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Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, Martinsried/Munich, Germany
Correspondence: Address reprint requests to Peter Fromherz, Dept. of Membrane and Neurophysics, Max Planck Institute for Biochemistry, Martinsried/Munich, Germany 82152. E-mail: fromherz{at}biochem.mpg.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 cm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at 
1.5 G. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 cm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Pine, 1980; Thomas et al., 1972; Regehr et al., 1988). Neurons were also coupled to electrically insulated semiconductors (Fromherz et al., 1991; Fromherz and Stett, 1995) which offer an interfacing without electrochemical currents that may damage electrodes and cells. Crucial for the strength of coupling between neurons and insulated silicon or metallic contacts is the seal resistance. It is determined by the thin sheet of electrolyte between cell membrane and oxidized silicon. This sheet of electrolyte together with the adjacent insulating films of membrane and oxide define a planar core-coat conductor that is open at its periphery to the bath (Weis et al., 1996; see also this article, Fig. 1 a). The sheet resistance and the capacitance of membrane and silicon oxide determine the electrical coupling between cells and chip in both directions—for stimulation and for detection of neuronal excitation.
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; Lo and Ferrier, 1998; Wegener et al., 2000) which allows to infer only global electrical properties of confluent cell layers with ambiguous interpretation of the seal resistance. We use fluorescent amphiphilic dyes which are well established to record voltage transients in neurons at low to medium resolution (Cohen and Salzberg, 1978; Fluhler et al., 1985; Grinvald et al., 1983; Gross et al., 1986; Hibino et al., 1991; Meyer et al., 1997; Rohr and Salzberg, 1994; Windisch et al., 1995). We show how a lock-in approach can image cellular response maps at the limit of optical resolution in three dimensions.
Voltage in cell-solid junction
We consider a cell on an electrically conductive solid with no Faradayic current across the solid-electrolyte interface (Fig. 1 a). Cell membrane and solid/electrolyte interface have area-specific capacitances cM and cS. We assume that the cleft between cell and substrate has a constant sheet resistance rJ and a constant width dJ, small compared to the diameter of the junction, with a negligible transition region at the periphery (Braun and Fromherz, 1998). The cell-silicon contact is a planar core-coat conductor. Ionic conductances of the membrane can be neglected for intact cells with closed ion channels (Weis and Fromherz, 1997). We apply a changing electrical potential VS(t) to the substrate with the bulk electrolyte at constant potential VE = const. The junction reacts with a potential profile VJ(x,y,t) and the cytoplasm with a potential VM(t). This gives rise to voltages VM–VJ and VM–VE across attached and free membrane.
Plate contact model
The balance of current in each area element of the junction is symbolized by the circuit of Fig. 1 a. The current along the cleft and through the attached membrane is driven by capacitive stimulation from the solid (Eq. 1). In the cell, the current through the total attached membrane is balanced by the current through the free membrane according to Eq. 2, where 
VJ
is the spatially averaged potential in the junction and ßM = AJM/AFM is the ratio of attached and free membrane area.
 | (1) |
 | (2) |
). The left-hand side describes the diffusion of charge along the core-coat conductor and the right-hand side accounts for nonlocal coupling by the cytoplasm and for capacitive stimulation. The boundary condition is VJ(x,y,t) = VE at the periphery of the attached membrane.
 | (3) |
Time constant
We disregard the nonlocal coupling in Eq. 3 by replacing the average with the local potential VJ = VJ and assume a circular contact of radius aJ. After a voltage step 
we find a leading term VJ(t) 
exp(–t/
J) with a characteristic time constant 
where (
1aJ)2 = 5.783 is defined by the first zero of the Bessel function J0 (1aJ) = 0 (Crank, 1975). We use J and its related frequency fJ for global characterizations of junctions of area AJM according to Eq. 4:
 | (4) |
AC stimulation
For sinusoidal stimulation with a frequency f we consider the complex Fourier amplitudes VJ and VS. From Eq. 3 we obtain for the transfer function hJ = (VJ–VE)/(VS–VE):
 | (5) |
We solve it for two dimensions with boundary condition hJ = 0 by an overrelaxation algorithm. The transfer functions between chip stimulation and free and attached membrane are defined by hFM = (VM–VE)/(VS–VE) and hJM = (VM–VJ)/(VS–VE). They are obtained as hFM = hJ ßM/(1 + ßM) and hJM = hFM – hJ. For illustration, they were calculated for a circular junction of radius aJ using cS/cM = 0.33 and ßM = 0.5. Fig. 2 shows radial amplitude and phase profiles of hJM (a/aJ) along with hFM in the surrounding for six relative frequencies f/fJ.
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1 there is a shallow phase trough across the junction. The phase profile is shifted from –90° toward –180° with increasing frequency. The amplitudes at high frequency are 
and 
determined by the capacitive voltage divider. At intermediate frequencies f/fJ 1 there is a distinct cupola of the amplitude in the junction with a narrow minimum near the periphery. |
MATERIALS AND METHODS |
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). For chip type B, we used highly p-doped silicon wafers (4" diameter, 400 µm thickness, 100 crystal surface, polished one side, 2–4 m, cm, Aurel, Landsberg, Germany). A field oxide of 1070 nm was grown. A circular stimulation spot (diameter 500 µm) was opened on the front side and covered with 16 nm or 50 nm silicon oxide. The back of the chip was contacted by 200 nm chromium. Chips were cut to octagons of 4 mm diameter and attached to 35-mm plastic dishes (Falcon 3001, Becton-Dickinson, Heidelberg, Germany) with a silicone glue (MK3, Sulzer Medica, Köln, Germany).
Cells
Erythrocyte ghosts were prepared according to a protocol of Schwoch and Passow (1973), as described previously (Braun and Fromherz, 1997). Chips were wiped with a detergent (2% Tickopur RP100, Bandelin, Berlin, Germany) at 60°C, rinsed with Milli-Q water (Millipore, Billerica, MA), dried with nitrogen and sterilized by ultraviolet (30 min). Several hours before the measurement a solution of 0.5 mg/ml poly-L-lysine (MW 10,000, Sigma, Heidelberg, Germany) in TRIS buffer was applied and rinsed three times with TRIS buffer. Human embryonic kidney (HEK293) cells (Graham et al., 1977) were obtained from ATCC (Manassas, VA; CRL-1573). Before culturing, cleaned chips were coated with fibronectin (Sigma, 25 µg/ml in PBS overnight) or poly-L-lysine (Sigma, MW 10,000, 100 µg/ml in PBS overnight). Trypsinated cells were cultured on the chips for 2–3 days at 37°C in 5% CO2 to a confluency of 50% in 3 ml medium (13.9 mg/ml DMEM (074-02100A, Gibco, Invitrogen, Karlsruhe, Germany) with 10% heat-inactivated fetal bovine serum (FBS, Seromed, Berlin, Germany), 3.7 mg/ml NaHCO3 (Sigma), 4 mM L-glutamine, 25 U/ml penicillin and streptomycin (Gibco) at pH 6.8. Madine Darby Canine Kidney (MDCK) cells were obtained from European Collection of Cell Cultures (Cambridge, UK; 85011435). The cleaned chips were coated with poly-L-lysine (Sigma, MW 10000, 100 µg/ml in PBS overnight). Trypsinated cells were cultured on the chips in EMEM (21885-025, Gibco) with 2 mM glutamine, 1% non-essential amino acids (11140-035, Gibco), and 10% fetal bovine serum (10270098, Gibco). Other treatments were identical to HEK cells. Culturing neurons from embryonic rat brain followed previously described protocols (Banker and Cowan, 1977; Vassanelli and Fromherz, 1999).
Voltage-sensitive dye
We used the amphiphilic hemicyanine dye dibutylamino-naphtalene-butylsulfonato-isoquinolinium, i.e., BNBIQ (this article, Fig. 1 b; see also Ephardt and Fromherz, 1993). Voltage-sensitive fluorescence in a cell membrane is due to identical spectral shifts of excitation and emission that are caused by interaction of the electrical field with intramolecular charge-displacement (Kuhn and Fromherz, 2003). The response time of fluorescence changes induced by such a molecular Stark effect is limited by the fluorescence lifetime which is in a range of 1 ns (Röcker et al., 1996). The relative change of fluorescence intensity F is proportional to a change of transmembrane voltage as 
F/F = SDYE VM with a sensitivity SDYE 
–10%/100 mV in leech neurons (Fromherz and Müller, 1993; Kuhn and Fromherz, 2003). We prepared a saturated dye solution in a 1:1 mixture of fetal bovine serum and Milli-Q water and added 40 µl to the culture medium. The experiments started after a few minutes to avoid internalization of the dye.
Stimulation
The bath was contacted with a 3 x 30 mm platinum electrode. An AC voltage from a function generator (model 33120A, Hewlett-Packard, Böblingen, Germany) with an amplitude 
was applied to the chip against the bath potential. It was superposed by a DC bias to the chip of 
The frequency was chosen near the presumed characteristic frequency fJ. In some experiments we applied a train of rectangular voltage pulses with an amplitude of 
a chip bias of +7 V, and a pulse duration of 4 µs.
Fluorescence
A confocal microscope (FluoView 1.26, Olympus, Hamburg, Germany) was used to achieve optical recording in three dimensions. We used a water objective (UApo/340, NA = 1.15, 40x) with built-in coverslip and no plan correction. On the basis of the spectral sensitivity of BNBIQ (Kuhn and Fromherz, 2003) we used an excitation at 488 nm (Ar laser, Stabilite 2017, Spectra Physics, Irvine, CA) and an emission filter between 590 and 700 nm (AHF, Tübingen, Germany). Fluorescence was detected with a photomultiplier (R928, Hamamatsu, Bridgewater, NJ; built-in amplifier, Olympus) and sampled at 5 MHz with 12 bits (PCI 6110E, National Instruments, Austin, TX). For each pixel we correlated the photomultiplier signal VPM to the stimulation voltage VSTIM using a lock-in algorithm. The in-phase and out-of-phase amplitudes Vx, Vy were obtained from the scalar product of the time average according to Eq. 6 with the appropriate phase of the reference VSTIM.
 | (6) |
The complex relative signal 
was defined by VPM = Vx + iVy and 
with background Vback. A typical scan included 64 x 64 pixels, with a dwell time of 4 ms per pixel to limit bleaching and phototoxic effects. 100 to 500 periods were considered at frequencies f = 25–125 kHz. The signal/noise ratio was 3%. It was further reduced by binning several pixels.
Phase corrections
The signal transfer from the function generator stimulus VSTIM to the relative output of the photomultiplier was determined by the product of transfer functions 
They are hCHIP = (VS–VE)/VSTIM from generator to chip, hJM (or hFM) from chip to membrane, SDYE = (F/F)/VM–VJ) from membrane voltage to relative fluorescence, and 
from relative fluorescence to relative photomultiplier signal. The product of transfer function hJM (or hFM) with dye sensitivity SDYE equals the relative modulation of fluorescence F/[F(VS–VE)] per chip voltage. It is obtained from dividing the measured signal by the transfer functions hPM and hCHIP and by the stimulus 
according to Eq. 7.
 | (7) |
The transfer function of the chips hCHIP was obtained by monitoring the current across a 12 resistance using a digital storage oscilloscope. It was modeled by a lowpass filter hCHIP (f) = 1/(1 + i2
fCHIP) with time constants CHIP = 0.3–1.2 µs, depending on oxide thickness and cell confluency. The transfer function of the photomultiplier hPM was determined from AC illumination by a LED. It was fitted by a filter of fourth order hPM = [(1 + a1P + b1P2) (1 + a2P + b2P2)]–1 with P = i f/fB and fB = 615 kHz with a1 = 0.7743, b1 = 0.3890, a2 = 1.3397, and b2 = 0.4889.
Data fit with plate contact model
The sheet resistance rJ was obtained as follows. Since the phase signal is not affected by the local dye sensitivity, we can fit the phase of the measured map –F/[F(VS–VE)] hJM and –F/[F(VS–VE)] hFM at the contact and in its surrounding using the plate contact model. We solved Eq. 5 on the lattice of the experimental pixels and fitted hJM and hFM to the phase map with varied sheet resistance rJ and given values of the capacitances. In a second step, the amplitude was fitted with a constant sensitivity SDYE < 0. Crucial for a solution of Eq. 5 was the definition of the border of adhesion where the boundary condition hJ = 0 holds. We defined it with a phase threshold of –45°. This approach was justified by the steep slope of the phase profile between 0° and –90° near the boundary (Fig. 2 a). Pixels at the periphery with phase values larger than the threshold were attributed to the upward bulging free membrane and are fitted with hFM. We visualized all experiments in terms of transfer functions hJM and hFM, which means that the amplitude signal was scaled with a fitted global dye sensitivity SDYE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). We stained erythrocyte ghosts on chip type A with 10 nm thickness of oxide and imaged the fluorescence with 64 x 64 pixels each probing 0.17 µm x 0.17 µm (Fig. 3 a). The adhesion area is marked by a bright rim where the membrane bulges upwards. We applied an AC voltage between silicon and bath with amplitude 
and frequency f = 25 kHz. The modulation of fluorescence was recorded in amplitude and phase. As described in Materials and Methods, we corrected for delays of chip and photomultiplier amplifier and scaled with the sensitivity of the dye to infer the transfer functions hJM and hFM. The signal quality was improved by a sliding average across 3 x 3 pixels with a final resolution of 0.5 µm.
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(hJM) –180° in the center and (hJM) –150° toward the periphery. In the surrounding, it jumps to (hFM) +20° where the signal is dominated by the upward bulging free membrane. The amplitude exhibits a broad maximum in the area of adhesion that decays to low values in the periphery. There it forms a narrow valley before it rises to higher values given by the amplitude of the free membrane.
The electrical properties of the contact were fitted from the phase map which revealed a sheet resistance rJ = 1500 ± 200 M with the given constants cS = 0.34 µF/cm2, cM = 1 µF/cm2, and ßM = 0.5. For illustration, phase and amplitude profiles are also plotted for a sheet resistance of rJ = 500 M with dashed lines in Fig. 3, d–e. We estimate a systematic error of 20% based on uncertainties of 0.3 nm for the oxide thickness, 0.15 µF/cm2 for the membrane capacitance, 0.05 for ßM, and 10% for the adhesion area. The characteristic frequency is fJ = 6.4 kHz, based on the adhesion area AJM = 30 µm2. The amplitude was fitted with a sensitivity SDYE = –2.5%/100 mV. The theoretical amplitude and phase are shown as solid lines in the profiles of Fig. 3, d–e, and as two-dimensional maps in Fig. 3, f–g.
HEK293 cells
HEK293 cells have been used for interfacing transistors and recombinant ion channels (Straub et al., 2001). They have a smooth membrane with low intrinsic ion conductance (Zhu et al., 1998). A large HEK293 cell on chip type A with 50 nm oxide is shown in Fig. 4 a with a resolution of 0.66 µm per pixel. Again a bright rim indicates where the membrane bulges upwards. We observed a distinct staining of the cytoplasm. Fig. 4, b–d, depicts phase and amplitude maps of the transfer functions hJM and hFM for f = 125 kHz and 
The phase map shows again two regions separated by a sharp boundary: a shallow trough in the adhesion area ( hJM) –150° and a surrounding with (hFM) +30°. Fig. 4 d indicates a cupola of the transfer amplitude in the adhesion area. In contrast to the phase signal, the amplitude map looks rather erratic, suggesting that the effective dye sensitivity decreases due to enhanced inhomogeneous background of insensitive dye bound to intracellular structures.
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with the constants previously used for erythrocyte ghosts. The theoretical phase profile is plotted in Fig. 4 c. To illustrate the accuracy, a profile with rJ = 15 M is drawn with a dashed line. We infer fJ to be 76 kHz based on an adhesion area AJM = 470 µm2. We find a maximal dye sensitivity of SDYE = –6%/100 mV. Measurements with HEK293 cells of different shape yielded a mean sheet resistance of rJ = 8.8 ± 0.6 M (N = 10) on chips coated with fibronectin and slightly higher values of rJ = 12.6 ± 1.3 M (N = 8) on poly-L-lysine.
MDCK cells
MDCK cells are known for their high cell-cell resistance in confluent cultures (Lo et al., 1995). For that reason they are interesting candidates for bioelectronic devices. Using the same approach as with HEK293 cells, we found for single MDCK cells on polylysine almost the same features as for HEK293 cells (data not shown) with a similar sheet resistance of rJ = 9.3 ± 1 M (N = 2).
Rat neurons
The sheet resistance between nerve cells and silicon is crucial for the interfacing in neuroelectronic devices (Weis and Fromherz, 1997). We cultured rat neurons on type A chips with 50-nm oxide coated with poly-lysine under the conditions used for neuron-transistor junctions (Vassanelli and Fromherz, 1999). A neuron is shown in Fig. 4 e with a resolution of 0.42 µm, 3 x 3-binned to 1.2 µm. The phase and amplitude maps of the transfer function hJM and hFM for f = 125 kHz and 
are depicted in Fig. 4, f–h. The patterns of response are similar to HEK293 cells. The phase map shows two regions with a plateau in the adhesion area (hJM) –120° and a surrounding with (hFM) +80°. In the center of the cell, where the phase map is smooth, the amplitude signal is locally reduced. Those indentations correlate with high total fluorescence. The intracellular staining leads to an lower effective voltage sensitivity.
We fitted the phase map with a sheet resistance rJ = 14 ± 4 M for cS = 0.07 µF/cm2, cM = 1 µF/cm2, and ßM = 0.5 as shown in Fig. 4 g (solid line). We infer fJ to be 250 kHz based on an area AJM = 110 µm2. For illustration, a phase map was computed also for rJ = 23 M as shown in Fig. 4 g (dashed line). The maximum dye sensitivity was found to be SDYE = –8%/100 mV.
Vertical scan of cell assembly
The presented method can be used to map cell-solid junctions in three dimensions. As an example, we performed a vertical scan through a bundle of HEK293 cells grown on chip type B with 50-nm-thick oxide. We can distinguish the attached membranes, the upper membranes, and the contacts of the cells as shown in the fluorescence image in Fig. 5 a. A section of phase and amplitude of the transfer function is shown in Fig. 5, b–c, for f = 125 kHz and an amplitude with a sliding average over 2 x 2 pixels (resolution 0.8 µm horizontal, 0.3 µm vertical). The amplitudes were scaled with SDYE = –6%/100 mV. We found that the phase is (hJM) –140° for the attached membranes and the lower parts of the cell-cell contacts. For the upper parts of the cell-cell contacts and the outer membranes we found (hFM) +20°. There is a phase inversion between attached and free membrane at the periphery of the cell bundle as with individual cells. However, between two adjacent cells the phase change occurs at half the cell height. Further analysis reveals a linear drop in membrane potential along the cell-cell contact.
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; Zeck and Fromherz, 2001). Optical recordings of the transient voltage in the attached membrane were reported for HEK293 cells (Braun and Fromherz, 2001). We tested the compatibility of AC stimulation and pulse stimulation for the cell already measured in Fig. 4, a–d. A train of voltage pulses was applied with a width of 4 µs, a period of 8 µs, and an amplitude of –6 V. Along the vertical white line in Fig. 4 a, we recorded the fluorescence over time with a resolution of 0.2 µs as shown in Fig. 6 a. Time traces from the attached membrane show a drop of fluorescence followed by a rise in the second half of the stimulation period. Toward the periphery, the transients fall off faster. The neighboring free membrane shows an inverted characteristic.
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as determined by the AC measurements, we computed the membrane potential as a function of time using the area contact model. It was convoluted with the transfer functions of chip and photomultiplier amplifier according to Eq. 7 (Braun and Fromherz, 2001) to obtain the expected fluorescence signal, as shown in Fig. 6 b, matching the measurement in all details. As expected, the AC measurements were sufficient to predict the behavior of the contact over time. It can be seen how the diffusive nature of the planar core-coat conductor leads to a faster falloff near the periphery. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Measurements with voltage-sensitive dyes are plagued by inhomogeneous sensitivities due to background staining. Since our technique relies only on the phase of the fluorescence signal, it can cope with large variations in voltage sensitivity (Fig. 4). Bleaching of the dye did not play a role and several scans of a cell were possible (Fig. 4, a–d, and Fig. 6). By increasing the stimulation voltage, large membrane potentials can be applied. Above 800 mV, cell death is indicated within half a minute by extracellular vesiculation, probably initiated by electroporation (Benz et al., 1979).
Seal resistance
Since we used the shape of each individual cell we could minimize systematic and statistic errors in the determination of the sheet resistance rJ. In cell-silicon systems the distance dJ between the insulating films of lipid bilayer and silicon dioxide is known from independent measurements by fluorescence interference contrast microscopy (Braun and Fromherz, 1998, 1997; Lambacher and Fromherz, 1996, 2002). So the sheet resistance rJ could be interpreted in terms of a specific resistance 
J = rJdJ in the cleft between cell and chip. For HEK293 cells on fibronectin we infer from rJ = 11 M and dJ = 50 nm (data not shown) a specific resistance of J = 55 cm, close to the bulk electrolyte J = 75 cm. For rat neurons on polylysine we infer from rJ = 14 M and dJ = 60 nm (Braun and Fromherz, 1998) a specific resistance of J = 84 cm, close to the bulk electrolyte J = 115 cm. Similar relations were found for HEK cells on polylysine and single MDCK cells. We conclude that for all these cells, the cleft of 50 nm between the lipid bilayer of the plasma membrane and silicon dioxide is filled with electrolyte from the surrounding culture medium. However, for erythrocytes on chips coated with polylysine, the sheet resistance of rJ = 1.5 G is higher by two orders of magnitude. Together with the close distance dJ = 11 nm (Braun and Fromherz, 1997), we infer a specific resistance J = 1650 cm, which is 20-fold higher than = 75 cm of the bulk electrolyte.
We therefore see that a surface of cells, which flow freely like red blood cells, gives different seal characteristics than cells of brain, kidney, and epithelium which are usually embedded in a tissue. Erythrocytes have a glycocalix with a thickness of 6 nm (Linss et al., 1991). We assume that here the narrow cleft induces polyelectrolyte effects in the glycocalix/polylysine layer which affect mobility and concentration of ions.
Relation to literature
Previous approaches to study the seal resistance of cells and electrodes lacked high spatial resolution. For that reason a global seal resistance RJ of the junction was considered (Regehr et al., 1988). A relation between global resistance and sheet resistance RJ = rJ/5 was proposed (Weis and Fromherz, 1997). From Eq. 4 we obtain a modified relation RJ = rJ/5.783. The area-specific conductance of the junction is 
for a radius aJ. A similar parameter 
was used for confluent cell layers (Lo et al., 1995).
For erythrocytes on polylysine, gJ = 132 mS/cm2 was reported from extracellular AC stimulation and transistor recording (Kiessling et al., 2000). With aJ = 3 µm the sheet resistance is rJ = 0.5 G. For neurons on polylysine, gJ 1000 mS/cm2 was determined by intracellular AC stimulation and transistor recording (Ephardt and Fromherz, 1993). With aJ = 6 µm the sheet resistance is rJ = 16 M. These values are similar to our recordings, although they were obtained from a single transistor probe at an unknown location. Impedance measurements of confluent cell layers on gold were evaluated with 2 = 49 cm2 and 12.3 cm2 (Lo et al., 1995), 2 = 324 cm2 and 400 cm2 (Lo et al., 1995), 2 = 4.84–13.0 cm2 (Lo and Ferrier, 1998), and 2 = 522 cm2 (Wegener et al., 2000). The given radii were aJ = 7–16 µm. The data of Lo and Ferrier (1998) lead to sheet resistances rJ = 9.9–26.5 M, which are similar to our results. However, the high 2 values of Lo et al. (1995) and Wegener et al. (2000) would lead to extremely high sheet resistances. The separation of sheet resistance and seal resistance between the cells may be ambiguous in the evaluation of global impedance measurements. By pulse stimulation and optical recording (Braun and Fromherz, 2001) with MDCK cell layers we observed a time constant of several milliseconds, as it is expected for a high seal resistance between the cells (data not shown).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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In the present study we focused on electrical imaging of single cells to demonstrate the resolution of the method and to confirm microscopic details of electrical coupling between cells and electrodes. We find the novel method to be most useful to screen for better electrode coatings and to study other improvements of cell-electrode contacts. Such microscopic circuit information is crucial for both the stimulation and recording of electrical signals of nerve cells and neuronal assemblies. The microfluorometric approach is also able to reveal details of more complex geometries such as in tissue on electrodes. For example, details of voltage spread in brain slices could be revealed (Fromherz, 2002). In that case, two-photon excitation may be applied to achieve sufficient resolution in the tissue. A bottleneck here, however, is the staining with amphiphilic hemicyanines as their extracellular application is effective only a few cell layers deep. Cell-selective methods are required such as microinjection or genetic labeling, both not yet developed for these dyes.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The project was supported by a generous grant of the Bundesministerium für Bildung und Forschung.
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FOOTNOTES |
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Submitted on January 15, 2004; accepted for publication April 1, 2004.
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durations and intervals 4 µs). (a) Relative change of photomultiplier response for one period along the vertical bar shown in