J. Phys. Chem.

B 2003, 107, 7903-7913 7903

Anellated Hemicyanine Dyes in a Neuron Membrane: Molecular Stark Effect and Optical
Voltage Recording

Bernd Kuhn and Peter Fromherz*

Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry,
Martinsried/München, Germany

ReceiVed: March 6, 2003

The voltage sensitivity of hemicyanine dyes ANNINE-6 and ANNINE-5 with anellated benzene rings and
without free CC single and double bonds is studied in Retzius neurons from Hirudo medicinalis. For
comparison, biaryl hemicyanine BNBIQ and styryl hemicyanines di-4-ANEPBS and RH-421 are investigated.
Fluorescence spectra are recorded by an independent variation of the wavelengths of excitation and emission
at two defined membrane voltages. With extracellular staining, a positive change in the intracellular voltage
shifts all excitation spectra to the blue. That modulation is assigned to a molecular Stark effect that increases
in the series RH-421, di-4-ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6 with displacements of elementary
charge by 0.24, 0.43, 0.51, 0.65, and 0.81 nm across the membrane. For BNBIQ, ANNINE-5, and ANNINE-
6, an almost identical blue shift is observed for the emission that is also assigned to a Stark effect. The
ANNINE dyes are the most efficient fluorescent probes of neuronal activity on the basis of a well-defined
physical mechanism. The implications with respect to the optical recording of voltage transients are considered.

Introduction
Optical recording of neuronal activity with high spatial and
temporal resolution is a crucial prerequisite to the elucidation
of the electric function of nerve cells, neuronal networks, and
the brain.1-3 After an initial attempt by Tasaki et al.,4 in a heroic
effort Cohen, Salzberg, and co-workers screened hundreds of
organic dyes for their suitability to the optical recording of fast
voltage transients in neuron membranes.5,6 On that foundation,
the styryl class of hemicyanine dyes was optimized for the
maximum response of their fluorescence change by neuronal
activity by Loew et al.7-9 and Grinvald et al.10,11 In particular,
the dyes di-4-ANEPPS9 and RH-42111 (Figure 1) were used in
numerous studies with individual nerve cells, neuronal networks,
and the brain.
Styryl hemicyanines exhibit striking solvatochromism in bulk
solvents of increasing polarity with a blue shift of absorption
and a red shift of fluorescence.12 This symmetrically divergent
spectral shift reflects an intramolecular displacement of electrical
charge by electronic excitation from the pyridinium moiety
toward the aniline moiety of the chromophores (Figure 1). Such
a charge displacement was postulated to provide a sound basis
for the development of optical probes in nerve cells because of
a linear Stark effect.7 A changing electric field across an
asymmetrically stained cell membrane would induce an identical Figure 1. Voltage-sensitive amphiphilic hemicyanine dyes. (1-4)
shift in the excitation and emission spectra, leading to a Homologous series with electron-pushing aniline, electron-pulling
pyridinium, and two intervening conjugated CC double bonds: styryl
modulated fluorescence intensity for appropriately chosen
dye RH-160 (RH-421 with pentyl substitutents), styryl dye di-4-
wavelengths of illumination and detection. However, measure- ANEBPS (di-4-ANEPPS with propylsulfonate), biaryl dye BNBIQ,
ments of excitation and emission spectra in a neuron membrane and anellated hemicyanine dye ANNINE-5. The conformation at the
revealed that the spectral shifts of excitation and emission were single bonds is matched to the structure of ANNINE-5. (5) Anellated
not identical and that other changes in the spectral shape played hemicyanine dye ANNINE-6.
a significant role.13,14 The mechanism of the voltage sensitivity
of the styryl dyes appeared to be rather involved.15,16 There are two effects that may interfere with a Stark-effect
mechanism: (i) The styryl chromophores are not rigid. Elec-
* Corresponding author. E-mail: fromherz@biochem.mpg.de. Phone: tronic excitation gives rise to photoisomerism of CC double
+49 89 8578 2820. Fax: +49 89 8578 2822. bonds and to photorotamerism of CC single bonds.17 Both
10.1021/jp0345811 CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/02/2003
7904 J. Phys. Chem. B, Vol. 107, No. 31, 2003 Kuhn and Fromherz

processes may be related to an intramolecular displacement of

an electrical charge shift and may be affected by an electrical
field. In particular, rotamerism was shown to lead to a twisted
internal charge-transfer (TICT) state with a reduced fluorescence
quantum yield.18 (ii) The styryl chromophores are not im-
mobilized in a membrane. Their amphiphilic structure guarantees
an accumulation at the membrane surface19 and some alignment
along the membrane normal.20,21 But a changing electric field
may affect the position or inclination of the chromophores, and
the inhomogeneous environment at the membrane surface may
give rise to a field-induced solvatochromic effect.16,22
In earlier work, the dynamics of the hemicyanines was
simplified by incorporating the CC double bonds in aromatic
rings.23 Biaryl dye BNBIQ (diButyl-Naphthylamino-Butyl-
sulfonato-IsoQuinolinium) (Figure 1) exhibited symmetrically
divergent solvatochromism that was distinctly stronger than with
the homologous di-4-ANEPBS and RH-421.12 Correspondingly,
its voltage sensitivity in a neuron membrane was higher.14 In a
second stage, the free CC single bonds were also eliminated, Figure 2. Experimental setup. A nerve cell (N) stained with a voltage-
with chromophores consisting of anellated benzene rings.24 The sensitive dye is kept at a defined intracellular voltage by a micropipet
solvatochromism of anellated hemicyanines ANNINE-5 and using a patch-clamp amplifier. The cell is illuminated in a microscope
ANNINE-6 (Figure 1) was also significantly enhanced compared through the monochromator, shutter, dichroic mirror (DM), and
to that of the styryl dyes, indicating a large intramolecular objective. The fluorescence passes the dichroic mirror and a long-pass
displacement of electric charge. filter (LP) and is detected by a diode array spectrometer. The complete
fluorescence spectrum is recorded for a wide range of excitation
In the present paper, we describe the voltage sensitivity of wavelengths at two different transmembrane voltages.
anellated hemicyanines ANNINE-6 and ANNINE-5 and, for
comparison, that of BNBIQ, di-4-ANEPBS, and RH-421. Two-
dimensional fluorescence spectra of excitation and emission are dyes in water decreases in the series RH-421, di-4-ANEPBS,
measured in leech neurons at defined membrane voltages. The BNBIQ, ANNINE-5, and ANNINE-6. Correspondingly, the
voltage-induced changes in fluorescence are parametrized in fluorescence intensity of cells stained with RH-421 and di-4-
terms of changes in spectral parameters. We find that the ANEPBS dropped after exchanging the medium with a dye-
ANNINE dyes are far more voltage-sensitive than the classical free solution because of desorption from the cells, whereas the
styryl dyes and that their sensitivity can be assigned almost intensity with ANNINE-5 and ANNINE-6 remained constant
completely to an identical spectral shift of excitation and for at least 1.5 h. With respect to bleaching and phototoxicity,
emission that is caused by a molecular Stark effect. we did not observe significant differences under comparable
conditions of staining and illumination.
Materials and Methods Electrophysiology. Patch pipets with a tip diameter of
5-10 µm were made from microhaematocrite tubes (Assistent,
Dyes. Styryl dye RH-42111 was obtained from Molecular Karl Hecht, Sondheim/Rhön, Germany) using an all-purpose
Probes (Eugene, OR). Styryl dye di-4-ANEPBS9 and Biaryl dye puller (DMZ-Universal Puller, Zeitz-Instrumente, Augsburg,
BNBIQ23 were synthesized by Gerd Hübener. The syntheses Germany). They were filled with 140 mM KCl, 1.5 mM MgCl2,
of dyes ANNINE-5 and ANNINE-6 are described in ref 24. 10 mM Hepes, and 10 mM EGTA, pH 7.3. The resistance of
Neurons. Ganglia of the leech Hirudo medicinalis the pipets was around 0.4 MΩ. The pipets with Ag/AgCl
(Moser, Schorndorf, Germany) were dissected and pinned on a electrodes were connected to a single electrode patch-clamp
Sylgard-coated dish in Leibowitz-15 medium (L-5520, Sigma, amplifier (SEC-10L, npi, Tamm, Germany). The patching of
Deisenhofen) with 5 mg/mL glucose, 0.3 mg/mL glutamine, Retzius cells led to a seal resistance of about 500 MΩ. Whole
and 3 µg/mL gentamycin sulfate (G-3632, Sigma).25 After open- cell contact27 was achieved by breaking the membrane with
ing the tissue capsules, we incubated the ganglia in dispase/ suction using a water-jet vacuum pump. The bath was held at
collagenase (Boehringer, Mannheim, 2 mg/mL L-15 medium) ground potential with a Ag/AgCl electrode. We kept the cells
for 1 h at room temperature. Retzius cells (soma diameter at an intracellular voltage of VM ) -40 mV. The fluorescence
60-90 µm) were dissociated by aspiration into a fire-polished spectra were measured at a hyperpolarized voltage of VM )
micropipet and washed with Leibowitz-15 medium.25 The cells -70 mV and a depolarized voltage of about VM ) 10 mV.
were seeded on an uncoated glass cover slip in a silicone Optical Setup. The spectrometer is sketched in Figure 2. It
chamber (Flexiperm-mikro 12, Vivascience AG, Hannover, is built on the basis of an inverted microscope (Axiovert 35,
Germany) with Leibowitz-15 medium and 2.5% fetal bovine Zeiss, Oberkochen, Germany) with a high numerical aperture
serum (10106, Gibco, Eggenstein) and kept for 1 or 2 days at oil-immersion objective (Neofluar 100×/1.3 oil). The light of
20 °C. a 75-W xenon short arc lamp (Ushio, Hyogo, Japan) is spectrally
Staining. The dyes were solubilized in Leibowitz-15 medium resolved (resolution 16 nm) by a grating monochromator (J&M,
with sodium cholate (Sigma, St. Louis, MO).26 We used 4.3 Aalen, Germany). It is fed into the microscope with an optical
mM RH-421 with 10 mM cholate, 4.3 mM di-4-ANEPBS with multimode fiber (diameter 1.2 mm) and collimating optics
10mM cholate, 10 mM BNBIQ with 10 mM cholate, 1 mM (J&M). Nerve cells are illuminated through a dichroic mirror
ANNINE-5 with 23 mM cholate, and 1 mM ANNINE-6 with (AHF analysentechnik, Tübingen, Germany) with a splitting
25 mM cholate. After centrifugation, the staining solutions were wavelength of 520 nm for RH-421, di-4-ANEPPS, BNBIQ, and
added to the culture chambers with the neurons in a volume ANNINE-6 and 460 nm for ANNINE-5. The illumination is
ratio of 1:1000 30 min before patching. The solubility of the controlled by a shutter and a field diaphragm. The light emitted
Anellated Hemicyanine Dyes in a Neuron Membrane J. Phys. Chem. B, Vol. 107, No. 31, 2003 7905

by the stained cell is collected by the objective. It passes through for ANNINE-5 with an integration time of 200 ms, (5) after a
the dichroic mirror and a long-pass filter (AHF analysentechnik, delay of 100 ms, depolarization to about VM ) 10 mV, (6)
Tübingen) with a cutoff at 520 nm for RH-421, di-4-ANEPBS, recording of two complete emission spectra, (7) after a delay
BNBIQ, and ANNINE-6 and 470 nm for ANNINE-5 and is of 100 ms, hyperpolarization to VM ) -70 mV, (8) recording
guided by an optical fiber (0.6 mm) to a diode array spectrom- another complete emission spectrum, and (9) closing the shutter.
eter (J&M) with a spectral range from 307 to 1135 nm at a Steps 2-8 are repeated for various wavelengths of excitation
resolution of 3.1 nm. in the range of λex ) 360-510 nm for RH-421, di-4-ANEPBS,
Calibrated Fluorescence Spectra. To derive molecular BNBIQ, and ANNINE-6 and λex ) 360-460 nm for ANNINE-5
parameters from the experimental data, the 2D spectra of at a step width of 5 nm. At each excitation wavelength, the
excitation and emission were calibrated. The number of excita- two spectra of the hyperpolarized cell as well as the two spectra
tions per unit time in a membrane area Amem with ndye molecules of the depolarized cell are averaged to obtain spectra P(λex,λem)
per unit area is Amemndye(λex)Iillλ (λex)∆λex with the molecular
at the two voltages. The 2D fluorescence spectra Fνj(νjex,νjem)
cross section of absorption (λex) and the quantum intensity of are computed according to eq 2 up to a constant factor using
illumination per wavelength interval Iill λ (λex) at a bandwidth
the spectra Iill rec
λ (λex)∆λex and T (λem)∆λem given by calibration.
∆λex. The number of detected quanta per unit time P(λex, λem) At the hyperpolarized voltage, the spectrum is scaled to its
is given by eq 1 with the quantum yield Φem, the normalized maximum as Fhyp hyp,max
νj /Fνj . At the depolarized voltage, the
quantum spectrum of fluorescence per wavelength interval spectrum is scaled by the same factor as Fdep hyp,max
νj /Fνj .
fλ(λem), and the efficiency Trec(λem) and bandwidth ∆λem of the
recording system. Results
P(λex,λem) ) Two-Dimensional Spectra. The relative fluorescence
AmemndyeIill rec spectra per wavenumber interval of emission Fνj(νjex,νjem)/Fmax
λ (λex)∆λex(λex)Φem fλ(λem)T (λem)∆λem (1) νj
in Retzius neurons at a voltage VM ) -70 mV are plotted in
We obtain a 2D fluorescence spectrum that is defined by the left column of Figure 3 for RH-421, di-4-ANEPBS, BNBIQ,
molecular parameters when we divide eq 1 by the number of ANNINE-5, and ANNINE-6. They are limited at the red end
dye molecules Amemndye and by the spectra and bandwidths of of excitation and the blue end of emission by the dichroic mirror
illumination Iill rec of the microspectrometer. The maxima reflect the position of
λ (λex)∆λex and recording T (λem)∆λem. The
resulting fluorescence spectrum of excitation and emission per the upward and downward vibroelectronic transitions between
unit wavenumber of emission Fνj(νjex,νjem) ) (νjex)Φem fνj(νjem) the S0 and S1 states of the dyes. They are shifted to higher
is given by eq 2, considering the relations fνj(νjem) ) λ2em fλ(λem) wavenumbers of excitation and emission in the homologous
and (νjex) ) (λex). series RH-421, di-4-ANEPBS, BNBIQ, and ANNINE-5 and also
in ANNINE-6. With respect to excitation, a second band appears
Fνj(νjex,νjem) ) in the ANNINEs at high wavenumbers because of the S0/S2
transition.
λ2em
P(λex,λem) (2) One-Dimensional Spectra. To study the interdependence of
AmemndyeIill rec
λ (λex)∆λexT (λem)∆λem excitation and emission, we consider emission spectra per
wavenumber of emission Fνj(νjem) at various wavenumbers of
Trec(λem)∆λem is obtained up to an arbitrary factor by excitation νjex and excitation spectra Fνj(νjex) per wavenumber
calibrating the recording system (objective, dichroic mirror, of emission for various wavenumbers of emission νjem in the
long-pass filter, 0.6-mm fiber optics, monochromator, and 2D spectra. These 1D spectra are fitted by log-normal functions28
diode array). We place a standard lamp (OL245M, Optronic according to eq 3 with an amplitude Fmax νj , a spectral maximum
Laboratories, Orlando, FL) with a known quantum spectrum νjmax, a spectral width W, and a spectral asymmetry b, excluding
λ (λ) on the microscope and measure the response P (λ) )
Ical cal the contribution of the S0/S2 transition.
cal rec

{ }
Iλ (λ)T (λem)∆λem of the photodiode array. We probe the
illumination (Xe lamp, monochromator, optical fiber, broadening Fνj(νj) ln2[1 + 2b(νj - νjmax)/W]
optics, dichroic mirror, and objective) with a calibrated detector ) exp - (3)
of efficiency Tcal(λ) and bandwidth ∆λ. For that purpose, we Fmax
νj b2/ln 2
use part of our recording systemsthe 0.6 nm optical fiber
with monochromator and diode arraysthat is again calibrated Figure 4 shows the parameters of the emission spectra νjmaxem ,
by illuminating the fiber end with the standard lamp. With Wem, and bem as a function of the excitation wavenumber νjex
the fiber end on the microscope, we measure Pill(λ) ) and the parameters of the excitation spectra νjmax
ex , Wex, and bex
Iill nom cal nom
λ (λex ,λ)T (λ)∆λ for each λex set at the monochromator. as a function of the emission wavenumber νjem. The maximum
The resulting illumination spectra Iill nom
λ (λex ,λ) are fitted with of excitation is shifted to the blue at high wavenumbers of
Gaussians with a maximum defining the excitation wavelenth emission, and the maximum of emission is shifted to the blue
λex and an integral that represents the intensity Iill
λ (λex)∆λex up for higher wavennumbers of excitation. Such effects are
to a constant factor. expected for hemicyanine dyes when the solvent shell is
Protocol. The measurements were started 30 min after incompletely relaxed in the excited state.18 However, these shifts
staining. Under a voltage clamp, the voltage-sensitive fluores- of the spectral maxima as well as all other changes of spectral
cence is investigated with the following protocol: (1) opening width and spectral asymmetry displayed in Figure 4 are rather
of the shutter, (2) selection of an excitation wavelength λex, small compared with the width of the spectra and the difference
(3) after a delay of 100 ms, hyperpolarization of the cell to in excitation and emission maxima. Considering these observa-
VM ) -70 mV, (4) recording of a complete emission tions with the 1D spectra, we use in the following evaluation
spectrum in the range of λem ) 510-833 nm for RH-421, di- of the 2D spectra the approximation that excitation and emission
4-ANEPBS, BNBIQ, and ANNINE-6 and λem ) 460-833 nm processes are independent of each other.
7906 J. Phys. Chem. B, Vol. 107, No. 31, 2003 Kuhn and Fromherz

TABLE 1: Spectral Parameters of Excitation and Emission

Spectra for the S0/S1 Transition of Hemicyanine Dyes in
Neuron Membranesa
νjmax
ex νjmax
em Wex Wem
[cm-1] [cm-1] [cm-1] [cm-1] bex bem
RH-421 20 249 15 417 4185 3239 0.340 -0.025
di-4-ANEPBS 21 199 16 390 4176 3292 0.404 -0.226
BNBIQ 22 298 17 125 4327 3471 0.413 -0.219
ANNINE-5 23 155 18 419 3986 3427 0.492 -0.289
ANNINE-6 23 673 17 456 4326 3688 0.321 -0.206
a
Spectral maxima νjmax jmax
ex and νem , spectral widths Wex and Wem, and
spectral asymmetries bex and bem are obtained from 2D fluorescence
spectra at a voltage of VM ) -70 mV by fitting the products of two
log-normal functions.

summarized in Table 1, and the fitted 2D spectra are displayed

in the left column of Figure 5. We note a distinct asymmetry
of the 2D spectra, in particular, for ANNINE-5 and ANNINE-6
with steep slopes toward the red in excitation and toward the
blue in emission. To illustrate the quality of the fit, we check
1D sections along the axes of excitation and emission. The upper
graphs of Figure 6 show the data and the fit function. The good
agreement, except for high wavenumbers of excitation where
the contribution of the S0/S2 transition is significant, confirms
the validity of the approach. Also, the 1D spectra exhibit steep
slopes in the red of the excitation band and in the blue of the
emission band.
Voltage Sensitivity. The spectrum of voltage sensitivity
SV(νj ex,νj em) is defined as the relative change in fluorescence
intensity per change in membrane voltage according to eq 5.

∆Fνj(νjex,νjem) 1
SV(νjex,νjem) ) (5)
Fνj(νjex,νjem) ∆VM

We obtain it by subtracting the relative spectrum Fhyp hyp,max

νj /Fνj
at a hyperpolarized voltage from the scaled spectrum Fdep νj /
Fhyp,max
νj at a depolarized voltage and dividing by Fhyp hyp,max
νj /Fνj
and the voltage difference ∆VM. The results, scaled to a voltage
∆VM ) 100 mV, are shown in the right column of Figure 3.
Figure 3. Experimental 2D fluorescence spectra. Left column: Color- All sensitivity spectra exhibit two regions: at high wavenumbers
coded relative fluorescence intensity Fνj(νjex,νjem)/Fmax
νj in Retzius cells of excitation and emission, the fluorescence is enhanced; at
at a voltage of VM ) -70 mV as a function of the wavenumbers of low wavenumbers, the fluorescence is reduced. The magnitude
excitation (abscissa) and emission (ordinate). Right column: Color- of this effect increases in the homologous series RH-421,
coded voltage sensitivity of fluorescence SV(νjex,νjem) ) ∆FνjFνj∆VM. The di-4-ANEPBS, BNBIQ, and ANNINE-5 and also in ANNINE-
diagonal lines mark equal wavenumbers of excitation and emission.
The dotted vertical and horizontal lines indicate 1D spectra of excitation 6. Within the limited spectral window of the measurements,
and emission. They are chosen through the 2D maxima with the the sensitivity is in the range of 10%/100 mV for RH-421,
exception of ANNINE-6, where the excitation is chosen in the red to 15%/100 mV for di-4-ANEPPS, 200/100 mV for BNBIQ, and
avoid a contribution of the S0/S2 transition. 25/100 mV for ANNINE-5 and ANNINE-6.
Parametrized Voltage Sensitivity. A parametrization of
Fit of 2D Spectra. To attain a well-defined parametrization voltage sensitivity is achieved by fitting the scaled spectra
of the fluorescence spectra, we fit the normalized 2D spectra
Fdep hyp,max
νj /Fνj and Fhyp hyp,max
νj /Fνj at the two voltages to the prod-
Fνj(νjex,νjem)/Fmax
νj shown in the left column of Figure 3 to the ucts of log-normal functions according to eq 4. The resulting
products of two log-normal spectra according to eq 4.
differences in spectral parameters ∆νjmax jmax
ex , ∆νem , ∆Wex, ∆Wem,

{ }
max
∆bex, ∆bem, and ∆Fνj are small for all dyes. For that reason,
Fνj(νjex,νjem) ln2[1 + 2bex(νj - νjmax
ex )/Wex]
) exp - × we can assume that the changes in the spectra are linear with
Fmax b2ex/ln 2 respect to changes in the spectral parameters, and we are allowed

{ }
νj
to scale the parameter changes to a standard voltage change of
ln2[1 + 2bem(νj - νjmax
em )/Wem]
exp - (4) ∆VM ) 100 mV, which is typical for neuronal excitation. The
b2em/ln 2 resulting differences are shown in Table 2.
Using the fit parameters from Tables 1 and 2, we reconstruct
Considering Fνj(νjex,νjem) ) (νjex)Φem fνj(νjem) with the cross the sensitivity spectra SV(νjex,νjem) according to eq 5. The results
section of absorption (νjex), the quantum yield Φem, and the are displayed in the central column of Figure 5 in a range where
normalized quantum spectrum of emission fνj(νjem), the product the relative change in intensity is above 10%. For ANNINE-6,
function of eq 4 accounts for the spectral shape of absorption ANNINE-5, and BNBIQ, the fitted sensitivity spectra exhibit a
and emission. The five sets of the six spectral parameters are distinct enhancement and reduction of fluorescence at high and
Anellated Hemicyanine Dyes in a Neuron Membrane J. Phys. Chem. B, Vol. 107, No. 31, 2003 7907

Figure 4. Mutual dependence of excitation and emission spectra. Left: Spectral parameters of the excitation spectra (maximum νjmax
ex , width Wex,
asymmetry bex) as a function of emission wavenumber νjem. Right: Spectral parameters of the emission spectra (maximum νjmax em , width Wem,
asymmetry bem) as a function of excitation wavenumber νjex.

low wavenumbers, respectively. In particular, we note the steep with the matrix. It may change the electronic 00 transition
increase in negative sensitivity with decreasing wavenumber energy, the Franck-Condon factors of excitation and emission,
of excitation in the red corner of the 2D spectrum and the steep the transition dipole moments, and the radiationless deactivation
increase in positive sensitivity with increasing wavenumber of channels. As a result, the 2D fluorescence spectrum Fνj(νjex,νjem)
emission in the blue corner of the 2D spectrum. For the two ) (νjex)Φem fνj(νjem) may be modulated through the absorption
styryl dyes, the sensitivity spectra are more involved. spectrum (νjex), the quantum yield of emission Φem, and the
To illustrate the quality of the sensitivity spectra SV(νjex,νjem) normalized quantum spectrum of emission fνj(νjem). The sensitiv-
reconstructed from the fit of the 2D spectra at two voltages, we ity spectrum defined by eq 5 reflects changes in all of these
select 1D spectra SV(νjex) and SV(νjem) across the sensitivity data molecular parameters according to eq 6.
(Figure 3) and across the fitted spectra (Figure 5). They are
shown in Figure 6 and exhibit good agreement, except for high SV(νjex,νjem) )
excitation wavenumbers where the S0/S2 transition contributes
1 ∆(νjex) 1 ∆Φem 1 ∆fνj(νjem)
and at low emission wavenumbers where the intensity is modest. + + (6)
(νjex) ∆VM Φem ∆VM fνj(νjem) ∆VM
Discussion
Voltage Sensitivity. An electric field across a cell membrane In fact, when we fit the 2D fluorescence spectra at two
may in principle affect the electronic structure of a bound voltages to products of log-normal functions, we find that the
chromophore, its vibroelectronic coupling, and its interaction joined amplitude Fmax
νj and all spectral parameters of excitation
7908 J. Phys. Chem. B, Vol. 107, No. 31, 2003 Kuhn and Fromherz

Figure 5. Two-dimensional fluorescence spectra parametrized by the product of log-normal functions. Left column: color-coded relative fluorescence
spectra Fνj(νjex,νjem)/Fmax
νj at VM ) -70 mV. Central column: color-coded sensitivity spectra SV(νjex,νjem) ) ∆Fνj/Fνj∆VM in a range where Fνj/Fmax
νj >
0.1 and where the S0/S2 transition plays a negligible role. Right column: color-coded relative response spectra RV ) ∆Fνj/Fmax
νj ∆VM. The diagonals
mark equal wavenumbers of excitation and emission. White lines in the sensitivity and response spectra indicate intensity levels Fνj/Fmax
νj ) 0.33 and
Fνj/Fmax
νj ) 0.67. Black lines mark the change in sign of sensitivity and response.

νjmax
ex , Wex, and bex and of emission ν jmax
em , Wem, and bem are
Electrochromism of Excitation. A typical feature of the
modified by an electric field as summarized in Table 2. An voltage sensitivity of all of the hemicyanines considered is a
assignment of these parameter changes to molecular mecha- blue shift ∆νjmax
ex of all excitation spectra induced by a positive
nisms, however, is difficult (e.g., spectral shifts ∆νjmax ex and change ∆VM in the membrane voltage (Table 2). This blue shift
∆νjmax
em may arise not only from a change in the electronic 00 increases in the homologous series RH-421, di-4-ANEPBS,
energy and amplitude changes ∆Fmax νj not only from a changed BNBIQ, ANNINE-5 and also in ANNINE-6. To confirm the
fluorescence quantum yield, but in both cases, changes in significance of this observation, we repeat the evaluation of the
spectral shape contribute, too). It is also a problem that parameter spectral data in terms of a product of log-normal functions (eq
changes ∆νjmax
ex , ∆νjmax
em , ∆Wex, ∆Wem, ∆bex, ∆bem, and ∆Fνj
max 4) with five parameters at constant spectral asymmetries bex and
are derived from a small difference between two spectra, a bem and also with three parameters at constant asymmetries and
procedure that implies a large error. For both reasons, we do constant spectral widths Wex and Wem. The resulting spectral
not attempt to provide a detailed mechanistic interpretation of shifts are summarized in Table 3. Apparently, the systematic
voltage sensitivity. We consider only one aspectsthe possible blue shift of excitation is rather insensitive to the fitting
contribution of a molecular Stark effect. procedure.
Anellated Hemicyanine Dyes in a Neuron Membrane J. Phys. Chem. B, Vol. 107, No. 31, 2003 7909

Figure 6. One-dimensional spectra of fluorescence and voltage sensitivity. Upper plots: spectra of the relative intensity of emission Fνj(νjem)/Fmax
νj
and excitation Fνj(νjex)/Fmax
νj across the 2D spectra as indicated in Figure 3. Lower plots: sensitivity spectra of emission SV(νjem) and excitation
SV(νjex). The drawn lines are 1D sections of the fit with the product of two log-normal functions taken from Figure 5. The wavenumbers of excitation
and emission are marked by arrows.

TABLE 2: Voltage Sensitivity of the Spectral Parameters of Excitation and Emission for the S0/S1 Transition of the
Hemicyanine Dyes in a Neuron Membranea
∆νjmax -1
ex [cm ] ∆νjmax -1
em [cm ] ∆Wex [cm-1] ∆Wem [cm-1] ∆bex ∆bem ∆Fmax
νj

RH-421 47 -18 -49 54 0.022 0.029 -0.039

di-4-ANEPBS 90 45 38 83 -0.024 0.006 -0.033
BNBIQ 103 99 8 24 -0.009 -0.014 -0.011
ANNINE-5 132 132 12 20 0.002 -0.016 -0.015
ANNINE-6 163 170 -19 0.022 0.018
a
Two-dimensional spectra of fluorescence are fitted with products of two log-normal functions at two different voltages. The changes in the
spectral parameterssof the maxima ∆νjmax
ex and ∆ν jmax
em , of the widths ∆Wex and ∆Wem, of the asymmetries ∆bex and ∆bem, and of the amplitude
∆Fνj sare scaled to a voltage change of ∆VM ) 100 mV. For ANNINE-6, the width and asymmetry of excitation was held constant because of
max

an overlap with S0/S2 excitation.

We assign the blue shift of excitation to a molecular Stark displacement ∆µEG by electronic excitation from the pyridinium
effect of the membrane-bound chromophores.29-32 Because of to the aniline moiety is directed against a change ∆E ) ∆VM/dM
their amphiphilic structure, the hemicyanines may be expected of the average electrical field in a membrane of thickness dM
to be bound to the surface of the neuron membrane with a as induced by a positive change ∆VM of the membrane voltage.
distinct orientation.20,21 In that case, an intramolecular charge The expected blue shift is expressed by eq 7 with a projection
7910 J. Phys. Chem. B, Vol. 107, No. 31, 2003 Kuhn and Fromherz

TABLE 3: Spectral Shifts of Excitation and Emission by a TABLE 4: Membrane Solvatochromism and
Voltage Change of ∆VM ) 100 mVa Electrochromisma
∆νjmax -1
ex [cm ] ∆νjmax -1
em [cm ] νjmem
00 νj00 ∆νj00 ∆νjmax
ex
[cm-1] [cm-1] [cm-1] [cm-1]
7 param 5 param 3 param 7 param 5 param 3 param
RH-421 17 833 16 500 1333 47
RH-421 47 46 47 -18 -3 -6
di-4-ANEPBS 18 795 16 800 1995 90
di-4-ANEPBS 90 78 78 45 43 42
BNBIQ 19 712 17 200 2512 103
BNBIQ 103 97 96 99 91 91
ANNINE-5 20 787 17 900 2888 132
ANNINE-5 132 132 134 132 123 123
ANNINE-6 20 565 17 400 3165 163
ANNINE-6 163 163 163 170 159 159
a
a Averages of the maxima of excitation and emission in the neuron
Two-dimensional fluorescence spectra at two different voltages are
fitted with products of two lognormal functions. The changes in the membrane νjmem
00 ) (νjmax
ex - ν jmax j00 in bulk solvents,
em )/2, 00 energies ν

spectral maxima ∆νjmax jmax solvatochromic blue shifts ∆νj00 ) νjmem - νj00, and electrochromic
ex and ∆ν em are shown for (i) a fit with seven
00
changing spectral parameters (maxima, widths, spectral asymmetries, blue shifts of excitation ∆νjmax
ex for an applied voltage change ∆VM )
amplitude), (ii) a fit with five changing spectral parameters (maxima, 100 mV in the neuron are shown. νj00 in bulk solvents are for RH-160
widths, amplitude), and (iii) a fit with three changing spectral parameters instead of 421 and for di-4-ANEPPS instead of di-4-ANEPBS.
(maxima, amplitude).

cos ϑ of charge displacement on the membrane normal (Planck’s

constant h, velocity of light c).

hc∆νjmax
ex ) ∆µEG∆E cos ϑ (7)

From the experimental shifts ∆νjmax ex , we estimate the charge

displacement ∆µEG cos ϑ along the membrane normal for ∆VM
) 100 mV and dM ) 4 nm. Considering the fit with seven
parameters (Table 2), we obtain for RH-421, di-4-ANEPBS,
BNBIQ, ANNINE-5, and ANNINE-6 values of 12, 21, 24, 31,
and 39 D. The effective displacements of an elementary charge
e0 along the membrane normal (∆µEG/e0)cos ϑ are 0.24, 0.43,
0.51, 0.65, and 0.81 nm. With respect to charge displacement, Figure 7. Electrochromism and membrane solvatochromism. Blue shift
ANNINE-6 is better by a factor of 3 than the classical styryl ∆νjmax
ex of the excitation spectra induced by a membrane voltage of
dye RH-421. The enhancement may be due either to a stronger ∆VM ) 100 mV versus a shift ∆νj00 ) νjmem00 - νj00 of the average
intramolecular charge shift ∆µEG or to a better orientation cos ϑ wavenumbers of excitation and emission νjmem00 ) (νjmax
ex - νjmax
em )/2
in the membrane. A discrimination of the two effects is not in the membrane with respect to the 00 energy νj00 in bulk solvents.
possible on the basis of the present data. It may be achieved by The dots refer to dyes 1-5 in Figure 1. The linear regression line
has a slope of 0.045.
systematic measurements of the chromophore orientation in the
neuron membrane and by quantum chemical computations of
the electronic structure of the chromophores. solvatochromic blue shifts ∆νj00 in the series RH-421, di-4-
Electrochromism and Membrane Solvatochromism. The ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6 reflect an
assignment of a Stark effect to the electrochromic blue shift increasing charge displacement ∆µEG cos ϑ. Because electro-
is confirmed by a consideration of solvatochromism in the chromism caused by a Stark effect and solvatochromism induced
neuron membrane. Because of their orientation, the chromo- by a polarity gradient are both proportional to ∆µEG cos ϑ, we
phores at the membrane/water interface are in an extremely expect a proportionality of the electrochromic shift ∆νjmax
ex and
inhomogeneous environment that may give rise to specific the solvatochromic shift ∆νj00. In fact, Figure 7 shows such a
solvatochromic effects with amphiphilic hemicyanines.33 We linear correlation with ∆νjmax
ex ) 0.045∆ν j00 and confirms the
have to distinguish the effect of local polarity and the effect of consistency of our interpretations.
the local polarity gradient.34 In homogeneous solvents, changing Ideal Stark-Effect Probes. A molecular Stark effect may
polarity gives rise to a symmetric opposite shift of excitation affect not only the excitation but also the emission of a dye.
and emission spectra with an invariant average wavenumber The resulting spectral shift is described by eq 9.
νj00 ) (νjmax
ex + ν jmax
em )/2 of the 00 energy.
12 As a consequence,

a shift ∆νj00 ) νjmem - j

ν of the average wavenumber in hc∆νjmax
em ) ∆µEG∆E cos ϑ
00 00 (9)
the membrane may be considered to be a quantitative indicator
for an effect of the polarity gradient. The wavenumbers νjmem 00 In fact, for BNBIQ, ANNINE-5, and ANNINE-6, we observe
in the neuron membrane and the 00 energies νj00 in bulk
electrochromic blue shifts of emission that are almost identical
solvents12,24 are summarized in Table 4. There is indeed a
to those of excitation (Table 3). We assign that correspondence
large blue shift for all dyes that increases in the series RH-421,
to an identical molecular Stark effect for excitation and emission
di-4-ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6.
with an identical charge shift ∆µEG cos ϑ due to an identical
We may expect that the solvatochromic effect of the polarity
electronic transition and a similar orientation of the chromophore
gradient is related to the intramolecular charge displacement
for excitation and emission.
∆µEG cos ϑ across the polarity gradient. Thus, we express the
To determine how far the Stark effect of excitation and
blue shift ∆νj00 by eq 8.
emission dominates the voltage sensitivity of BNBIQ, ANNINE-
hc∆νj00 ) E0∆µEG cos ϑ (8) 5, and ANNINE-6, we compute the sensitivity spectrum
SV(νjex,νjem) for an identical blue shift of excitation and emission
The effective internal electric field E0 characterizes the at invariant spectral shape and amplitude with ∆Wex )
strength of the gradient. On the basis of eq 8, the increasing ∆Wem ) ∆bex ) ∆bem ) ∆Fmax νj ) 0 using eqs 4 and 5.
Anellated Hemicyanine Dyes in a Neuron Membrane J. Phys. Chem. B, Vol. 107, No. 31, 2003 7911

vibroelectronic transitions. The relative slope of the excitation

spectrum for ANNINE-5 and ANNINE-6 is highest and positive
in the red, whereas the relative slope of the emission spectrum
is highest and negative in the blue, as shown in Figures 5 and
6. The relative slopes are distinctly smaller in the blue of
excitation and in the red of emission. However, because of their
different signs, optimal sensitivitites with respect to excitation
and emission cannot be combined. For optical recording, we
may choose the red corner of the 2D sensitivity spectra as
illustrated in Figure 5. In accordance with eq 10, the sensitivity
increases dramatically in the red of excitation but only modestly
in the red of emission, as illustrated also by the 1D spectra of
Figure 6.
Styryl Dyes. For styryl dyes di-4-ANEPBS and RH421, the
spectral shifts of excitation and emission are not identical (Table
2). The blue shift of emission for di-4-ANEPBS is about 50%
of the blue shift of excitation, and RH-421 even exhibits a red
shift of emission. These results do not depend on the fitting
procedure (Table 3). Apparently, with these dyes, the emission
is affected by other effects besides a Stark effect. Possibly, the
electrical field changes the position and orientation of the excited
chromophores, resulting in a field-induced solvatochromic red
shift of emission that is superimposed on a Stark effect.
Optical Recording. The sensitivity function SV(νjex,νjem)
reflects molecular features of the membrane-bound dye accord-
ing to eq 6. However, the photodiode signal ∆P(νj ex,νj em)
due to a voltage change ∆VM of neuronal activity in an
experimental setup of optical recording depends not only on
voltage sensitivity but also on staining, illumination, and
detection.35,36 Considering eqs 1 and 2, we obtain eq 11: a high
response requires a large observed membrane area Amem, a high
density of dye molecules per unit area ndye, a high quantum
ill
intensity of illumination Iνj (νjex)∆νjex, a high efficiency of
the detection system T (νjem)∆νjem, a high voltage sensitivity
rec

SV(νj ex,νj em) and, considering Fνj(νj ex,νj em) ) (νj ex) fνj(νj em)Φem,
a high cross section of absorption (νjex) and a high yield of
Figure 8. Voltage sensitivity for the molecular Stark effect. Color- fluorescence fνj(νjem)Φem.
coded sensitivity spectra SV(νjex,νjem) ) ∆Fνj/Fνj∆VM in the range of
relative intensity Fνj/Fmax
νj > 0.1 where the S0/S2 transition plays a
negligible role. The spectral shifts of excitation and emission by a ∆P(νjex,νjem) ) ∆VMAmemndye‚Iill jex)∆νjex‚SV(νjex,νjem)‚
νj (ν
voltage change of ∆VM ) 100 mV are ∆νjex,em ) 101 cm-1 for BNBIQ,
∆νjex,em ) 132 cm-1 for ANNINE-5, and ∆νjex,em ) 166 cm-1 for Fνj(νjex,νjem)‚Trec(νjem)∆νjem (11)
ANNINE-6. The diagonals mark equal wavenumbers of excitation and
emission. White lines in the sensitivity and response spectra indicate
We consider an illumination with a narrow spectral band-
intensity levels Fνj/Fmax
νj ) 0.33 and Fνj/Fmax
νj ) 0.67. Black lines mark
the change in sign of sensitivity and response. width at a wavenumber νj/ex with an integral intensity Iill )
ill
∫Iνj (νjex) dνjex and a detection with constant efficiency
Trec(νjem) ) Trec within the spectral limits νjAem and νjBem of
Choosing the averages ∆νjmax ex,em ) (∆ν jmax
ex + ∆ν jmax
em )/2 of the recording. The total photodiode signal is given by eq 12.
blue shifts from Table 2, we obtain Figure 8. The computed
voltage sensitivities are most similar to the fitted spectra in
the central column of Figure 5: BNBIQ, ANNINE-5, and
∆P )
∫νjνj
B
ANNINE-6 are almost ideal Stark probes for voltage changes ∆VMAmemndyeIillTrec A
em
Fνj(νj/ex,νjem) SV(νj/ex,νjem) dνjem (12)
in nerve cells. em

When we express the voltage sensitivity for a pure Stark effect

by eq 6, considering a small spectral shift ∆νjex,em, we obtain An optimal signal is achieved by good staining, high
eq 10 in terms of the derivatives of the absorption and emission illumination intensity, high detector sensitivity, and the proper
spectra ′ ) d/dνex and f ′ ) df/dνjem. selection of the excitation wavenumber νj/ex and the emission
filters νjAem and νjBem to attain a large integral over the product of
SV(νjex,νjem) )
[
∆νjex,em ′(νjex) fνj′(νjem)
∆VM (νjex)
+
fνj(νjem) ] (10)
the sensitivity spectrum and the fluorescence spectrum. For
illustration, the normalized response spectrum RV(νjex,νjem) )
SV(νjex,νjem)Fνj(νjex,νjem)/Fmax
νj is plotted in the right column of
The voltage sensitivity SV(νjex,νjem) depends not only on the Figure 5. The highest negative integrals are found in the
spectral shift caused by the intramolecular charge displacement red corner of the spectrum with an excitation wavenumber
but also on the shapes of the absorption and emission spectra νj/ex at the minimum of RV(νjex,νjem) and with emission filters
that are determined by the Franck-Condon factors of the νjAem and νjBem excluding the region of positive response. The
7912 J. Phys. Chem. B, Vol. 107, No. 31, 2003 Kuhn and Fromherz

highest reponse increases significantly in the series RH-421,

di-4-ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6.
Signal-to-Noise Ratio. The total response expressed by eq
12 reflects the signal-to-noise ratio only if the noise is constant
(e.g., dominated by the electronic amplifier). If the shot noise
of photons dominates,37 then we have to consider a noise level
N ) xP/2τD that depends on the total photon count P and the
time constant of the detector τD. With

∫νjνj
B
P ) AmemndyeIillTrec A
em
Fνj(νj/ex,νjem) dνjem
em

we obtain eq 13.
Figure 9. Weighted voltage sensitivity |〈SV(νjex)〉| of ANNINE-6 versus
) ∆VMx2τDAmemIillndyeTrecΦem ×
∆P
wavenumber of monochromatic excitation (s) νjex. |〈SV(νjex)〉| is
N proportional to the signal-to-noise ratio with the constraint of a constant
∫νjνj
B
number of excitation processes per unit time. The detection ranges from
A
em
Fνj(νj/ex,νjem) Sνj(νj/ex,νjem) dνjem 12 000 to 17 600 cm-1. For comparison, the relative excitation spectrum
em
(13) is plotted (- -).
x∫ νjBem
νjAem
Fνj(νj/ex,νjem) dνjem
These features of the signal-to-noise ratio for Iill(νj/ex) )
In optimizing the signal-to-noise ratio, we have to take into const are expected for a Stark effect. When we insert eq 10
account the roles of photobleaching and phototoxicity of the into eq 15, we obtain eq 16.
dyes. These effects impair optical recording because dyes and
∆νjex,em
x∫
cells fade away by illumination. Both photochemical processes νBem
〈SV(νj/ex)〉 ) fνj(νjem) dνjem ×

{ }
are determined by the number of excitations per unit time. The ∆VM νjAem
signal-to-noise ratio must be optimized with the constraint of a
∫νjνj
B
certain number of excitations per unit time that is tolerable for ′(νj/ex) A
em
fνj′(νjem) dνjem
+
em
a certain experiment. When we express the fluorescence (16)
(νj/ex) ∫νjνj
B
spectrum by Fνj(νj/ex,νjem) ) (νj/ex)Φem fνj(νjem), we obtain eq 14. A
em
fνj(νjem) dνjem
em

) ∆VMx2τDAmem xIillndye(νj/ex) xTrecΦem ×

∆P
When we choose an excitation wavenumber in the red where
N the relative slope of the excitation spectrum is steep, we expect
∫νjνj
B

A
em
fνj(νjem) Sνj(νj/ex,νjem) dνjem an increasing contribution of the Stark effect of excitation at a
em
(14) rather invariant contribution of the Stark effect of emission.

x∫ νjBem Thus, we expect an increasing signal-to-noise ratio for an

f (νj ) dνjem
νjAem νj em illumination νj/ex at the red end of the excitation spectrum. Of
course, the signal-to-noise ratio is proportional to the spectral
Here the signal-to-noise ratio is expressed in terms of (i) shift by the Stark effect.
the spatio-temporal resolution τDAmem of the setup, (ii) the
number of excitations per area and time Iillndye(νj/ex), (iii) the Conclusions
yield of detected quanta TrecΦem, and (iv) the sensitivity function
Sνj(νj/ex,νjem) weighted by the fluorescence spectrum fνj(νjem) Anellated hemicyanine dyes ANNINE-5 and ANNINE-6
within the spectral limits of detection. With the constraint exhibit voltage sensitivities in a neuron membrane that are
distinctly higher than those of the classical styryl dyes. These
Iill(νj/ex) ) const, the choice of an optimal wavenumber of
novel probes rely on a well-defined physical mechanism, the
excitation νj/ex and the choice of optimal emission filters molecular Stark effect. Their excellence in the optical recording
νjAem and νjBem are determined by a weighted sensitivity function of neuronal excitation is due to a large intramolecular charge
〈SV(νj/ex)〉, defined by eq 15. shift in connection with suitable Franck-Condon factors of
vibroelectronic transitions and a high quantum yield of fluo-
∫νjνj
B

A
em
fνj(νjem) Sνj(νj/ex,νjem) dνjem rescence. Further improvements to the voltage-sensitive dyes
〈SV(νj/ex)〉 )
em
(15) must be directed toward higher photochemical stability and

x∫ νjem
B
fνj(νjem) dνjem lower phototoxicity and toward the selective staining of
νjAem individual cells in a tissue.
For the ANNINE dyes, the sensitivity Sνj(νj/ex,νjem) (Figure 5) Acknowledgment. We thank Gerd Hübener, Birgit Haringer,
reaches its largest negative values in the red corner of the and Michaela Morawetz for the synthesis of the dyes, Armin
spectrum. We expect a high signal-to-noise ratio when we excite Lambacher for discussions, and Marlon Hinner and Armin
monochromatically at a rather low wavenumber νj/ex. To keep Lambacher for critically reading the manuscript. This project
Iill(νj/ex) constant, we have to compensate the low cross section was supported in part by the Fonds der Chemischen Industrie.
of absorption by an enhanced illumination intensity. Of course,
we have to limit the detection to the range of negative response. References and Notes
For illustration, the weighted sensitivity function 〈SV(νj/ex)〉
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