BODIPY Fluorophores for Membrane Potential Imaging

Article abstract of DOI:10.1021/jacs.9b05912

Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based, voltage-sensitive fluorophores (VF). The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water-soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.

Full text of DOI:10.1021/jacs.9b05912

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Article
BODIPY Fluorophores for Membrane Potential Imaging
Jenna M. Franke, Benjamin K Raliski, Steven C. Boggess, Divya V. Natesan, Evan T.
Koretsky, Patrick Zhang, Rishikesh U Kulkarni, Parker Edward Deal, and Evan W. Miller
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05912 • Publication Date (Web): 24 Jul 2019
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Jenna M. Franke,‡ Benjamin K. Raliski,‡ Steven C. Boggess,‡ Divya V. Natesan,‡ Evan T. Koretsky,‡
Patrick Zhang,‡ Rishikesh U. Kulkarni,‡ Parker E. Deal‡ and Evan W. Miller‡§†*
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Departments of ‡Chemistry and §Molecular & Cell Biology and †Helen Wills Neuroscience Institute. University of Califor-
nia, Berkeley, California 94720, United States.
ABSTRACT: Fluorophores based on the BODIPY scaffold are
prized for their tunable excitation and emission profiles, mild synthe-
ses, and biological compatibility. Improving the water-solubility of
BODIPY dyes remains an outstanding challenge. The development
of water-soluble BODIPY dyes usually involves direct modification
of the BODIPY fluorophore core with ionizable groups or substitu-
tion at the boron center. While these strategies are effective for the
generation of water-soluble fluorophores, they are challenging to im-
plement when developing BODIPY-based indicators: direct modifi-
cation of BODIPY core can disrupt the electronics of the dye, com-
plicating the design of functional indicators; and substitution at the
boron center often renders the resultant BODIPY incompatible with
the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated
aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new
sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To high-
light the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based voltage-sensitive
fluorophores. The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional
BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action
potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in
the context of a water soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY
dyes for functional indicators.
Introduction
Scheme 1. Design of H2O-soluble BODIPYs
Synthetic chemistry has long been a source of colorful com-
pounds1-3 whose ability to absorb light enable applications in
far-ranging fields. Fluorescent dyes find wide-spread use in the
modern research laboratory where features such as visible exci-
tation and emission profiles, large molecular brightness values,
and photostability are highly prized, along with biologically-
compatible properties like water-solubility. Since the late 19th
century, xanthene dyes like fluoresceins4 and rhodamines5-6 of-
fered a fertile source of inspiration as scaffolds for biologically-
useful dyes and indicators.7-9 More recently, BODIPY, or 4,4-
difluoro-4-bora-3a,4a,-diaza-s-indacene, (Scheme 1) dyes have
emerged as a versatile complement to xanthene dyes. Owing to
the relatively mild reaction conditions for the generation of
BODIPY fluorophores,10 a number of flexible synthetic routes
afford the opportunity to install a range of substituents directly
to the BODIPY core, tuning both the color and electronic prop-
erties of BODIPY dyes.
species,14 electron transfer reactions,30 and membrane viscos-
ity.31 Because of the broad tunability of BODIPY-based scaf-
folds, we thought these fluorophores would make an excellent
choice for incorporation into a molecular wire-based, photo-in-
duced electron transfer (PeT) membrane potential sensing
framework.32 Previous work in our lab showed that tuning the
relative electron affinities between a fluorescein-based reporter
and electronically-orthogonal phenylenevinylene molecular
wire voltage-sensing domain profoundly altered the voltage
sensitivities of fluorescein based dyes. However, the limited
synthetic scope of sulfonated fluorescein only allowed access to
a narrow range of substituents (H, F, Cl, Me).33
Since the initial report of BODIPY in 1968,11 a proliferation
of synthetic methods10, 12-13 and conceptual understanding14-15
enabled the application of BODIPYs as indicators for a number
of important, biologically-relevant analytes and properties,16-17
22
including pH,18-19 cations like Na+,20-21 K+,20,
Mg2+,23 and
Here, we introduce new, water-soluble sulfonated BODIPYs
with substituents ranging from highly electron donating (R =
Et) to
Ca2+;24-25 transition metals;26-28 reactive oxygen29 and nitrogen
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Scheme 2. Synthesis of BODIPY VoltageFluor dyes with Et, H and CN at the 2,6-positions
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withdrawing (R = CN). We incorporate the new, sulfonated
BODIPYs into a molecular wire voltage-sensing scaffold to
provide the first examples of PeT-based voltage-sensitive
BODIPYs. The most sensitive of these dyes displays a 48%
ΔF/F per 100 mV in HEK cells, and two others possess ≥24%
ΔF/F, making them useful for voltage sensing applications in
both neurons and cardiomyocytes.
benzaldehyde precursor, 9 (Scheme 2, and related para-isomer,
1, Scheme 2), was completely insoluble in CH2Cl2 and toluene,
the most commonly used solvents for BODIPY condensations.
10, 14, 20, 31, 42-45 We screened polar solvents for the TFA-catalyzed
condensation of aldehyde 9 (or 1) with kryptopyrrole 2
(Scheme 2). DMF gave the best conversion to the dipyrro-
methane. Oxidation with DDQ to form the corresponding di-
pyrromethene followed by BF2 chelation with boron trifluoride
diethyl etherate (BF3·OEt2) in CH2Cl2 solvent gave ortho-sul-
fonated BODIPY 3 (Br para to BODIPY, Scheme 2) in 49%
yield and 11 (Br meta to BODIPY, Scheme 2) in 33% yield.
Design of water soluble BODIPYs
We prepared a total of 13 BODIPY-based Voltage-sensitive
Fluorophores, or BODIPY-VF dyes. All of the BODIPY com-
pounds feature a common ortho-sulfonic acid substituted meso
aromatic ring (8-position, Scheme 1) and substitution patterns
at the 2,6 positions that include hydrogen, ethyl, carboxylate,
amide, and cyano functionalities (Scheme 1). Our initial at-
tempts to access BODIPY-based VoltageFluor indicators cen-
tered around the development of water-soluble tetramethyl, di-
ethyl BODIPY fluorophores. Ionizable groups, such as sul-
fonates or carboxylates, are essential for the proper orientation
of VF-type dyes in cellular membranes.34-35 We first sought to
introduce water-solubilizing groups centered on substitution at
boron.36-38 However, in our hands, these modifications proved
incompatible with the Pd-catalyzed Heck coupling for installa-
tion of voltage-sensing phenylenevinylene molecular wires.
Functionalization of the 2 and 6 positions of the BODIPY core
offered a route to the installation of water-solubilizing groups
like sulfonates39-40 or carboxylates,41 but direct functionaliza-
tion of the BODIPY core can profoundly alter redox properties,
confounding the tuning of fluorophore redox potential15, 33 with
installation of water solubilizing groups. One solution is to in-
clude a sulfonate on the meso aromatic ring (Scheme 1), which
we hypothesized would improve solubility, be generalizable
across a range of 2,6-substitution patterns on the BODIPY core,
and aid in the proper orientation within cellular plasma mem-
branes.
A Pd-catalyzed Heck coupling between BODIPY 3 and sub-
stituted styrenes 4 and 5 gave two different 2,6-diethyl, para
molecular wire BODIPY VoltageFluors: EtpH (6) and EtpOMe
(7) in 92 and 25% isolated yield, respectively (Scheme 2). The
naming convention represents the ethyl groups at the 2,6-posi-
tions, molecular wire para from the fluorophore, and the iden-
tity of the R1 substituent. Derivatives with the molecular wire
meta from the fluorophore were prepared via a similar route
from BODIPY 11 (Scheme 2; EtmH 15, 26% yield, and Et-
mOMe 16, 29%). Tetramethyl BODIPY VoltageFluors 17-19
(R = H) were prepared first by reacting 2,4-dimethyl-1H-pyr-
role 10 with sulfonated aldehyde 9, resulting in a 38% yield of
ortho-sulfonated tetramethyl BODIPY 12. Heck coupling with
substituted styrene 4, 13, or 14 then gave TMmH (17), TMmMe
(18), and TMmOMe (19) in 35-62% yield after silica gel chro-
matography.
Synthesis of CN-BODIPY VoltageFluor
Access to electron-withdrawing BODIPY derivatives pro-
vide a useful counterpoint to H- and ethyl-substituted BODIPYs
1
and may produce lower levels of reactive O2 than more elec-
tron-rich derivatives.41 Synthesis of cyano VoltageFluor deriv-
ative 22 was more challenging than either H- or Et-substituted
BODIPY VoltageFluors. Because of the poor nucleophilicity of
2,4-dimethyl-1H-pyrrole-3-carbonitrile (20), no reaction with
sulfonated benzaldehyde 9 was observed unless heated to 60 °C.
The heated condensation resulted in only an 8% isolated yield
of 21. Switching the solvent to a 2:3 DMF:CH2Cl2 mixture and
Synthesis of H- and Et-BODIPY VoltageFluors
Owing to the commercial availability of the 3-ethyl-2,4-di-
methyl-1H-pyrrole precursors (kryptopyrrole), we first synthe-
sized BODIPY 3 and 11 (Scheme 2) for use in subsequent cou-
pling with phenylenevinylene molecular wires. The sulfonated
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Scheme 3. Synthesis of BODIPY VoltageFluor dyes with CO2H and CONHR at the 2,6-positions.
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adding an excess of TFA (100 µL, 6 equiv.) allowed the syn-
thesis to proceed at room temperature and increased the isolated
yield to 29% (Scheme 2).
Benzyl ester protected pyrrole 23 is less nucleophilic than its
carboxylic acid precursor. We performed the BODIPY conden-
sation in 2:3 DMF:CH2Cl2, providing benzyl-protected
BODIPY 24 in a 61% isolated yield (Scheme 3). Benzyl-pro-
tected BODIPY 24 proceeds cleanly through Heck coupling,
even in the presence of NEt3. Benzyl-protected intermediates 26
and 27 were isolated in a 30 and 43% yield following column
chromatography. Cleavage of the benzyl groups with Pd/C un-
der hydrogen atmosphere also reduced one of the alkenes of the
molecular wire, evidenced by a mass 2 m/z higher than the de-
sired product (Figure S1) and increased brightness of the re-
sulting dye. A Birkofer reduction47-48 with Pd(OAc)2, Et3SiH,
NEt3 in CH2Cl2 at room temperature gave the cleanest conver-
sion to the free carboxylate product with minimal over-reduc-
tion of the alkenes of the molecular wire. BODIPY VF dyes 29
and 30 were isolated in 31 and 14% yield after pTLC.
BODIPY 21 is less stable than BODIPYs 11 and 12, possibly
due to the lower effective charges on the dipyrromethene nitro-
gen atoms.46 When subjected to the Pd-catalyzed Heck coupling
conditions that afforded previous BODIPY VF dyes, BODIPY
21 decomposed before conversion to product. Lowering the re-
action temperature from 100 °C to 70 °C did not prevent de-
composition. By exposing BODIPY 21 to Heck reaction condi-
tions and systematically removing single reaction components,
we determined that the presence of trimethylamine (NEt3) was
initiating decomposition of BODIPY 21. Replacing NEt3 with
inorganic bases (Cs2CO3, K2CO3) or bulky amine bases (1,8-
bis(dimethylamino)naphthalene) resulted in scant improvement
in conversion; decomposition of 21 remained a problem. To cir-
cumvent the sensitivity of BODIPY 21, we attempted a base-
free Heck coupling, relying only on the substituted aniline of
styrene reactant 4 to buffer generated HBr. The resulting Heck
coupling was low yielding (6%), but provided sufficient 22 to
purify and characterize (Scheme 2).
BODIPY VoltageFluors 35 and 36 were synthesized via
Heck coupling between styrenes 4 or 13 and BODIPY 34,
which was accessed in 82% yield from a HATU-mediated am-
ide bond formation between BODIPY 32 and glycine methyl
Synthesis of dicarboxy- and diamido-BODIPY VoltageFlu-
ors
The 2,6-dicarboxy VoltageFluor series was synthesized via
two different routes. Initially, BODIPY 32 was synthesized in a
49% yield from aldehyde 9 and 2,4-dimethylpyrrole-3-carbox-
ylic acid 31 (Scheme 3), then subjected to the same base-free
Heck coupling conditions as the BODIPY 21, giving the 2,6-
dicarboxylic acid VoltageFluor, 28 in a 6% yield after prepara-
tive thin layer chromatography (pTLC). Subsequent Heck cou-
plings with unprotected BODIPY 32 gave inconsistent results:
either unmodified starting material or decomposition. We sus-
pected the carboxylates could be chelating the palladium cata-
lyst and decided to switch to a protecting group approach, which
would likely improve the Heck coupling and allow for more
facile purification of intermediates by normal phase chromatog-
raphy.
Figure 1. Plots of normalized a) absorption and b) emission of sul-
fonated BODIPY fluorophores 3, 11, 12, 32, 34, and 21. Spectra
were acquired in PBS pH 7.4. Dye concentration is 1 μM.
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Table 1. Spectral properties of sulfonated BODIPYs
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2
3
ε
R
λmax abs
λmax em
ϕfl
(M-1 cm-1)
4
5
6
7
8
9
3
Et
530
530
503
517
544
545
515
532
42000
44000
57000
56000
0.72
0.70
0.99
0.95
11
12
32
Et
H
CO2H
CONHCH2
CO2Me
34
21
507
502
519
517
76000
38000
0.92
0.93
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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CN
All spectral properties acquired in PBS pH 7.4
ester 33. Like benzyl-protected BODIPY 24, the amide-sub-
stituted BODIPY withstands the presence of NEt3 in the Pd-cat-
alyzed cross-coupling, which returns 35 and 36 in 21% and 34%
isolated yields, respectively (Scheme 3).
Spectroscopic characterization of sulfonated BODIPYs
The absorption and the emission of BODIPY fluorophores
(Figure 1, Table 1) and VoltageFluors (Figure 2a, Figure S2,
Table 2) varied with the 2,6-substituents. Consistent with a
Dewar formalism,49-51 electron-withdrawing groups at the 2,6-
positions result in a hypsochromatic shift (λmax = 502 nm for
BODIPY 21) and electron donating groups like Et (BODIPY 3
and 11) yield bathochromic shifts (λmax = 530 nm). Emission
trends mirror the absorption profiles, with the electron-rich
BODIPYs 3 and 11 emitting around 544 nm, and BODIPY 21,
the most electron-poor, emitting at 517 nm. The absorption and
emission profiles of the complete BODIPY VF dyes (acquired
in ethanol to improve solubility of the complete BODIPY VF
dyes) closely match the spectra of the parent BODIPY fluoro-
phores, with absorption profiles centered at 502 to 528 nm and
the phenylene vinylene molecular wire absorbing near 400 nm
(Figure 2a, Figure S2, and Table 2). The ortho-sulfonated
BODIPY fluorophores have impressive fluorescence quantum
yields (ϕfl) of 0.70—0.99 (Table 1), but after the addition of the
phenylene vinylene molecular wire the quantum yields drop
dramatically, supporting the presence of PeT within the com-
pounds (Table 2). The absorption spectra of BODIPY VF dyes
17, 18, and 19 closely match the excitation spectra (Figure S3a-
c). The BODIPY region of the absorbance spectra of 17-19 does
not change from pH 2.5 to 10 (Figure S3d-f). However, the
emission of 17-19 increases up to 20-fold at pH 2.5, consistent
with a PeT-based mechanism of fluorescence enhancement
(Figure S3g-i).
Figure 2. Spectroscopic, cellular, and functional characterization
of BODIPY VF 19. a) Plot of normalized absorbance and emission
intensity for BODIPY VF 19 (1 μM, ethanol). Excitation is pro-
vided at 470 nm. b) Widefield fluorescence micrograph of HEK
cells stained with BODIPY VF 19 (1 μM). c) Plot of fractional
change in fluorescence of BODIPY VF 19 (ΔF/F) vs. time for 100
ms hyper- and depolarizing steps (±100 mV in 20 mV increments)
from a holding potential of -60 mV in a single HEK cell under
whole-cell voltage-clamp mode. d) Plot of fractional change in flu-
orescence (ΔF/F) vs final membrane potential. Data represent the
mean ΔF/F, ±S.E.M. for n = 3 separate cells. Grey line is the line
of best fit.
cell voltage-clamp electrophysiology in tandem with epiflu-
orescence microscopy (Figure 2c,d Figure S4c,d-S9c,d). We
stepped the membrane potential of a single HEK cell stained
with 2 µM BODIPY VF from a holding potential of -60 mV to
±100 mV while recording dye fluorescence intensity. BODIPY
VF dyes 6, 7, 15, and 16 demonstrate little to no voltage sensi-
tivity. BODIPY VF dyes 6 and 7, with a para molecular wire
configuration, show no voltage sensitivity (Figure S4c), while
BODIPY VF dyes 15 and EtmOMe 16 display modest voltage
sensitivities of 1.5 and 5 % ΔF/F per 100 mV (Figure S5c,d and
Table 2).
We hypothesized replacing the 2,6-diethyl BODIPY with
progressively more electron-poor BODIPYs would increase
PeT and therefore increase % ΔF/F. Gratifyingly, we see a 67%
increase in voltage sensitivity from 16 to 17, from 1.5 to 2.5 %
ΔF/F (Figure S6c,d and Table 2). Strengthening the electron-
donating ability of the aniline by addition of a methyl or meth-
oxy group increased the voltage-sensitivity to 6.2 % for 18 and
33 % ΔF/F for 19 (Figure 2).
Cellular performance of BODIPY VF Dyes
All BODIPY VF dyes localize to cell membranes (Figure 2b,
Figure S4a,b-S9a,b) and display different cellular brightness
(Table 2, Figure S10a). Despite having the highest ϕfl, 6 was
one of the dimmest dyes in cells (relative brightness in cells =
0.4, compared to 19), likely due to its poor solubility in aqueous
buffer even in the presence of detergent (Table 2). On the other
hand, BODIPY VF dyes 28 – 30 possessed the largest cellular
brightness (relative brightness up to 12x, Table 2, Figure
S10a). We speculate that the increased anionic character of
BODIPY VFs 28 – 30, with three negative charges, improves
the water solubility of the dyes, enabling more efficient delivery
to cellular membranes.
More electron-deficient BODIPY VF 22 displayed extremely
low cellular brightness (Table 2, Figure S7b, S10a) and re-
quired increasing both illumination intensity and camera expo-
sure time in order to obtain a reasonable estimate of its voltage
sensitivity, which was low: 3.8 % ΔF/F per 100 mV (Table 2,
Figure S7c,d). While BODIPY VF 22 was slightly more volt-
age sensitive than its analogous precursors, 15 and 17, its ex-
tremely low cellular brightness prohibited further use as a volt-
age-sensitive dye in cells.
After confirming BODIPY VF dyes localize to the cell mem-
brane, we next investigated their voltage sensitivity using whole
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Table 2. Properties of BODIPY VoltageFluor (VF) dyes
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3
4
5
6
7
8
a
Name
R
R1
H
isomer λmax absa
λmax ema ϕfl
% ΔF/Fb,c
Cell brightnessc,d
6
EtpH
Et
para
528
527
528
527
503
504
504
503
503
509
508
509
502
541
541
541
541
518
517
512
516
517
522
521
521
519
0.14
0.07
0.15
0.05
0.11
0.07
0.05
0.07
0.03
0.06
0.06
0.03
0.08
0
0.43 ± 0.02f
0.76 ± 0.03f
4.4 ± 0.3f
0.60 ± 0.03f
0.62 ± 0.08
1.5 ± 0.2
7
EtpOMe
Et
OMe para
meta
OMe meta
0
15
16
17
18
19
28
29
30
35
36
22
EtmH
Et
H
1.8 ± 0.1
5.4 ± 0.6
2.5 ± 0.1
6.2 ± 0.4
33 ± 0.7
4.4 ± 0.2
9.9 ± 0.4
24 ± 0.5
48 ± 2
EtmOMe
TMmH
Et
H
H
meta
meta
9
TMmMe
TMmOMe
carboxymH
carboxymMe
carboxymOMe
amidemH
amidemMe
cyanomH
H
Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
OMe meta
1.0 ± 0.1
COOH
H
meta
meta
12 ± 2
COOH
Me
5.1 ± 0.9
COOH
OMe meta
7.1 ± 0.8
CONHCH2CO2Me
H
meta
meta
meta
1.0 ± 0.1
CONHCH2CO2Me Me
CN
5.1 ± 0.4
3.8e
1.0 ± 0.1
H
0.34 ± 0.002
a Determined in ethanol. b Per 100 mV depolarization. c Determined in HEK cells. d Relative to 19, set to a relative brightness of 1.0. e
Increased exposure time and light intensity required to make measurement. f Pluronic F-127 (0.01%) used during loading.
Figure 3. Voltage imaging in mammalian neurons with BODIPY VF 30. a) Transmitted light and b) widefield epifluorescence image of
cultured rat hippocampal neurons stained with 500 nM BODIPY VF 30. Scale bar is 20 μm. c) Widefield epifluorescence image of neurons
stained with 1 μM BODIPY VF 30 and imaged at 500 Hz. Image is a single frame from this high-speed acquisition. Scale bar is 20 μm. d)
Plot of fractional change in fluorescence (ΔF/F) for the cells identified in panel (c) or for the entire field of view (FOV).
We then evaluated the 2,6-dicarboxy and diamido BODIPY
series, hoping to find an electronic “sweet spot” between the
tetramethyl and cyano series. The BODIPY VF dyes 28, 29, and
30 had voltage sensitivities of 4.4%, 9.9%, and 24% ΔF/F per
100 mV, respectively (Figure S8c,d and Table 2). While dicar-
boxy BODIPY VF dyes display a similar range of voltage sen-
sitivities to their tetramethyl precursors, the most striking qual-
ity of the dicarboxy BODIPY VF dyes was their cellular bright-
ness—they were 5-12x brighter compared to the cellular fluo-
rescence intensity of 19 (Table 2, all cellular brightness values
normalized to 19). The in vitro fluorescence quantum yields of
the carboxy BODIPY VF dyes are slightly lower than the tetra-
methyl BODIPY VF dyes, so this increase in brightness is likely
due to improved hydrophilicity and cell loading efficiency. We
found that amide-substituted BODIPY VF 35 (Figure S9c,d)
possesses voltage sensitivity 10x greater than the corresponding
28, with a fractional sensitivity of 48% ΔF/F per 100 mV in
HEK cells (compared to 4.4% for 28). Introduction of a more
electron-rich molecular wire (methyl substitution) results in a
loss of voltage sensitivity for 36, which displays only nominal
voltage sensitivity (5.1% ΔF/F per 100 mV).
neurons and human induced pluripotent stem cell-derived car-
diomyocytes (hiPSC-CMs). Three BODIPY VoltageFluors
stood out as good candidates for functional imaging: 19 and 35
because of their high ΔF/F (33 and 48%, respectively), and 30
because of its combination of cellular brightness (7x brighter
than 19 and 35) and good sensitivity (24% ΔF/F).
We evaluated the photostability of these BODIPY VF dyes
in HEK cells. Based on previous reports,41 we predicted photo-
stability would decrease from 35 > 19 > 30. Indeed, we find
BODIPY VF 35 to be the most photostable in HEK cells, main-
taining near 100% fluorescence after 6 min of constant illumi-
nation, although with some photobrightening (Figure S10b).
BODIPY VF 19 displays photostability comparable to fluores-
cein-based VF2.1.Cl32 (Figure S10b). Carboxy-substituted
BODIPY 30 bleaches the most rapidly, dropping to about 20%
of original fluorescence values after 2 minutes (Figure S10b).
However, because of the high starting brightness of BODIPY
VF 30, this indicator retains its utility for functional voltage im-
aging.
In cultured rat hippocampal neurons stained with BODIPY
VFs, both 19 and 35 were too dim to capture evoked neuronal
action potentials with sufficient signal-to-noise. BODIPY VF
30 displayed bright, membrane-localized staining in neurons
isolated from rat hippocampi (Figure 3a and b). BODIPY VF
Functional Imaging in Neurons and Cardiomyocytes
We evaluated the ability of BODIPY VF dyes to report on
voltage dynamics in electrically excitable cells: mammalian
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Figure 4. Voltage imaging in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with BODIPY VF 19. a) Wide-
field, epifluorescence micrograph of hiPSC-CMs stained with 500 nM BODIPY VF 19. Scale bar is 50 µm. b) Single frame of a movie
collected at 200 Hz for functional imaging of hiPSC-CM spontaneous action potentials. Scale bar is 50 µm. c) Trace of mean pixel intensity
(arbitrary fluorescence units, a.u.) from full region of interest (ROI) in panel (b) plotted vs time during 10 second acquisition, corrected for
photobleach. d) Averaged action potential trace (black) from three 10 second recordings from 3 separate ROIs over individual AP events
from each recording (blue).
30 responded to electrically-evoked neuronal action poten-
tials (Figure 3c and d), which could be detected when analyz-
ing single cell regions of interest (ROIs) or when viewing the
entire field of neurons.
imaging in cardiomyocytes or neurons, where they can each re-
port on action potential dynamics in single trials.
The voltage sensitivity of the BODIPY VF dyes correlates
with the electron-withdrawing character of the 2,6-substitution
pattern in the BODIPY fluorophore. More electron-withdraw-
ing substituents increase voltage sensitivity in the order of -Et
< -H < -CO2H < -CONHR > -CN. The extremely electron-with-
drawing character of nitrile substitution makes for a poorly sen-
sitive BODIPY VF. We find that calculated values of HOMO
energies (Figure S13) for the BODIPY fluorophores—lacking
the molecular wire—correlate extremely well with either meta
or para Hammett constants (σm or σp),55 validating the use of
tabulated Hammett constants for analysis of the relative elec-
tron density of a particular BODIPY fluorophore (Figure S14a
and b). Correlation between calculated HOMO energies and σm
or σp values is best when evaluating neutral BODIPYs (Et, H,
CONHR, or CN), with correlation coefficients (R2) >0.99 for
both σm and σp compared to HOMO. If carboxy-substituted
BODIPYs are included, the correlation (R2) between HOMO
level and Hammett parameter drops to 0.92 (σm) and 0.78 (σp)
(Figure S14a and b).
We also evaluated the performance of BODIPY VF dyes 19,
30, and 35 in human induced pluripotent stem cell-derived car-
diomyocytes (hiPSC-CMs). All three BODIPY VFs stain the
membranes of hiPSC-CMs (Figure 4a, Figure S11). High-
speed fluorescence microscopy (200 Hz frame rate) demon-
strates that all three BODIPY VFs can report on spontaneously-
generated cardiac action potentials in hiPSC-CMs (Figure 4
and Figure S11) during 10 second bouts of imaging.
We found BODIPY VF 19 was the best suited voltage re-
porter because of its robust 33% ΔF/F and good signal-to noise
(Figure 4c). Additionally, under longer-term imaging (60 sec
of continuous imaging), 19 displays the least phototoxic effects
among the BODIPYs tested under constant illumination. Under
these conditions, the photostability of BODIPY VF 19 was
comparable to VF2.1.Cl,32 although with lower signal to noise
than either VF2.1.Cl or fVF 252 (Figure S12). Like VF2.1.Cl,
BODIPY VF 19 also slightly alters the shape and magnitude of
cardiac action potentials under prolonged illumination (Figure
S12). These effects are not seen during shorter imaging sessions
(Figure 4d).
The average ΔF/F for a class of BODIPY fluorophore (R =
Et, H, CO2H, CONHR, or CN) displays a parabolic relationship
with calculated HOMO energy levels (Figure S14c), with max-
imum voltage sensitivity at around -4.75 eV (or σ = 0.2 - 0.4).
Discussion
14
BODIPY VF dyes that have very large and negative ΔGPeT
,
We designed and synthesized 5 new sulfonated BODIPY
dyes with variable 2,6-subsitution patterns. These sulfonated
fluorophores represent a generalizable solution to improving
BODIPY water solubility while simultaneously avoiding mod-
ification to the boron center or fluorophore core. We incorpo-
rated these fluorophores into 13 new BODIPY voltage-sensitive
fluorophores for evaluation in live-cell imaging, but the tunable,
water-soluble BODIPYs presented here may have applications
beyond voltage sensing. BODIPY VF 35 is the most sensitive
BODIPY-based voltage indicator to date,53-54 but its low cellular
brightness precludes its ready adoption for functional imaging
in electrically excitable cells like neurons or cardiomyocytes.
Two other indicators developed in this study, BODIPY VF 19,
with its slightly lower sensitivity (33% ΔF/F per 100 mv), but
good brightness, and BODIPY VF 30, which retains good volt-
age sensitivity (24% ΔF/F per 100 mV) and exceptional bright-
ness (~7x brighter than 19 or 35) are better suited for functional
either by a combination of electron deficient fluorophores (R =
CN) with mildly donating anilines (R = H) as in the case of
BODIPY VF 22, or by with moderately withdrawing fluoro-
phores (R = CONHR) with electron-rich anilines (R = Me) in
the case of BODIPY 35, will have low voltage sensitivity.
These results suggest that 35 occupies a “sweet spot” of PeT to
optimize the voltage sensitivity for BODIPY VoltageFluors,
and any further lowering of the fluorophore HOMO (such as
amide BODIPY to cyano BODIPY) or raising the HOMO of
the aniline PeT donor (unsubstituted aniline to methyl-substi-
tuted aniline) is detrimental to the voltage sensitivity.
Despite its impressive 48% ΔF/F of BODIPY 35 in HEK
cells, its low cellular brightness (12x less bright than dicarboxy
BODIPY VFs) precludes its direct use in functional imaging.
The low cellular brightness likely results from poor solubility
of the dye, because the highly charged dicarboxy BODIPY VFs
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from Hammett constants: estimating the driving force of photoinduced
electron transfer. J Phys Chem A 2014, 118 (45), 10622-30.
16. Boens, N.; Leen, V.; Dehaen, W., Fluorescent indicators
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ing the electronics of the 2,6-diamide substitution pattern. Ex-
periments are underway to expand the utility of amide-substi-
tuted BODIPYs in the context of voltage imaging and beyond.
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Supporting Information. Support figures, spectra, synthetic
methods and imaging details. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Research in the Miller lab is supported by the National Institutes of
Health (R35GM119855). J.M.F, B.K.R., S.C.B., and R.U.K. were
supported in part by a training grant from the National Institutes of
Health (T32GM066698). We thank Dr. Kathy Durkin and Dr. Da-
vid Small for assisting with calculations performed in the Molecu-
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