rh-Ser, Ton = 63.3 ms) exhibit extended on-time in a
uniform fashion, compared to their esterified analogues
-1
compared analogs show much lower rates of k
b
(< 4 s )
-1
d
than the rates of k (> 12 s ), the transformation to dark
(
Rh-dMAsp, Ton = 41.3 ms; Rh-MSer, Ton = 36.5 ms). This
spirolactam constitutes a more significant effect than the
photobleach process on on-time (discussions on other
obtained rates are presented in supporting information
section 4.4). Therefore, it is the intramolecular proton
transfer that stabilizes the zwitterionic structure of bright
state and further extends the fluorescence on-time of Rh-
Gly.
result confirms the stabilization effect of intramolecular
proton transfer on the zwitterionic structure of
rhodamine spirolactams. In addition, the on-times of Rh-
Asp and Rh-Ser, are in proximity to the on-time of Rh-
Gly (as listed in Figure 3d and Table S1), suggesting that
extra hydrophilicity of the substituents does not prolong
the lifetime of zwitterionic bright state. On the other side,
as listed in Figure 3d, the single molecule brightness of
Enhanced single molecule fluorescence signals are also
observed following the on-time increase. As listed in
Figure 3d, Rh-Gly exhibits robust single molecule
4
these acid installed spirolactams (Rh-Asp, 3.34 × 10
4
photons/s; Rh-Ser, 3.55 × 10 photons/s) is enhanced than
4
4
brightness (average photon emission rate: 3.57 × 10
their esterified analogues (Rh-dMAsp, 2.82
×
10
4
photons/s), significantly higher than the compared
photons/s; Rh-MSer, 2.62 × 10 photons/s), further
proving the stabilization effect through intramolecular
hydrogen binding. Furthermore, the stabilization leads to
strengthened SNR (Rh-Asp, 5.14; Rh-Ser, 5.41), enlarged
total collected photons before photobleach (Rh-Asp, 1.76
× 10 ; Rh-Ser, 2.27 × 10 ) and improved localization
uncertainties (Rh-Asp, 22.6; Rh-Ser, 22.0) of acid
installed spirolactams. Therefore, the rigidification of
zwitterion, caused by the proximate acid group,
significantly extends the on-time and enhances the single
molecule fluorescence of rhodamine spirolactams.
4
4
analogs (2.44 × 10 photons/s for Rh-Pht and 2.85 × 10
photons/s for Rh-MGly). The enhancement of the
brightness for Rh-Gly is consistent with its prolonged on-
time, owing to the rigidification of the zwitterion through
intramolecular hydrogen bonding. Following the
enhancement of single molecule brightness and the
lengthening of on-time, Rh-Gly also exhibits the highest
SNR (5.48). On the other side, the prolonged on-time and
the enhanced emission rate increases the times of
excitation to unstable excited states during one switching
cycle. The integration of excitations further increases the
possibilities of photobleach during each cycle, leading to
a decreased number of switching cycles of Rh-Gly (7.7,
Table S2), compared to the other analogs (Rh-Pht: 9.3;
Rh-MGly: 9.8). Nevertheless, these numbers are still
comparable to previous reported values of fluorophores,
5
5
Rh-Gly for PALM imaging of mitochondria in live
cells
1
1,50
which require excessive imaging enhancing additives.
Although the number of switching cycles of Rh-Gly is
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cells. The majority of fluorescent signals of Rh-Gly
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overlap well with the signals of Mitotracker Deep Red
(commercial mitochondrial tracker) in both cell lines as
revealed by merged images in Figure 4a and b. Plots from
Figure 4a and 4b right column show synchronous
intensity distributions between two fluorophores along
the yellow line marked in the merged images, proving the
co-occurrence of fluorescence of the two dyes. In addition,
more fields of view from colocalization experiments are
presented in the supporting information (Figure S5). The
correlations between two stainings are further quantified,
yielding high average Pearson’s coefficients at 0.86±0.07
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MCF-7 cells, n = 52) and 0.83±0.06 (HeLa cells, n = 67).
All results demonstrate that Rh-Gly specifically stains
mitochondria in live cells with few nonspecific signals.
The accumulation of the dye inside mitochondria is
probably caused by the protonation equilibrium on
spirolactam (Figure 2a). Under neutral cytosolic
5
1
conditions (pH = 7.2) , Rh-Gly molecules partially
protonate and exist in forms of positively charged
zwitterions; therefore, they are attracted into
mitochondria by the negative potential of inner
Figure 4. Two-color colocalization analysis of Rh-Gly in
HeLa (panel a) and MCF-7 (panel b) cell lines. In each panel,
from left to right: confocal fluorescence image of Rh-Gly
after UV irradiation, colored in red; confocal fluorescence
image of Mitotracker Deep Red (Mitotracker), colored in
blue; merged image of two fluorescent images; intensity
profiles from two fluorophores along the yellow line marked
in the merged image. Scale bar: 25 μm.
5
2
membranes.
Once they enter mitochondria, the
hydrophobic microenvironment will force them to
transform back into the colorless spirolactam form of
uncharged and hydrophobic nature, which also leads to
their continual sequester within mitochondria. Therefore,
Rh-Gly accumulates in mitochondria mostly in its ring-
closed form and could be photoactivated to bright
zwitterion form by UV irradiation.
Before applying Rh-Gly to live cell PALM imaging, we
analyzed the distribution of Rh-Gly in different cell lines.
After UV photoactivation, fluorescence of Rh-Gly exhibits
mitochondrial networks in both HeLa cells and MCF-7
Figure 5. Super-resolution imaging of mitochondria-enriched regions in live HeLa (panel a) and MCF-7 (panel b) cells stained
with Rh-Gly. Inside each panel, the top left image is a wide-field fluorescence image of the region of interest and the right image
is a PALM image of the same region reconstructed from ~2000 consecutive frames (10 s total duration) under inclined
illumination ; yellow arrows mark the regions with high localization density, whereas green arrows indicate domains with low
localization density; bottom row, from left to right: histogram of single molecule brightness, histogram of localization precisions
and time trace analysis of Nyquist resolutions (orange color band shows the standard deviations of the resolutions estimated
from >16 domains during the PALM imaging). Scale bars: 2 μm.
5
3,54
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Finally, we performed PALM imaging of mitochondria
in live HeLa and MCF-7 cells stained with Rh-Gly under a
precisions and ~50 nm Nyquist resolutions are obtained
for super-resolution imaging of mitochondria in live cells
within 10 s.
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total internal reflection fluorescence (TIRF) microscopy in
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inclined illumination mode.
The fluorophore exhibits
Despite the tradeoff between spatial and temporal
good photoswitching performance (shown in Movie S1
and S2) in live cells upon simultaneous irradiation by
green and UV light. The blink events are generated in
normal culture media free from additional oxygen
1
resolutions during super-resolution imaging, the PALM
imaging of mitochondria stained with Rh-Gly does not
sacrifice the imaging speed to obtain high quality. The
temporal resolution of mitochondrial super-resolution
imaging is shortened to 10 s, which is comparable to the
best reported single molecule localization super-
1
1,55,56
depletion regents and toxic reductants,
which inhibit
5
7
ATP generation in live cells. Though PALM imaging
certainly requires UV irradiation, the intensity of the laser
is relatively low (~2 W/cm ) and causes no apparent cell
1,5,25,46,59,65,66
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resolution imaging results.
(Table S3) Although
2
previous imaging of mitochondria has obtained
a
5
8,59
damage during the short imaging time.
In sharp
58
temporal resolution of 2 s, the imaging demand toxic
blinking enhancing additives or heavily intensified laser
power (Detail comparison is discussed in supporting
information section 5.4). The high temporal resolution is
probably a consequence of the on-time improvement of
Rh-Gly. The combination of prolonged on-time and the
stabilization effect on the zwitterion of Rh-Gly increases
photon emission rate and enhances the single molecule
brightness (Figure 3d). The brightness improvement
further enlarges the number of localizations obtained
during imaging, leading to improved spatial resolution
within shortened time. Therefore, the inherent nature of
on-time extension of the zwitterion makes Rh-Gly a good
dye for PALM imaging of mitochondria in live cells. The
dye exhibits a balanced tradeoff between preserving the
localization precision and improving the temporal
resolution of PALM imaging. In addition, the prolonged
on-time also provides applicable potential for obtaining
extended single molecule trajectories in live cells.
contrast to the blurred wide-field images of mitochondria,
the PALM images reveal detailed morphologies in Figure
a and b (top row). Moreover, the super-resolution
images indicate mitochondrial activity. Because of the
prolonged on-time of protonated Rh-Gly and the acid-
5
induced ring-opening tendency of
a
rhodamine
spirolactam, the number of obtained localizations
increases in regions where active proton pumping
happens. Thus, the corresponding localization density
also reflects the mitochondrial activity. The yellow arrows
indicate typical dense regions with intense mitochondrial
respiration, whereas the green arrows point to low density
domains with lower activity. To further demonstrate the
capability of mitochondrial super-resolution imaging with
Rh-Gly, six additional results are shown in Figure S6 for
each cell line, respectively. Therefore, Rh-Gly offers great
potential for super-resolution imaging of mitochondria in
live cells.
Two core parameters to quantify the quality of PALM
imaging are the localization precision and the localization
density (ρ) of rendered molecules. The precision describes
the uncertainty of each localization.60,61 As shown in
Figure 5a, b (second plot in the bottom row of each panel)
and Figure S7, the average localization precision of Rh-
Gly in each mitochondrial imaging is approximately 25
nm, corresponding to average photon numbers of 400-
8
00 per single molecule per frame (first bottom plot in
Figure 5a, 5b; Figure S7). On the other hand, the
localization density defines the sampling interval of
reconstructed images. According to the Nyquist-Shannon
1
,62
criterion,
intervals of discrete sampling (localization
spaces) for target structures must be less than half of the
desired resolution of the image. Hence, the Nyquist
criterion-based resolution (Nyquist resolution) of a 2D
localization image is calculated as 푅푁푦푞푢푖푠푡 = 2/
휌
and is
utilized for the evaluation of the overall spatial resolution
of reconstructed images. Through the equation above,
Nyquist resolutions (bottom right column of Figure 5a, b
and Figure S7) were consequently determined at each
second during PALM imaging. Following the elongation
of imaging time, more localizations are gathered until
Nyquist resolutions improve to ~50 nm at 10 s. The
63,64
corresponding Fourier ring correlation (FRC) analysis
of these images at 10 s also shows a relatively high spatial
resolution of 128 nm (for HeLa cells in Figure 5a) and 122
nm (for MCF-7 cell in Figure 5b) in Figure S8. Therefore,
through staining of Rh-Gly, balanced ~25 nm localization
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Right: model of HaloTag protein covalently bounded to Rh-
Gly-Halo. Image of Rh-Gly-Halo coupled haloalkane
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dehalogenase (1BN6) is designed with chimera. (b) PALM
image of H2B-Halo fusion proteins labeled with Rh-Gly-
Halo in live HeLa cell under inclined illumination. Inset
shows the wide-field image of the same H2B-Halo fusion
proteins labeled with Rh-Gly-Halo. (c) Histogram of single
molecule brightness. (d) Histogram of localization
uncertainties. Scale bars: 3 μm.
Rh-Gly labelled HaloTag for PALM imaging of
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fusion proteins in live cells
Through established technique of HaloTag fusion
69
proteins, we further obtained the super-resolution
imaging of histones with Rh-Gly in live HeLa cells. To
achieve the labeling of target enzymes (HaloTag protein),
we prepared a chloroalkane ligand conjugated derivative
of Rh-Gly, i.e. Rh-Gly-Halo, as shown in Figure 6a
(
details of the synthetic procedures are presented in
supporting information). This probe is cell-permeable,
and covalently binds to H2B-Halo fusion proteins in live
HeLa cell as presented in Figure 6b. The reconstructed
image of histone H2B proteins exhibits a significant
enhancement of resolution compared to the wide-field
image (Figure 6b inset), with an average photon number
of 1040 per single molecule per frame (Figure 6c) and an
average localization precision of 21 nm (Figure 6d).
Moreover, additional four cases of super-resolution
imaging of H2B proteins are exhibited in Figure S9,
validating the efficiency of Rh-Gly-Halo in imaging Halo-
H2B fusion proteins. Therefore, Halo ligand conjugated
derivative of Rh-Gly demonstrates an extended
functionality for specific labeling other cellular structures
besides mitochondria, and retains the capacity for PALM
imaging in live cells.
Figure 6. Super-resolution imaging of histones in nucleus of
live HeLa cell. (a) Left: molecular structure of Rh-Gly-Halo;
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Figure 7. Super-resolution imaging of microtubules in fixed HeLa cell. (a) Molecular structure of Rh-Gly-NCS. Super-resolution
image (b) and corresponding wide-field image (c) of microtubules immunolabeled with primary antibody against α-tubulin and
secondary antibody labeled with Rh-Gly-NCS under inclined illumination. Super-resolution image was reconstructed from
~15000 frames. The imaging media is phosphate buffer saline (PBS). (d) Histogram of the localization precisions. (e) The
intensity profiles of microtubule filament in super-resolution image (b) and wide-field image (c) indicated with blue lines. A
single gaussian function was fitted to the data, estimating that the full width at half maximum (FWHM) of microtubule filament
was 62 nm in the super-resolution image and 396 nm in the wide-field image. Scale bar: 3 μm.
resolution imaging of microtubules are shown in Figure
S12. In short, owing to its capability of PALM imaging,
bioconjugates labeled with Rh-Gly enable super-
resolution imaging of microtubules in fixed cells.
Rh-Gly labelled antibody for PALM imaging of
microtubules
To further extend the functionality of Rh-Gly, a
derivative with an isothiocyanate group, i.e., Rh-Gly-NCS,
was prepared for super-resolution imaging of
microtubules in HeLa cells (details of the synthesis are
provided in the supporting information). Through
immunolabeled fixed HeLa cells with primary antibodies
against α-tubulin and secondary antibodies labeled with
Rh-Gly-NCS, the super-resolution image of microtubules
was successfully obtained in simple PBS solution, without
the aid of special imaging enhancing additives (Figure 7b).
The reconstruction image demonstrates a notable clarity
improvement versus the wide-field image (Figure 7c),
which is blurred by diffraction. Quantitative
measurement of the PALM imaging provides an average
single molecule brightness of 981 photons per frame of
Rh-Gly-NCS (Figure S11) and an average precision of 15
nm of the corresponding localizations (Figure 7d). In
addition, the super-resolution image of tubulin reveals a
Conclusion
In conclusion, we have proposed a simple and effective
strategy to lengthen the on-time of photochromic
rhodamine spirolactam, which proves to benefit PALM
imaging. Our idea is to introduce a carboxyl group close
to the lactam site in order to exert an inner acidity effect
to stabilize photogenerated zwitterions. Rh-Gly shows
repeatable and UV-controllable activation ability with a
prolonged on-time of the zwitterion at the single
molecule level, through comparing with both its esterified
product (Rh-MGly) and another inspiring analog (a
18
pioneering PALM dye, Rh-Pht) reported by Hell et al.
The stabilization effect of the carboxyl group on
zwitterion not only increases total photon collection but
also enhances single molecule brightness. Furthermore,
Rh-Gly also exhibits a specific mitochondrial staining in
different cell lines (HeLa and MCF-7). As a consequence
of the lengthened on-time, the temporal resolution of
PALM imaging of mitochondria in live cells is shortened
to 10 s, preserving localization precisions of approximately
6
2 nm width of antibody coated microtubule, close to
70
previously reported value, and the result demonstrates a
robust improvement on resolution versus the crude
estimation of 396 nm width of microtubule in the wide-
field image (Figure 7e). Two appended cases of super-
25 nm. Because of the balanced tradeoff between spatial
and temporal resolutions, a series of robust super-
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resolution images of mitochondria in live cells are further
(4)
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Imaging by Stochastic Optical Reconstruction Microscopy
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obtained by Rh-Gly. The functionality of Rh-Gly for
specific cellular labeling is extended with a HaloTag
ligand derivative and an isothiocyanate derivative;
through self-labeling protein tags and immunostaining
method, these derivatives enable super-resolution
imaging of histones and microtubules in HeLa cells.
Therefore, our simple design paves a new path towards
prolonging on-time for the development of rhodamine
spirolactams to improve PALM imaging in the future.
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(
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Supporting Information.
The supporting information is available free of charge on
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Experimental methods, synthesis procedures and
characterization spectra, Table S1-S3 and Figure S1-13.
Movie S1. Blink behaviors of Rh-Gly in Figure 5a.
Movie S2. Blink behaviors of Rh-Gly in Figure 5b.
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AUTHOR INFORMATION
Corresponding Author
*
*
xiaoyi@dlut.edu.cn (Y. Xiao)
yuhaibo@lnu.edu.cn (H. Yu)
(
12)
Author Contributions
‡
These authors contributed equally to this work.
(13)
ACKNOWLEDGMENT
This work is supported by National Natural Science
Foundation of China (Nos. 21421005, 21576040, 21776037 and
(
14)
21804016), the Fundamental Research Funds for the Central
(15)
(16)
(17)
Universities (No. DUT17LK43), Natural Science Foundation
of Liaoning Province (20180510044), Program Funded by
Liaoning Province Education Administration (No. L2014010)
and National Water Pollution Control and Treatment
Science and Technology Major Project (2015ZX07202012).
Special thanks to Prof. Xinfu Zhang, at State Key
Laboratory of Fine Chemicals, Dalian University of
Technology, for providing the comparison analog Rh-Pht;
to Prof. Wei Ji, at Institute of Biophysics, Chinese
Academy of Sciences, for help in building up the single
molecule localization microscopy; and to Prof. Youjun
Yang at East China University of Science and Technology,
for inspiring discussions on the mechanism of prolonged
on-time and for his help in improving the English of this
manuscript.
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