Vinyl-Fluorene Molecular Wires for Voltage Imaging with Enhanced Sensitivity and Reduced Phototoxicity

Article abstract of DOI:10.1021/jacs.1c04543

Fluorescent voltage indicators are an attractive alternative for studying the electrical activity of excitable cells; however, the development of indicators that are both highly sensitive and low in toxicity over long-term experiments remains a challenge.

Full text of DOI:10.1021/jacs.1c04543

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Vinyl-Fluorene Molecular Wires for Voltage Imaging with Enhanced
Sensitivity and Reduced Phototoxicity
Steven C. Boggess, Shivaani S. Gandhi, Brittany R. Benlian, and Evan W. Miller*
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ABSTRACT: Fluorescent voltage indicators are an attractive alternative for studying the electrical activity of excitable cells;
however, the development of indicators that are both highly sensitive and low in toxicity over long-term experiments remains a
challenge. Previously, we reported a fluorene-based voltage-sensitive fluorophore that exhibits much lower phototoxicity than
previous voltage indicators in cardiomyocyte monolayers, but suffers from low sensitivity to membrane potential changes. Here, we
report that the addition of a single vinyl spacer in the fluorene molecular wire scaffold improves the voltage sensitivity 1.5- to 3.5-fold
over fluorene-based voltage probes. Furthermore, we demonstrate the improved ability of the new vinyl-fluorene VoltageFluors to
monitor action potential kinetics in both mammalian neurons and human-induced pluripotent stem cell-derived cardiomyocytes.
Addition of the vinyl spacer between the aniline donor and fluorene monomer results in indicators that are significantly less
phototoxic in cardiomyocyte monolayers. These results demonstrate how structural modification to the voltage sensing domain have
a large effect on improving the overall properties of molecular wire-based voltage indicators.
Scheme 1. Hybrid Vinyl-Fluorene Wires for Voltage Sensing
ells expend a large proportion of their energy budget to
maintain tight control over the electrical potential across
C
the plasma membrane. In excitable cells, membrane potential
(Vmem) can change quickly, and these action potentials govern
the unique physiology of neurons and cardiomyocytes. Patch-
clamp electrophysiology is the gold-standard for measuring
electrical impulses in live cells, but is highly invasive and low
throughput.1 To complement traditional electrophysiology, we
have been developing voltage-sensitive fluorophores, or
2
VoltageFluors, that optically measure rapid changes of Vmem
.
Using fluorescence to monitor Vmem dynamics is attractive
because it circumvents many of the issues associated with
electrode-based methods. VoltageFluor (VF) indicators
increase their fluorescence in response to membrane
depolarizations due to attenuation of fluorescence quenching
by photoinduced electron transfer (PeT).3 Within the
VoltageFluor scaffold, the identity of the fluorescent reporter,
the aniline electron donor, and the molecular wire can be
modified to tune the spectral and voltage sensing properties of
the probe.4−8
Previously, we showed that 9′,9′-dimethylfluorene (fVF,
Scheme 1) can replace the phenylene-vinylene-based molec-
ular wire in the VoltageFluor scaffold (VF2.1.Cl, Scheme 1).7
Fluorene VoltageFluors (fVFs) are less phototoxic than other
voltage indicators in cardiomyocyte monolayers, making them
useful for reporting cardiac action potential kinetics over
prolonged experiments. However, fVFs are 2- to 5-fold less
sensitive to Vmem than their phenylenevinylene-based counter-
parts.7 We sought to improve the sensitivity of the fluorene
molecular wire scaffold while harnessing its reduced photo-
toxicity characteristics.
compared to the phenylene-vinylene scaffold of VF2.1.Cl (β
= 0.04 Å−1).10−12 Previous studies showed that oligo-
fluorenevinylene molecular wires, which combine aspects of
Received: April 30, 2021
Published: July 29, 2021
We hypothesize that the voltage sensitivity of fluorene
molecular wire fVF dyes is reduced due to the higher
attenuation factor (0.09 Å−1),9 or β value, of fluorenes
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fluorenes and vinyl spacers, possess a β value of 0.075 Å.13
Therefore, we hypothesized that incorporation of a vinylene
spacer into the fluorene VoltageFluor scaffold would boost
voltage sensitivity while retaining the low phototoxicity of fVF
dyes (Scheme 1). These new indicators, vinylene-fluorene
VoltageFluors (v-fVFs), represent a structural hybrid between
the two wire scaffolds reported by our laboratory.7,14
We designed indicators with vinyl spacers in two different,
regioisomeric locations: between the fluorene subunit and the
dichlorofluorescein fluorophore (2v-fVFs, Scheme 1) and
between the aniline donor and the fluorene (7v-fVFs, Scheme
1). The numbers 2 and 7 indicate the substituent position of
the vinyl group on the fluorene ring. Indicators with the 2v-fVF
substitution are accessible in two steps from previously
reported compounds (Schemes 2, S1).7 Cross-coupling using
isomers could not be separated by chromatography. Nitro-
styryl-fluorene wires are converted to anilines 11 and 12 by
reduction with SnCl2, followed by reductive amination with
formaldehyde to yield 13 and 14 (Scheme 3). Pd-catalyzed
cross-coupling with bis(pinacolato)diboron provides boronic
esters 15 and 16 for coupling with bromo-2,7-dichlorosulfo-
fluorescein. Pd-catalyzed cross-coupling of 15 or 16 with
halogenated sulfonofluorescein furnishes 7v-fVF indicators 17
(7v-fVF 1) and 18 (7v-fVF 2). As control compounds, we
synthesized indicators that lack an aniline donor (Scheme S2,
2v-fVF 0 and 7v-fVF 0).
The position of the vinyl spacer has no effect on the
absorption or emission of the xanthene fluorophore (Figure S1,
Table S1). However, relative to fVF dyes, both of the vinyl-
fluorene substitutions result in bathochromic shifts in the
molecular wire absorbance. This shift is larger for 7v-fVF
substitution (Figure S1, Table S1). The red shift of the
molecular wire absorbance is due to the increased conjugation
in the molecular wire system, which is electronically decoupled
from the xanthene chromophore in the ground state. In
agreement with a PeT mechanism for voltage sensing,
indicators lacking an aniline exhibit quantum yields two to
three times higher than the donor-containing counterparts
(Table S1).
Scheme 2. Synthesis of 2-Vinyl Fluorene VF Dyes
To test the effect of vinyl spacers on voltage sensitivity, we
recorded the fluorescence intensity of voltage-clamped
HEK293T cells stained with vinyl-fluorene VoltageFluors. 2-
Vinyl indicators (2v-fVF 1 and 2v-fVF 2) exhibit a 7.4% and
15.2% ΔF/F per 100 mV, respectively (Figure 1, Table 1).
Compared to the analogous fVF dyes,7 this modification
produces a modest 60% and 40% improvement in sensitivity
(fVF 1, 4.5%; fVF 2, 10.5% ΔF/F; Figure S2).7 The 7-vinyl
spacer improves voltage sensitivity to a greater extent; 7v-fVF 1
has a 16.5% ΔF/F (350% improvement), and 7v-fVF 2 exhibits
a 30.6% ΔF/F (290% improvement) per 100 mV change in
Pd(OAc)2 and tri-o-tolylphosphine gives vinyl-fluorene MIDA
boronate esters 1 and 2 in good yield. Suzuki−Miyaura
coupling with modified conditions15 provides 2v-fVF indica-
tors 3 (2v-fVF 1) and 4 (2v-fVF 2).
Construction of 7v-fVFs begins by converting substituted 4-
nitrobenzaldehydes to styrenes 7 and 8 by Wittig olefination
(Scheme 3). Pd-catalyzed cross-coupling with 2-bromo-7-iodo-
9,9-dimethyl-9H-fluorene yields nitrostyryl-fluorene wires 9
and 10 as orange solids. Starting from the 4-nitrostyrenes
(Scheme 3) was essential: use of the corresponding 4-
aminostyrenes in the cross-coupling reaction yields the desired
linear isomer in a 2:1 ratio with the branched isomer. The
V
mem. Indicators lacking an aniline donor (2v-fVF 0 and 7v-fVF
0) were not sensitive to changes in Vmem, consistent with a
PeT-based mechanism of voltage sensing (Figure S2g−i, p−r,
Table 1)
In rat hippocampal neurons, all of the new v-fVF indicators
have higher ΔF/F and signal-to-noise (SNR) per action
potential than fVF 2, the best of the fluorene-only fVF series.7
Of the new indicators, 7v-fVF 1 (17) has the highest SNR per
action potential, at 18:1, while 7v-fVF 2 (18) has the highest
Scheme 3. Synthesis of 7-Vinyl Fluorene VF Dyes
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ΔF/F, at 9% per action potential (Table S2, Figure S3).
However, under identical conditions, phenylenevinylene-based
VF2.1.Cl retains the best SNR per action potential, at 35:1.
In human-induced pluripotent stem cell-derived cardiomyo-
cyte (hiPSC-CM) monolayers, all of the new v-fVF indicators
report cardiac action potential kinetics with high SNR. Vinyl-
fVF indicators have larger SNR per action potential than either
fVF 2 or VF2.1.Cl, albeit with a lower ΔF/F (Table S2, Figure
2, Figure S4). Previously, we found that substituting the
phenylene-vinylene molecular wire (VF2.1.Cl) with a fluorene
monomer (fVF) in the VF scaffold substantially reduces
Figure 1. Characterization of vinyl-fluorene VoltageFluors in
HEK293T cells. Live cell fluorescence images of (a) 2v-fVF 1, (c)
2v-fVF 2, (e) 7v-fVF 1, and (g) 7v-fVF 2 loaded at 0.5 μM in
HEK293T cells. Scale bar is 20 μm. Plots of ΔF/F versus membrane
potential (millivolts) in voltage-clamped HEK293T cells for (b) 2v-
fVF 1, (d) 2v-fVF 2, (f) 7v-fVF1, and (h) 7v-fVF 2. The red line is the
line of best fit; error bars are SEM for a minimum of 3 independent
determinations.
Table 1. Voltage Sensitivity of v-fVF Dyes
a
b
entry
brightness
%ΔF/F
SNR
2v-fVF 1 (3)
7v-fVF 1 (17)
2v-fVF 2 (4)
7v-fVF 2 (18)
2v-fVF 0 (20)
7v-fVF 0 (23)
1.6
1.0
1.5
0.4
5.8
4.4
7.4 0.3
16.5 0.2
15.2 0.2
30.6 0.7
−0.3 0.01
−0.3 0.01
41 10.5
67 8.5
49 3.4
37 5.6
1.7 0.2
Figure 2. Performance of vinyl-fluorene VoltageFluors in hiPSC-CM
monolayers. Representative fluorescence micrograph of hiPSC-CMs
loaded with 0.5 μM of either (a) 2v-fVF 1 (3), (b) 2v-fVF 2 (4), (c)
7v-fVF 1 (17), or (d) 7v-fVF 2 (18). Scale bar is 50 μm. (e) Plot of
SNR per action potential for each indicator. Data are mean SEM.
(f) Plot of ΔF/F per action potential in hiPSC-CMs for each
indicator. Data are mean SEM from n = 15 recordings. (g−j) (Left)
Plots of fluorescence intensity vs time for either (g) 2v-fVF 1 (3), (h)
2v-fVF 2 (4), (i) 7v-fVF 1 (17), or (j) 7v-fVF 2 (18). (Right)
Concatenated action potentials.
0.8 0.2
a
b
Measured in HEK 293T cells, relative to brightness of 17. Per 100
mV, recorded at a 0.5 kHz optical sampling rate.
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phototoxicity in cardiomyocytes, allowing up to 10 min of
continuous illumination without disrupting the waveform of
action potentials.7 Thus, we were curious whether the new
vinyl-fluorene hybrids, with their enhanced voltage sensitivity
compared to fluorene-only indicators, would behave more like
fVF 2 or VF2.1.Cl in terms of phototoxicity. We continuously
illuminated hiPSC-CM monolayers stained with indicators,
making 10 second recordings every minute to monitor action
potential morphology. Indicators with 2v-fVF substitution
appear extremely phototoxic: action potential morphology
changes after just 1 (2v-fVF 1, Figure 3a) to 3 (2v-fVF 2,
Figure 3b) minutes of illumination. Shortly after, the hiPSC-
CMs stopped beating (Figure 3a,b). This degree of toxicity was
similar to VF2.1.Cl (Figure S5).
fVF 2 throughout the experimentsabove 100:1 after
illuminating 7v-fVF 2 for 9 minpermitting long-term
recordings of electrical activity in cardiomyocyte monolayers
(Figure S5).
The precise molecular mechanisms governing the reduced
phototoxicity of the 7v-fVF scaffold compared to VF2.1.Cl
remain elusive, but experimental evidence points to the
involvement of reactive oxygen species (ROS). One hypothesis
is that differential dye accumulation in cellular membranes
leads to a range of effective dye concentrations and different
levels of ROS production and toxicity. However, v-fVF dyes
show only small differences in cellular brightness (Figure S6,
Table 1), and fluorescence intensity alone cannot be used as a
quantitative measure of dye concentration.8 Rates of photo-
bleaching in cell membranes are complex (Figure S7),
consistent with studies of fluorescein photobleaching, which
indicate multiple photobleach pathways, including dye−oxygen
and dye−dye interactions, and show that bleach rates depend
strongly on whether the dye is in solution or localized on a
surface (such as a plasma membrane).16 7v-fVF indicators have
slower rates of photobleaching and an initial plateau phase
compared to other dyes (in HEK293T cells, Figures S7 and
S8). Under low O2 environments, the initial photobleach rate
for VF2.1.Cl, 2v-fVF, and 7v-fVF increases, while the rate for
fVF 2the only dye lacking a vinyl spacerremains
unchanged, but large (Figure S8). Origins of phototoxicity
seem unlikely to be photothermal because irradiation of VF
dyes does not increase solution temperature (Figure S9).
Instead, phototoxicity may arise from ROS production. Both
2v-fVF and 7v-fVF induce lower levels of ROS than VF2.1.Cl,
whether measured after illumination (using the nonspecific
ROS indicator dihydroethidium,17 Figure S10) or during
illumination (using lipid-associated BODIPY 581/591 C11,18
Figure S11). Currently, we lack the resolution to conclusively
say whether cellular toxicity is mediated by ROS produced by
cells in response to illumination in the presence of VF dyes
(Figure S10) or whether it is ROS produced directly by VF
dyes within the lipid membrane (Figure S11) that drive
toxicity. Studies are underway to investigate these elementary
steps in higher detail.
In summary, we developed four new voltage indicators. All
four vinylene-fluorene VoltageFluor indicators show higher
voltage sensitivity than their cognate fluorene VoltageFluor
dyes. We found that a vinyl spacer between the electron-
donating aniline and the fluorene in the molecular wire (7v-
fVFs) increased sensitivity by about 3-fold compared to fVF
dyes. For 7v-fVF 2 (18), voltage sensitivity was higher (30%
ΔF/F) than the sensitivity of VF2.1.Cl (25% ΔF/F). More
importantly, 7v-fVF indicators remain less phototoxic in
cardiomyocytes relative to VF2.1.Cl, while maintaining higher
SNR throughout extended recordings. Vinyl-fluorene Voltage-
Fluors represent an improvement over the fluorene Voltage-
Fluor scaffold and are an attractive alternative to the traditional
VF2.1.Cl for prolonged recordings of electrical activity in cells.
Figure 3. Extended imaging with vinyl-fluorene VoltageFluors in
hiPSC-iCM monolayers under constant illumination. Monolayers
were stained with v-fVF indicators and illuminated constantly. Every
minute, 10 s optical recordings of voltage dynamics were acquired.
Representative traces are shown for (a) 2v-fVF 1 (3), (b) 2v-fVF 2
(4), (c) 7v-fVF 1 (17), and (d) 7v-fVF 2 (18). (e) Plot of the ratio of
cAPD90/cAPD30, which is the action potential duration at 90% and
30% of the repolarization, corrected for beat rate by Fridericia’s
formula. Initial recordings start with a ratio between 1.2 and 1.4,
regardless of the indicator used. Increases in the ratio indicate a
change in action potential morphology. (f) Plot of SNR per action
potential vs total minutes of illumination.
ASSOCIATED CONTENT
■
sı
* Supporting Information
However, indicators with the vinyl spacer at the 7-position
are much less phototoxic than 2v-fVF or VF2.1.Cl (Figure 3c−
f). With either 7v-fVF 1 (Figure 3c) or 7v-fVF 2 (Figure 3d),
recordings can be made for up to 10 min without changes to
action potential shape (Figure 3, Figure S5). Unlike fluorene-
only fVF 2, the SNR remains high for both 7v-fVF 1 and 7v-
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c04543.
Supplementary data, including supporting figures,
spectra, procedures, and analysis (PDF)
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(11) Eng, M. P.; Albinsson, B. Non-exponential distance dependence
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AUTHOR INFORMATION
Corresponding Author
Evan W. Miller − Department of Chemistry, Department of
Molecular & Cell Biology, and Helen Wills Neuroscience
Institute, University of California, Berkeley, California 94720,
United States; orcid.org/0000-0002-6556-7679;
Email: evanwmiller@berkeley.edu
■
̊
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B.; Bauer, T.; Clark, T.; Guldi, D. M.; Martín, N. Vinyl spacers
tuning electron transfer through fluorene-based molecular wires.
Energy Environ. Sci. 2011, 4 (3), 765−771.
Authors
(14) Kulkarni, R. U.; Yin, H.; Pourmandi, N.; James, F.; Adil, M. M.;
Schaffer, D. V.; Wang, Y.; Miller, E. W. A Rationally Designed,
General Strategy for Membrane Orientation of Photoinduced
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2017, 12 (2), 407−413.
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Photobleaching kinetics of fluorescein in quantitative fluorescence
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Steven C. Boggess − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Shivaani S. Gandhi − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Brittany R. Benlian − Department of Molecular & Cell
Biology, University of California, Berkeley, California 94720,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c04543
(17) Zielonka, J.; Kalyanaraman, B. Hydroethidine- and MitoSOX-
derived red fluorescence is not a reliable indicator of intracellular
superoxide formation: Another inconvenient truth. Free Radical Biol.
Med. 2010, 48 (8), 983−1001.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH for support of this research
(R35GM119855). S.C.B. and B.R.B. were supported in part
by a grant from the NIH (T32GM066698). We thank Dr.
Hasan Celik at the UC Berkeley NMR Facility, Dr. Jeff Pelton
at the Central California 900 MHz NMR Facility, and Dr. Ulla
Andersen at the UC Berkeley/QB3 Mass Spectrometry Facility
for expert technical assistance.
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