Quaternary Piperazine-Substituted Rhodamines with Enhanced Brightness for Super-Resolution Imaging

Article abstract of DOI:10.1021/jacs.9b04893

Insufficient brightness of fluorophores poses a major bottleneck for the advancement of super-resolution microscopes. Despite being widely used, many rhodamine dyes exhibit sub-optimal brightness due to the formation of twisted intramolecular charge transfer (TICT) upon photoexcitation. Herein, we have developed a new class of quaternary piperazine-substituted rhodamines with outstanding quantum yields (φ = 0.93) and superior brightness (? × φ = 8.1 × 10⁴ L·mol^-1·cm^-1), by utilizing the electronic inductive effect to prevent TICT. We have also successfully deployed these rhodamines in the super-resolution imaging of the microtubules of fixed cells and of the cell membrane and lysosomes of live cells. Finally, we demonstrated that this strategy was generalizable to other families of fluorophores, resulting in substantially increased quantum yields.

Full text of DOI:10.1021/jacs.9b04893

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Communication
Quaternary Piperazine Substituted Rhodamines with
Enhanced Brightness for Super-Resolution Imaging
Zhiwei Ye, Wei Yang, Chao Wang, Ying Zheng, Weijie Chi,
Xiaogang Liu, Zhenlong Huang, Xiaoyuan Li, and Yi Xiao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b04893 • Publication Date (Web): 05 Sep 2019
Downloaded from pubs.acs.org on September 5, 2019
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Quaternary Piperazine Substituted Rhodamines with Enhanced
Brightness for Super-Resolution Imaging
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†,§
†,‖,§,*
‡,§
†,§
‡
‡,*
Zhiwei Ye , Wei Yang
, Chao Wang , Ying Zheng , Weijie Chi , Xiaogang Liu , Zhenlong
†
†
†,*
Huang , Xiaoyuan Li , and Yi Xiao
†
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, 116024, China.
‡
Singapore University of Technology and Design, 8 Somapah Road, Singapore 487372.
‖
Chemical Analysis and Research Center, Dalian University of Technology, Dalian 116024, China
Supporting Information Placeholder
Owing to these widespread applications, significant
efforts have been taken to further enhance the performance
ABSTRACT: Insufficient brightness of fluorophores poses a
major bottleneck for the advancement of super-resolution
microscopes. Despite being widely used, many rhodamine
dyes exhibit suboptimal brightness due to the formation of
twisted intramolecular charge transfer (TICT) upon
photoexcitation. Herein, we have developed a new class of
quaternary piperazine substituted rhodamines with
outstanding quantum yields (Φ = 0.93) and superior
brightness (ε×Φ = 8.1 × 10 L∙mol ∙cm ), by utilizing the
electronic inductive effect to prevent TICT. We have also
successfully deployed these rhodamines in the super-
resolution imaging of the microtubules of fixed cells, and
cell membrane and lysosomes of live cells. Finally, we
demonstrated that this strategy was generalizable to other
families of fluorophores, resulting in substantially increased
quantum yields.
26
of rhodamines.
Previous experimental studies have
indicated some rhodamine’s non-radiative decays match the
27–30
formation of TICT states,
although other mechanisms,
e.g., hydrogen-bonding interactions, could not be ruled out.
Therefore, one of the important strategies in the
development of new rhodamine derivatives is to suppress
31
4
-1
-1
the probable TICT formation (Figure 1a). In the TICT state,
the donor moiety undergoes a ~90° rotation with respect to
the fluorophore core, thus becoming non-radiative and
highly reactive. To inhibit TICT, chemists used to rigidify
dialkylamino donor moieties by forming ring structures.
Unfortunately, this method often leads to large molecular
structures with poor biological compatibility. Alternatively,
11,13,32
Lavis et al.
replaced the N,N-dimethylamino
substituent in tetramethylrhodamine (TMR) with an
33
azetidine ring. Later, Xu and Liu et al. further showed that
an aziridine ring effectively suppresses TICT formation.
Recent years have witnessed
advanced fluorescence imaging techniques, such as single-
molecule localization microscopy (SMLM)
unprecedented resolution beyond the Abbe diffraction limit.
In contrast to the rapid evolution of imaging techniques, the
development of dyes with sufficient brightness and
photostability remains slow, which poses a significant
constraint to in-vivo cellular dynamic studies. Therefore, it
is urgent to develop bright and photostable dyes on the
basis of rational molecular design strategies.
a rapid evolution of
Herein, we employ a simple quaternary piperazine moiety
to replace the dimethylamino group in TMR. The resulted
N,N-dimethylpiperazine substituted rhodamine (MPR)
doubles the brightness of TMR and maintains excellent
biocompatibility.
1–3
that affords
Besides environmental conditions, TICT formation in a dye
is intrinsically governed by two factors: steric hindrance
(Lavis’ and Xu’s work) and electronic (or push-pull) effect
(this work). We reason that reducing the electron-donating
strength of the amino group would destabilize the TICT
state and increase the energy barrier to enter the TICT state,
thus suppressing TICT formation. This reasoning brings our
attention to an electron-withdrawing quaternary piperazine
moiety. Positively-charged ammonium exerts an inductive
effect and reduces the electron donating capability of the
adjacent amino group. Consequently, replacing the
dimethylamino group with a quaternary piperazine moiety
should minimize the TICT formation.
4,5
Rhodamines have been extensively utilized in many
6
–8
super-resolution imaging studies.
Ever since (probably)
the first implementation of rhodamine spiroamide for
9
SMLM in 2007, photoswitchable rhodamines have been
developed into versatile markers for localization-based
10–12
10,13–
19–22
super-resolution imaging of nucleus,
mitochondria,
16
14
16–18
endoplasmic reticulum,
lysosomes
cells.
tubulins,
actins,
19,23
and plasma membrane24,25 in live or fixed
Our reasoning is supported by calculating vertical
ionization energies (VIE) of a series of amino moieties
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(
Figure 1b). A large VIE corresponds to a weak electron-
a significantly enhanced quantum yield (Φ = 0.93) and
increased molar extinction coefficient (ε = 8.7 × 10 M cm
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donating strength. Our results show that quaternary
piperazine moieties have significantly larger VIE than other
amino groups, indicating weak electron-donating strength.
1
) in aqueous solution, in comparison to TMR (Φ = 0.47; ε =
4
-1
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7.8 × 10 M cm ). Similar enhancement was also found in
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Lyso-RH (Φ = 0.92, ε = 8.7 × 10 M cm ), which is a
protonated form of the piperazine rhodamine (Figure 1d; SI
Section 3.3). Furthermore, in a systematical photophysical
studies, the decreased non-radiative rates of TMR in less
polar and viscous solvents indicate the probable existence
of TICT states as well as other fluorescence quenching
We next computed the potential energy surface of TMR
and MPR in water, as a function of the amino group rotation
(
faces a considerably larger energy barrier in MPR (0.50 eV)
than that in TMR (0.42 eV). Moreover, the driving energy to
enter the TICT state from the local excited (LE) state in MPR
Figures 1c, S1-S5). Our results show the TICT formation
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mechanisms (e.g., hydrogen-bonding), while the near-
(
0.15 eV) is much less than that in TMR (0.60 eV). Therefore,
unity quantum yield of Lyso-RH in water demonstrates that
quaternary piperazine substitution suppresses this TICT
state and most other nonradiative decays (SI Section 3.4).
Overall, our strategy leads to more than one-fold increment
in the ensemble brightness.
TICT formation of MPR should be less likely than that of
TMR.
Figure 1. (a) Schematic illustrations of the potential energy
profile, which leads to both emissive LE state and non-
emissive TICT state. (b) Calculated vertical ionization
energies of various amino groups in vacuo. (c) Calculated
potential energy surfaces of TMR and MPR in water. (d)
Spectroscopic properties of quaternary piperazine
fluorophores and their dimethylamino analogs.
These theoretical calculations encouraged us to
experimentally verify the quaternary piperazine strategy in
enhancing the brightness of rhodamines. Therefore, we
synthesized MPR through an effective N-alkylation to our
previously reported piperazine rhodamine (SI Section 2.2).
Our spectroscopic studies demonstrate that MPR exhibited
34
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Figure 2. (a) Carboxylic derivatives of MPR and TMR were coupled to IgG molecules. (b) Single-molecule fluorescent signals of
MPR-IgG and TMR-IgG. (c) Single-molecule fluorescence trajectory of an MPR-IgG molecule. (d) Summary of single-molecule
characteristics of MPR-IgG and TMR-IgG (n = 3). (e) Single-molecule brightness of MPR-IgG and TMR-IgG as a function of
laser power in PBS (pH = 7.4) solution. (f) Super-resolution image of microtubules immunolabeled with a primary antibody
against α-tubulin and MPR-IgG. A conventional image is overlaid on the upper side. Scale bars: 10 μm (b); 2 μm (f).
We further compared the fluorescence of MPR and TMR
at the single-molecule level. Through carboxyl groups at the
same position, both MPR (synthesis in SI Section 2.10) and
TMR were coupled to immunoglobulin G (IgG; Figure 2a),
and adsorbed on the surface of a coverslip, thus allowing
the study of their single-molecule characteristics in aqueous
conditions. As shown in Figure 2b, the single-molecule
signals of both dyes are sparsely distributed, avoiding
resolution. Moreover, the comparison of imaging results
indicates an enhanced super-resolution imaging capability
of MPR than that of TMR (Figures S22-24).
To further investigate MPR’s applicability in live cells, we
designed a bipolar probe, Mem-R (Φ = 0.64, Figure 3a;
synthesis in SI Section 2.9), to specifically label plasma
membranes. Indeed, confocal imaging confirms this
specificity of Mem-R (Figure S19).
overlapping.
A
typical single-molecule fluorescence
trajectory of MPR-IgG is depicted in Figure 2c, with the
measurement of its single-molecule brightness (photons per
single molecule per frame) and total collected photons
before photobleaching. MPR-IgG demonstrates significant
improvements of single-molecule brightness (518 ± 68),
4
total collected photons (4.07 ± 0.26 × 10 ), signal-to-noise
ratio (SNR: 8.51 ± 0.32) and localization precisions (10.08 ±
0.19 nm), compared to TMR-IgG (Figure 2d). These results
match the ensemble spectroscopic studies as well as the
quantum-chemical predictions. In addition, the enhanced
photon statistics of MPR-IgG was confirmed at a series of
conditions both in PBS and imaging enhancing buffer
(Figure 2e; Table S4), and as indicated by a simulation study
(Figure S18), MPR-IgG demonstrates an improved
localization-based super-resolution imaging potential than
TMR-IgG does (SI Section 4.5). Accordingly, these results
demonstrate that quaternary piperazine strategy effectively
enhanced the brightness of rhodamines.
Figure 3. (a) Molecular structure of Mem-R. (b) Super-
resolution image of the plasma membrane of a live HeLa
cell stained with Mem-R. The bottom side is overlaid with a
conventional image from the same region. Histogram of
single-molecule brightness (c) and localization uncertainties
(d) in (b). (e) High-density molecular tracking of Mem-R in
the marked region in (b). Trajectories were colored
according to their diffusion coefficients. Scale bars: 4 μm (b);
2 μm (e).
To investigate utilities of the quaternary piperazine
strategy in super-resolution imaging, we immunostained
microtubules in HeLa cells. Reconstructed image (Figure 2f)
reveals the twists and crosses of microtubules, exhibiting a
considerable enhancement of clarity compared to
a
conventional image. Quantitative analysis of the
reconstruction (Figure S21) estimated a 14.9 nm localization
We then performed super-resolution imaging of cell
membranes using Mem-R. Sufficiently sparse single-
36,37
uncertainty and 96.3 nm FRC (Fourier ring correlation)
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molecule signals were collected, probably resulting from the
exchangeable binding of Mem-R on the cell membrane
Movie S1; SI Section 6.4). The reconstruction image (Figures
b, S25) reveals the detailed architecture of cell membrane
in stark comparison with a blurred conventional image.
Further analysis of the reconstruction estimated an average
single-molecule brightness of 984 (Figure 3c) and an
average localization precision of 20.9 nm (Figure 3d). We
further compared its super-resolution imaging results of
membrane with Mem-TMR (an analog membrane
targetable probe based on TMR; synthesis in SI section
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2.11). The results in Figures S26-27 indicate that Mem-R
hold a significantly enhanced capability of super-resolution
imaging than Mem-TMR. We noted that the single-
molecule brightness of Mem-R was comparable to that of
DiI (a Cy3 based commercial membrane tracker; Figure
S28a), which should be ascribed to the much higher
quantum yield of Mem-R than DiI in context of the much
higher absorption coefficient of the latter probe (ε = 148
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0
00 M cm ) . Moreover, as shown in Figure 3e, the
excellent photophysical properties of Mem-R enable high-
density single-molecule track experiment, which reveals a
heterogeneous diffusion of Mem-R on the plasma
membrane. These results show that Mem-R permits super-
resolution imaging of cell membrane with high quality.
Recently, we have reported the lysosomal targetability of
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Lyso-R . As shown in Figure 4a, the probe Lyso-R is mostly
non-protonated and exhibit low fluorescence at neutral pH,
whereas its protonation (pk1/2 = 6.56; Figure S10) results in
the formation of a highly fluorescent Lyso-RH, fulfilling our
quaternary piperazine strategy. We hypothesized that this
protonation induced bright-dark transformation and the
diffusion equilibrium of the probe between cytosol and
acidic organelles would develop a desirable photoswitching
behavior beneficial for super-resolution imaging. To confirm
the targetability of Lyso-RH, we performed a diffraction-
limited confocal colocalization analysis with a lysosomal
marker protein (Lamp-1) in live HeLa cells. Lyso-RH
demonstrates a good affinity towards lysosomes (Pearson
correlation coefficient: 0.84, n = 21; Figure S20).
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Figure 4. (a) Protonation of Lyso-R generates Lyso-RH in situ in acidic lysosomes. (b) Super-resolution and (c) conventional
images of lysosomes in a live HeLa cell stained with Lyso-RH. (d) Time colored super-resolution images of lysosomes. The
white box highlights the imaging region of (b). Magnified views of typical lysosomes are presented on the right. Images (e)
and (f) were magnified views of the highlighted regions in (b) and (c). (g) Intensity distributions across the blue lines in (e) and
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(
f). Histogram of single-molecule brightness (h) and localization uncertainties (i) in (b). (j) FRC analysis37 of (b). (k) The survival
fraction of molecules during imaging of (d). Scale bars: 4 μm (o); 1 μm (b, c); 300 nm (e,f); 200 nm (d 1-5).
We performed super-resolution imaging of lysosomes in
live HeLa cells with Lyso-RH. Temporal separated signals
repeatedly appeared in lysosomes of live cells under normal
culture media, probably owing to the protonation
equilibrium and the intracellular redox system (Movie S2;
SI Section 6.4). Statistical analysis showed that the
brightness of photoswitching events of Lyso-RH (Figure
S28b) surpasses that of the commercial Lysotracker Red. The
reconstruction images revealed the true shape and size of
globular lysosomes (Figure 4b, 4e) with significantly
improved clarity than diffraction limited images (Figure 4c,
The Supporting Information is available free of charge on
the ACS Publications website.
Experimental methods, synthesis procedures, compound
characterization, Figure S1-S53 and Table S1-S5. (PDF)
Movie S1. Single-molecule signals of Mem-R on the plasma
membrane. (AVI)
Movie S2. Single-molecule signals of Lyso-RH in lysosomes.
(AVI)
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AUTHOR INFORMATION
Corresponding Author
4f). The width of the lysosome is measured to be 70.3 nm in
xiaoyi@dlut.edu.cn; (Y. Xiao)
xiaogang_liu@sutd.edu.sg; (X. Liu)
yw917@dlut.edu.cn. (W. Yang)
the super-resolution image, matching the reported values
40,41
from electron microscopy,
whereas this width is
estimated to be 421 nm in a conventional image (Figure 4g).
Quantification analysis of the reconstruction demonstrates
high spatial resolution (average localization precision: 13.9
nm, Figure 4i; overall resolution: 37.6 nm, Figure 4j),
comparable with previously reported results (Table S5).
Besides, owing to the durable photoswitching of Lyso-RH in
lysosomes even without excessive imaging enhancing
agents (Figure 4k), a long-term super-resolution imaging
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work is supported by National Natural Science
Foundation of China (Nos. 21376038, 21421005, 21576040,
21776037 and 21804016), the Singapore University of
Technology and Design (T1SRCI17126) and SUTD-MIT
(
Figure 4d) was achieved, showing heterogeneous
movements of lysosomes with high spatial resolutions
(
Figure 4d 1-5).
Finally, we explored the generalizability of the quaternary
International
computational work is conducted with support from SUTD-
MIT International Design Center and National
Supercomputing Centre (NSCC) Singapore. The fluorescent
imaging is performed with support from Chemical Analysis
and Research Center, Dalian University of Technology.
Design
Center
(IDG31800104).
The
piperazine
strategy.
Quantum-chemical
calculations
predicted that this strategy was translational to other
fluorophores, such as naphthalimide and NBD dyes (Figures
S6-S7) and subsequent experiments demonstrate an
enhancement of brightness by up to 77 times (Figure 1d).
In conclusion, we have proposed and demonstrated a
new strategy to enhance the brightness of rhodamine dyes
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