GigaOhm Seal by Membrane Spreading - American Chemical Society

Langmuir 2004, 20, 11175-11182

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Giant Lipid Vesicles Impaled with Glass Microelectrodes: GigaOhm Seal by Membrane Spreading Christian H. Reccius and Peter Fromherz* Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, Martinsried/Mu¨ nchen, Germany 82152 Received July 14, 2004. In Final Form: September 6, 2004 Giant unilamellar lipid vesicles could be perfect systems to study ion channels in the environment of lipid membranes with defined chemical and physical properties. Prerequisite for electrical measurements is an intravesicular electrical contact. We describe the impalement of giant lipid vesicles by glass micropipet electrodes with a tight seal. To avoid displacement or burst during impalement, the vesicles are immobilized in relaxed conditions by microscopic picket fences of polyimide. The outer surface of the pipets is selectively coated with silanes or polylysine. Structurally, the impalement is verified by ejecting a fluorescent solution out of the pipet. For electrical characterization, current pulses are applied to the pipet and voltage transients are recorded. The data are evaluated in terms of the capacitance and effective resistance of the membrane. Directly after impalement, we observe a seal resistance up to 1.2 GΩ that continuously decays within a period of up to 20 min until it suddenly disappears without burst of the vesicle. During impalement, a spreading of the vesicle membrane along the outer surface of the pipets is observed using a fluorescent membrane-bound dye. We assign the tight pipet-vesicle contact to spreading of the lipid bilayer by a rolling mechanism and the loss of resistance to micro- and macropores that are induced by the resulting membrane tension. Limitation of spreading is attempted with barriers on the pipet.

Introduction Ion channels of biological membranes are embedded in the complex matrix of lipid bilayers. To investigate their interactions with the environment, they must be incorporated into membranes with defined composition. Two systems have been considered: (i) black lipid membranes (BLMs)1,2 that separate two compartments with Ag/AgCl electrodes and (ii) dispersions of small unilamellar vesicles (SUVs).3 BLMs allow electrical measurements with high sensitivity and time resolution.4-8 But usually they are not free of solvent and they suffer from a lack of a simple method for reconstituting ion channels. With SUVs (diameter < 100 nm), there is no solvent and reconstitution of membrane proteins can be achieved by dialysis. Slow transport processes have been studied with radioactive tracers and fluorescent dyes,9-11 but electrical measurements are not possible due to their small size. In principle, the advantages of BLMs and SUVs could be combined with giant unilamellar vesicles (GUVs).12-14 They are free of solvent, and their diameter (>1 µm) allows impalement with micropipets as applied for the injection of dyes, salt,15 * To whom correspondence should be addressed. E-mail: [email protected]. (1) Mueller, P.; Rudin, D. O.; Tien, H. T.; Wescott, W. C. Nature 1962, 194, 979-980. (2) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561-3566. (3) Huang, C. Biochemistry 1969, 8, 344-352. (4) Korenbrot, J. I.; Hwang, S. B. J. Gen. Physiol. 1980, 76, 649-682. (5) Schindler, H. FEBS Lett. 1980, 122, 77-79. (6) Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3057-3061. (7) Montal, M.; Darszon, A.; Schindlers, H. Q. Rev. Biophys. 1981, 14, 1-80. (8) Miller, C. Comm. Mol. Cell. Biophys. 1983, 1, 413-428. (9) Wu, W. C. S.; Moore, H. P. H.; Raftery, M. A. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 775-779. (10) Eytan, G. D. Biochim. Biophys. Acta 1982, 694, 185-202. (11) Miller, C. Annu. Rev. Physiol. 1984, 46, 549-558. (12) Reeves, J. P.; Dowben, R. M. J. Cell. Physiol. 1969, 73, 49-60. (13) Oku, N.; Macdonald, R. C. Biochemistry 1983, 22, 855-863. (14) Angelova, M. I.; Dimitrov, D. S. Faraday Discuss. 1986, 303311.

enzymes,16 DNA,17 latex beads, and SUVs.18 Electrical measurements have been described by impaling micropipets19 or by patching techniques in a whole-vesicle configuration.20 But very low effective resistances were found for the membrane-pipet system. Electrical measurements with ion channels were reported only for insideout patches on micropipets.19-21 In the present paper, we describe a novel method to impale giant vesicles with glass micropipets such that high seal resistances up to 1.2 GΩ are attained. Impalement without burst of the vesicles is achieved by immobilizing the vesicles in relaxed conditions (see Figure 1). High resistance is controlled by selective coating of the outer pipet surface. The time-dependent electrical properties of the systems are determined by repetitive currentvoltage measurements. Materials and Methods Vesicles. Giant vesicles were prepared by electroswelling14,16,22-25 using mixtures of palmitoyl-oleoyl-phosphatidylcholine (POPC, Avanti Polar Lipids, Alabaster, AL), dioleoylphosphatidylcholine (DOPC, Sigma-Aldrich, Steinheim, Ger(15) Menger, F. M.; Lee, S. J. Langmuir 1995, 11, 3685-3689. (16) Wick, R.; Angelova, M. I.; Walde, P.; Luisi, P. L. Chem. Biol. 1996, 3, 105-111. (17) Bucher, P.; Fischer, A.; Luisi, P. L.; Oberholzer, T.; Walde, P. Langmuir 1998, 14, 2712-2721. (18) Karlsson, M.; Nolkrantz, K.; Davidson, M. J.; Stromberg, A.; Ryttsen, F.; Akerman, B.; Orwar, O. Anal. Chem. 2000, 72, 5857-5862. (19) Higashi, K.; Suzuki, S.; Fujii, H.; Kirino, Y. J. Biochem. (Tokyo) 1987, 101, 433-440. (20) Keller, B. U.; Hedrich, R.; Vaz, W. L. C.; Criado, M. Pflugers Arch. 1988, 411, 94-100. (21) Tank, D. W.; Miller, C.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7749-7753. (22) Dimitrov, D. S.; Angelova, M. I. Bioelectrochem. Bioenerg. 1988, 19, 323-336. (23) Angelova, M. I.; Soleau, S.; Meleard, P.; Faucon, J. F.; Bothorel, P. Prog. Colloid Polym. Sci. 1992, 89, 127-131. (24) Mathivet, L.; Cribier, S.; Devaux, P. F. Biophys. J. 1996, 70, 1112-1121. (25) Fromherz, P.; Kiessling, V.; Kottig, K.; Zeck, G. Appl. Phys. A 1999, 69, 571-576.

10.1021/la048233h CCC: $27.50 © 2004 American Chemical Society Published on Web 11/02/2004

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Langmuir, Vol. 20, No. 25, 2004

Reccius and Fromherz

Figure 1. Electrical contact of a giant lipid vesicle. A vesicle is immobilized without strong adhesion in relaxed conditions by a fence of polyimide columns on a silicon chip. It is impaled with a glass micropipet with a Ag/AgCl electrode. Voltage or current is applied with respect to a Ag/AgCl electrode in the bath. many) and dioleoyl-phosphatidyl-methylester (DOPME, SigmaAldrich) with the lipoid cyanine dye DiIC18(3) (Molecular Probes, Eugene, OR). The lipids were dissolved in diethyl ether/methanol (volume ratio, 9:1). The solution (5 µL) was applied to two planar electrodes of indium tin oxide coated with 70 nm of silica.25 After drying in a vacuum overnight at a pressure below 0.1 mbar, we added 2 mL of 300 mM sucrose (Merck, Darmstadt, Germany) and applied an ac voltage of 10 Hz, ramping the amplitude from 50 mV to 3 V within 2 h. Attached giant vesicles were dissociated by applying an ac voltage of 3 V amplitude and frequencies of 4 Hz for 20 min, 1 Hz for 10 min, and 0.1 Hz for 10 min. We sucked 0.5 mL of suspension into a wide plastic pipet and added it to 2.5 mL of 300 mM glucose (Merck). The vesicle dispersion (100 µL) was applied to 2 mL of medium on the chips which contained 300 mM glucose, 40 mM NaCl, and 5 mM Tris/HCl buffer at pH 7.4. The higher density of intravesicular sucrose promoted sedimentation of the vesicles. In case of chips with picket fences (see below), positioning was carried out by slight movements of the chip. This method reduced stress on the vesicles and fences compared to positioning with a large pipet. The electrical and optical experiments were started immediately after positioning. Silicon Chips with Picket Fences. Silicon chips (25 mm × 25 mm) with natural oxide (1 nm) were cut from 100 n-type silicon wafers (diameter, 100 mm; resistance, 2-4 Ω cm). They were covered with 4600 picket fences consisting of 6 columns with a height of 80 µm and a diameter of 25 µm. The diameter of the cages was 70-150 µm. After the chips were dehydrated in an oxygen plasma for 5 min, they were covered by photosensitive polyimide (Durimide 7020, Arch Chemicals, Zwijndrecht, Belgium) by spin coating at 1500 rpm for 10 s under red light. After prebaking for 2.5 h, starting at 35 °C and ending at 105 °C, they were illuminated for 115 s in the near UV through a photographic mask. After 15 min, the chips were carefully spun for 17-22 min in a developer (HTR-D2, Arch Chemicals), rinsed in a 1:1 mixture of developer and rinser (RER 600, Arch Chemicals) for 10 s, and rinsed for 20 s in pure rinser. To avoid damage by the surface tension of evaporating rinser, the chips were dipped in ethanol for 30 s and diethyl ether for 30 s. The fences were cured under nitrogen up to a temperature of 300 °C that was reached in 160 min, held for 110 min, and lowered in 210 min. Before the experiments, the chips were cleaned by sonication for 5 min in a 2% solution of Tickopur (RP 100, Bandelin, Berlin, Germany) at 20 °C, rinsed with a Milli-Q (Millipore, Bedford, MA) water jet, and dried with nitrogen. Finally they were coated with bovine serum albumin (A-4503, Sigma-Aldrich) by adsorption from a 2 mg/mL solution in phosphate buffered saline (Life Technologies, Paisley, U.K.) for at least 3 h and intensively rinsed with Milli-Q water. A chip with picket fences is shown in Figure 2. In preliminary experiments, we used silicon chips without picket fences. A layer of silicon dioxide (20 nm) was formed by wet oxidation. The chips were wiped with an alkaline detergent (2%, Tickopur) at 80 °C and rinsed with Milli-Q water. Then they were coated with poly-L-lysine hydrobromide (P-6516, MW ) 4000-15000, Sigma-Aldrich) by adsorption from a 25 µg/mL aqueous solution for 30 min. Micropipets. Micropipets were fabricated from borosilicate glass capillaries with internal filament (no. 1403547, Hilgenberg,

Figure 2. Picket fences of polyimide on an oxidized silicon chip for giant vesicles. (A) Pattern of picket fences with cages of a diameter of 70-150 µm. Electron micrograph taken at an angle of 60° to the surface. The white rectangle marks a unit cell. The circle highlights a single cage. The columns are 80 µm high and have a diameter of 25 µm. (B) Blowup of a selected hexagonal picket fence with an inner diameter of 70 µm. Electron micrograph taken at an angle of 20° to the surface. Malsfeld, Germany) on a multipurpose puller (DMZ Universal Puller, Zeitz, Augsburg, Germany). The inner and the outer diameter of the micropipet tips was 360 and 380 nm as estimated from scanning electron micrographs. They were coated with dimethyldichlorosilane (no. D-3879, Sigma-Aldrich), aminosilane (3-(2-aminoethylamino)propyl-methyl-dimethoxysilane, no. 06667, Fluka, Neu-Ulm, Germany), or poly-L-lysine hydrobromide (no. P-6516, MW ) 4000-15000, Sigma-Aldrich). The main problem was the clogging of the micropipets. For coating with dimethyldichlorosilane, the micropipet tips were submersed in a 2% silane solution in toluene for 15 min and rinsed for 15 min in pure toluene and for 5 min in Milli-Q water. Clogging was prevented by using micropipets filled with electrolyte. For coating with aminosilane and polylysine, a high pressure of inert gas up to 34 bar was applied to the pipets to prevent inside clogging of the pipets by capillarity effects. The pressure holder is illustrated in Figure 3. Coating with aminosilane was performed similar to a method described by Weller et al.26 The micropipet tips were cleaned in ethanol for 30 min and in freshly prepared hot piranha solution (2:1 mixture of 96% sulfuric acid and 31% hydrogen peroxide) for 30 min at a nitrogen pressure of 33 bar. Then they were rinsed three times with Milli-Q water and dried for 30 min in a 140-320 °C air stream (hotwind S, Leister, Ka¨giswil, Switzerland). After cooling to room temperature, a pressure of 20 bar was applied and the micropipets were dipped for 22 h in a solution of 1 mL of aminosilane in 100 mL of toluene saturated with water under an argon atmosphere and rinsed with toluene for 30 min at 22 bar and Milli-Q water for 30 min at 33 bar. For coating with polylysine, the micropipet (26) Weller, M. G.; Schuetz, A. J.; Winklmair, M.; Niessner, R. Anal. Chim. Acta 1999, 393, 29-41.

GigaOhm Seal of Impaled Giant Vesicles

Figure 3. Surface coating of glass micropipets. Transverse section of pipet holder with two parts a and b joined by a rubber ring c. The holder is tightly pressed against a rubber gasket d on a glass vessel f that contains the reaction solution under nitrogen or argon. Nitrogen or argon up to a pressure of 40 bar is applied simultaneously to six micropipets that dip into the reaction solution and are attached to the holder with a silicone tube that is plugged into a holder-pipe and a screw-head as shown in the blowup. tips were cleaned in Milli-Q water at 34 bar for 30 min and then dipped in a 50 µg/mL aqueous solution of polylysine for 60-90 min. Finally they were either rinsed for 1-10 min in Milli-Q water or dried without rinsing. We made hydrophobic barriers on micropipets coated with polylysine using two methods. (i) Barriers of lipid were obtained from smaller giant vesicles (diameter, 5 µm; POPC/DOPME/DiI, molar ratios of 200:2:1). They were attached to the micropipets at a distance of 30 µm from the tip. After adhesion, they burst within a minute and generated a lipid ring with a length of 20 µm around the pipet. (ii) Barriers of poly(methyl methacrylate) (PMMA) were made from a 0.4 mg/mL solution of PMMA (Plexiglas GS, Ro¨hm, Darmstadt, Germany) in chlorobenzene mixed at a ratio of 6:1 with a 10 mM solution of DiIC18(5) (DiD, Molecular Probes) in ethanol. A small drop (diameter, 3.5 mm) was formed on a Teflon disk (diameter, 3 mm). The micropipet was horizontally dipped into the drop with a micromanipulator under visual control with a 10× objective of a microscope such that the pipet was coated by the polymer solution at a certain distance from the tip. Fluorescence Imaging. All experiments were performed under a microscope (BX-50-WI, Olympus, Tokyo, Japan) with a reflected light fluorescence attachment (BX-FLA, Olympus). For illumination, we used a xenon light source (LQX 1800, Linos, Go¨ttingen, Germany). The vesicles were positioned on the chip with a 10× air objective (LMPlanFL 10×, Olympus) and a brightfield module. To detect fluorescein ejection and DiI of the membrane during impalement, a 40× water objective (LUMPlanFL 40× W, Olympus) and a fluorescence module (filters: HQ 495/50, Q 525 LP, HQ 615/170, AHF Analysentechnik, Tu¨bingen, Germany) were used. The starting situation of every experiment was documented with a CCD camera (HV-C20E/K-S4, Hitachi, Tokyo, Japan) connected to a computer (miroVIDEO DC30 plus, Pinnacle Systems, Mountain View, CA). To investigate the spreading effect, single frames of recorded videos were analyzed by suitable software written in IMAQ Vision 5.0 (National Instruments, Austin, TX). To allow long-term exposure, the CCD camera was connected to a frame grabber (PCEYE 2, ELTEC, Mainz, Germany). This feature was also used to visualize the PMMA/DiD barriers on the micropipets with a second fluorescence module (filters: 615/70, Q 660 LP, LP 665). Electrophysiology. After coating, the micropipets were filled with a solution of 1 M KCl and 1 mM sodium fluorescein (no. 10399, Merck) (access resistance, 15-40 MΩ) and contacted by the chlorinated silver wire of a microelectrode holder mounted on a micromanipulator (Inject Man, Eppendorf, Hamburg, Germany). That holder had to allow electrophysiological measurements as well as injections through the microelectrode (transjector 5246, Eppendorf). Standard holders have one pressure connection perpendicular to the straight connection of the silver electrode. This asymmetric construction leads to bending of the holder and movements of the pipet tips if high pressures

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Figure 4. Giant lipid vesicle with an impaled micropipet in a hexagonal picket fence on a silicon chip. Micrograph with bright-field illumination at a small aperture. (p e 7 bar) are used. For that reason, we used a custom-made holder with a second pressure connection. This symmetric construction prevented bending of the holder. A pipet amplifier (SEC-10L, npi electronic, Tamm, Germany) was used in current clamp mode. The current-clamp command was provided by a PC-based multi-I/O board (AT-MIO-16E-2, National Instruments). The electrolyte was kept on ground potential by a chlorinated silver wire. No compensation protocol was used because the effective access resistance of the microelectrode was not constant during the experiments. Thus the voltage response between pipet and electrolyte was measured. Current and voltage were recorded and evaluated with LabVIEW 5.1 (National Instruments). For membrane breakthrough, usually current pulses of 100 pA and 100 ms were used. During an experiment, current pulses with amplitudes of 10-100 pA were continuously applied. The voltage responses were offset corrected and automatically fitted using a Levenberg-Marquardt algorithm. Thus the time course of the fit parameters could be observed. For very tight micropipetmembrane contacts, the discharging of the membrane was very slow and the effects of the current pulses (duration, 100 ms; interval, 125 ms) were overlapping. Therefore in the first seconds of the time course the discharging of the preceding pulse was extrapolated and subtracted from the voltage signal before fitting.

Results and Discussion Impalement. In a first set of experiments, we sedimented giant vesicles of POPC on a chip of oxidized silicon that was coated with polylysine. On that substrate, the vesicles are immobilized by adhesion.25 When we tried to impale them with a micropipet, they burst within 3 min due to the high membrane tension caused by the strong adhesion.27 In a second approach, we coated the chips with albumin. Such a surface is known to interact weakly with giant vesicles.28,29 However, the attachment is so weak that an approaching microelectrode pushed the vesicle along the surface such that impalement was prevented. To achieve immobilization of giant vesicles under conditions of weak adhesion, we fabricated microscopic picket fences of polyimide as shown in Figure 2. After coating chip and fences with albumin, we placed a vesicle into a cage of appropriate diameter. When a micropipet approached now, microscopic inspection suggested that the vesicle was successfully impaled as illustrated in Figure 4. We tested whether a real connection existed between the volumes of the micropipet and of the vesicle (27) Seifert, U. Adv. Phys. 1997, 46, 13-137. (28) Radler, J. O.; Feder, T. J.; Strey, H. H.; Sackmann, E. Phys. Rev. E 1995, 51, 4526-4536. (29) Zeck, G.; Fromherz, P. Langmuir 2003, 19, 1580-1585.

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Figure 5. Topology of impaled vesicles. Fluorescein solution is ejected from a micropipet after impalement of a giant vesicle. (A) Successful impalement. The dye spreads across the whole volume of the vesicle. (B) Failed impalement. The dye solution is localized in bubbles that move upward along the pipet and unload to the bath. Although the pipet tip is lowered far below the perimeter of the vesicle, it does not penetrate the membrane.


bubbles that moved upward along the pipet and unloaded to the bath as illustrated in Figure 5B. The homogeneous staining of the vesicle indicated successful impalement, whereas the second kind of staining revealed that the pipet induced only an indentation of the membrane without real impalement by a breakthrough. Electrical Circuit. The electrical properties of impaled giant vesicles were determined by current-voltage measurements. At first, we consider the equivalent circuit used to evaluate the data (Figure 6). The membrane is described by a capacitance CM and a resistance RM. A parallel resistance RL is introduced for leaks at the pipetmembrane contact. Thus the effective membrane resistance is R/M ) RMRL/(RM + RL) leading to a time constant τ/M ) R/MCM. The contribution of micropipet, electrode, and headstage of the amplifier are described by the access resistance RA and a stray capacitance CS. A serial resistance RI accounts for the inner resistance of the vesicle caused by the high resistivity F ) 260 kΩ cm of the sucrose solution. With an effective access resistance R/A ) RA + RI, we define a time constant τ/AS ) R/ACS. When a current step IP(t) ) I0Θ(t) is applied to the pipet, we expect a voltage response VP(t) with two exponentials as derived in the Appendix. For small stray capacitance CS , CM, we obtain

[

( )]

VP(t) ) I0R/A 1 - exp -

t

τ/AS

[

( )]

+ I0R/M 1 - exp -

t τ/M

(1)

Figure 6. Equivalent circuit of an impaled vesicle. The membrane is described by a resistance RM and a capacitance CM. Leaks of the membrane and the membrane-pipet contact are represented by a resistance RL. RA is the access resistance, and CS is the stray capacitance of the micropipet electrode and amplifier. RI describes the internal resistance of the vesicle. IP is the current clamped by the amplifier, and VP is the measured pipet voltage.

by intravesicular staining. We filled the micropipet with a solution of fluorescein and injected the solution after the impalement. For situations where the micrographs indicated a successful impalement (Figure 4), we observed two types of fluorescence patterns: (i) The fluorescent dye spread across the volume of the vesicle as shown in Figure 5A. (ii) The dye solution remained localized in

Considering a specific capacitance cM ) 0.6 µF/cm2 30 and a specific resistance rM ) 1 MΩ cm2 of giant lipid vesicles,25 we expect for a vesicle with a diameter of 50150 µm a capacitance of CM ) 50-400 pF, a resistance RM ) 12-1.4 GΩ, and without leaks a time constant around τ/M ) 0.6 s. With freshly pulled micropipets, the stray capacitance is around CS ) 10 pF and the access resistance is RA ) 15-40 MΩ. Neglecting the inner resistance of the vesicle, we expect a time constant around τ/AS ) 0.5 ms. Impalement with Uncoated Pipets. At first we studied the impalement of pure POPC vesicles with freshly pulled uncoated pipets. We applied current pulses with amplitudes of I0 ) 10-100 pA and durations of tP ) 0.1-2 s. Three kinds of signals VP(t) were observed as shown in Figure 7: (A) A biphasic response with a fast step of a few millivolts and a subsequent slow signal up to 30 mV. (B) A fast and small voltage response only. (C) A large and slow response only. After each electrical measurement, a fluorescent solution was ejected to verify the topology of the system. Signal types A and B were observed in cases

Figure 7. Current pulse and voltage response for giant vesicles impaled with uncoated pipets. The vesicles had a diameter of around 80 µm. Top: current. Bottom: voltage. (A) Two-phase response with fast and slow components indicating successful impalement with a small leak. (B) Fast response indicating impalement with a large leak. (C) Slow response indicating a vesicle-attached configuration without impalement.


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Figure 8. Dynamics of pipet-vesicle impalement with a gigaOhm seal. (A) Current clamp measurement shortly after impalement of the vesicle (diameter, 144 µm). A rectangular current pulse IP (upper graph) with an amplitude of 100 pA and a duration of 1 s resulted in a voltage signal VP (lower graph). A fit of the signal led to a membrane capacitance CM ) 0.49 nF, an effective membrane resistance R/M ) 1.2 GΩ, and an effective access resistance R/A ) 130 MΩ. (B) Time course of membrane capacitance, effective membrane resistance, and effective access resistance. For details, see the text.

where the fluorescence experiment indicated a successful impalement (Figure 5A). Signal type C was observed when fluorescence indicated a failed impalement (Figure 5B). The A-type signal was biphasic as expected for an impaled vesicle. Assuming CS ) 10 pF, we fitted the measurement with eq 1 and estimated a membrane capacitance CM ) 130 pF, an effective membrane resistance R/M ) 240 MΩ, and an effective access resistance R/A ) 80 MΩ. For the vesicle with a diameter of 80 µm, the capacitance was close to the value of CM ) 120 pF for an ideal bilayer. However, the effective resistance R/M is far lower than the expected membrane resistance of RM ) 5 GΩ. That discrepancy indicates a leak in the vesiclepipet system. The effective access resistance R/A is much higher than the pipet resistance of RA ) 15 MΩ determined before impaling. That difference is assigned to an internal resistance RI ) 65 MΩ of the vesicle with sucrose solution. In the B-type signal, there is no visible amplitude of a slow phase. Considering eq 1, the effective membrane resistance must be low due to leaks. By fitting the experiment with the first term of eq 1, we obtain CS ) 10 pF and R/A ) 44 MΩ. Also here the difference of the effective access resistance and the measured pipet resistance RA ) 20 MΩ before impaling is assigned to the intravesicular sucrose solution. Considering the fluorescence experiment, we assign signal type C to a vesicle-attached configuration of the pipet. Of course, that contact is dissociated by the ejection experiment. The equivalent circuit of Figure 6 cannot be applied. The situation is described by the capacitance CS and the access resistance RA of the micropipet and by a serial seal resistance Rseal of the pipet-vesicle contact. With a time constant τseal ) (RA + Rseal)CS, we obtain eq 2 for the voltage response to a stimulation by a current step.

[

(

VP(t) ) I0(RA + Rseal) 1 - exp -

)]

t τseal

(2)

With RA ) 20 MΩ of the pipet, we obtain CS ) 10 pF and a seal resistance of Rseal ) 8.8 GΩ. Probably that kind of C-type signal was observed in earlier studies31 and assigned there to successful whole-vesicle impalement with a high seal resistance.

We conclude that only the A-type signal is compatible with a successful impalement of vesicles. It was observed in 20% of the experiments (n ) 45) with uncoated pipets. The effective resistances were in a range of R/M ) 40-150 MΩ usually up to 3 min. The resistance was far lower than the resistance RM ) 1.4-12 GΩ for an ideal lipid bilayer. In a single experiment, we found R/M ) 0.43 GΩ for 3 s. The low resistance must be assigned to leaks in the vesicle membrane or at the micropipet-membrane contact. We found no significant differences for vesicles made of POPC, of DOPC, or of mixtures with POPC/ DOPME (molar ratios of 100:1), POPC/DOPME/DiI (molar ratios of 200:2:1), and POPC/DiI (molar ratios of 200:1). Impalement with Coated Pipets. To improve the resistance of the micropipet-membrane contact, we coated the pipets by three methods: (i) We made the glass surface hydrophobic with dimethyldichlorosilane. In that case, vesicles of almost electrically neutral lipid (POPC with 0.5% DiI) were used. (ii) We coated the pipets with an aminosilane. In that case, vesicles of negatively charged lipid were used (POPC with 1% DOPME and 0.5% DiI). (iii) We coated the glass with polylysine and used again a negatively charged lipid mixture. The procedures of coating are described in the methods section. The three coating methods showed a dramatically enhanced effective membrane resistance as derived from A-type signals. For illustration, the experiment with the highest resistance obtained by polylysine coating is shown in Figure 8. The first electrical test after impalement is plotted in Figure 8A. From a fit with eq 1, we obtained a capacitance CM ) 0.49 nF, an effective membrane resistance R/M ) 1.2 GΩ, and an effective access resistance R/A ) 130 MΩ. During the impalement, we performed repetitive current-voltage measurements. The resulting dynamics of the parameters CM(t), R/M(t), and R/A(t) are plotted in Figure 8B for constant CS ) 10 pF. The membrane capacitance CM stayed almost constant around 0.5 nF and disappeared after 1290 s. The effective membrane resistance slowly decayed to 70 MΩ and also (30) Dambacher, K. H.; Fromherz, P. Biochim. Biophys. Acta 1986, 861, 331-336. (31) Sandre, O.; Moreaux, L.; Brochard-Wyart, F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10591-10596.

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Figure 10. Membrane spreading along an impaled micropipet. (A) Brightness amplitude of the fluorescence micrograph after contrast enhancement for an impaled giant vesicle after subtracting a micrograph taken before impalement. The vesicle membrane has spread by a distance s along the pipet with a concomitant reduction of the apparent vesicle diameter. (B) Spreading distance s versus time after penetration with a fast rise and a subsequent slower phase.

Figure 9. Histograms of the maximum effective membrane resistance R/M and of the duration of impalements with tight seals for different coating materials: (A) dimethyldichlorosilane; (B) aminosilane; (C) polylysine.

disappeared after 1290 s. The access resistance R/A increased in the moment of impalement from 50 to 150 MΩ due to the sucrose solution in the vesicle and was fairly constant afterward. Some irregularities were observed near the end of the experiment in Figure 8B. At time t ) 1140 s, the capacitance dropped to 0.31 nF (point a in Figure 8B), while the access resistance R/A increased to 140 MΩ (point c). That transition was related to the appearance of a small vesicle at the pipet tip. The effects were reversed at time t ) 1270 s (points b and d) when we ejected the dye solution to test the topology of the pipetvesicle contact. That manipulation induced the destruction of the small vesicle and eventually also the end of the experiment at time t ) 1290 s. All three coating methods showed a dramatically increased yield of whole-vesicle contacts with signal type A as compared to uncoated glass pipets with 58% (n ) 52) for dimethyldichlorosilane, 60% (n ) 45) for aminosilane, 60% (n ) 42) for polylysine coating with rinsing, and 66% (n ) 35) without rinsing. Histograms of the maximum effective resistances R/M and the duration of impalement are shown in Figure 9. With respect to the resistance R/M, polylysine was the best coating material, while with aminosilane there was a higher yield of stability. Membrane Parameters. From the experiments with coated pipets, we estimated the area specific membrane parameters, determining the vesicle area AM ) 4πaV2 from the vesicle radius aV in microscopic images. The membrane capacitance cM ) CM/AM was determined only for experiments with an effective membrane resistance R/M g 200 MΩ and with standard deviations of R/M smaller than 10 MΩ and of CM smaller than 0.002 nF. As an average of 42 experiments, we obtained cM ) 0.76 ( 0.21 µF/cm2. This value is in good agreement with 0.6 µF/cm2 determined for solvent-free lipid membranes30 and 0.99 µF/ cm2 determined for asolectin vesicle membranes.19 The result provides evidence for the unilamellarity of the

vesicles. The maximum value for the specific effective membrane resistance r/M ) R/MAM was 0.79 MΩ cm2. This result is close to an estimated rM > 1 MΩ cm2 obtained from vesicle-transistor measurements25 and close to the resistance of quasi-solvent-free bilayers.2 This result demonstrates that perfect vesicles with perfect pipetvesicle seal can be obtained in principle. But the yield of such systems is low. Membrane Spreading. When giant vesicles were impaled, we frequently observed a spreading of the membrane along the outer surface of the micropipets. That spreading was visualized by subtracting images before and after impalement. A contrast-enhanced difference picture is shown in Figure 10A for a pipet coated with polylysine. Spreading was connected with a change of shape of the vesicle as illustrated in Figure 10A. The maximum spreading distance varied for the three coatings and was about 10 µm for dimethyldichlorosilane, 20 µm for aminosilane, and up to 300 µm for polylysine. We evaluated the position of the front of the spreading membrane for a pipet coated with polylysine as shown in Figure 10B. There is a fast initial phase followed by a slower process. We assign the fast phase to a spreading of the membrane at low tension until the membrane is stretched. The slow spreading may be related to tensioninduced micropores32 that give rise to the decaying resistance in the experiments as considered above. With increasing duration of impalement, we often observed transient macropores with a diameter of 1-25 µm near the impaled pipet with a lifetime of up to a second. Their appearance was correlated with a sudden drop of effective membrane resistance and a sudden advancement of spreading. Such macropores are known to be induced by tension.31,33 Their surprisingly long lifetime in the low viscosity medium of our experiments may be due to the continuous tension caused by the pipet.34 A quantitative discussion of the formation of tension-induced pores will (32) Taupin, C.; Dvolaitzky, M.; Sauterey, C. Biochemistry 1975, 14, 4771-4775. (33) Brochard-Wyart, F.; de Gennes, P. G.; Sandre, O. Physica A 2000, 278, 32-51. (34) Zhelev, D. V.; Needham, D. Biochim. Biophys. Acta 1993, 1147, 89-104.


Figure 11. Barriers for membrane spreading. Fluorescence micrographs after impalement of giant vesicles by micropipets. (A) Barrier of lipid. The white arrow marks the border of the weakly fluorescent barrier lipid and the front of the spreading membrane of the vesicle (orange fluorescence of DiI). (B) Barrier of PMMA. Two pictures are taken at different excitation wavelengths for the polymer (red fluorecence of DiD) and for the lipid (orange fluorescence of DiI) and displaced horizontally to better visualize the identical shape of the border of PMMA and the spread membrane. (A small vesicle was formed at the tip of the pipet.)

Langmuir, Vol. 20, No. 25, 2004 11181

process during coating.38,39 We only observed fast membrane spreading until the visible PMMA barrier in one experiment. In that case, the resistance was very stable (R/M ) 70-240 MΩ) with a duration of 21 min. That striking result suggests that a systematic improvement of polymer coating may lead to long-term high-resistance systems. Spreading Model. Spreading of lipid membranes on hydrophilic planar surfaces is well-known.40 Two mechanisms are distinguished: sliding and rolling. In the first type, a membrane lamella glides over the surface on a thin water film (about 1 nm),25 while in the second type the membrane folds back upon itself and rolls off in a carpet-like manner. The two mechanisms are illustrated in Figure 12 for membrane spreading on a microelectrode. Two arguments are in favor of rolling in our system: (i) An impaling microelectrode forms an extended tube-like indentation up to 30 µm long. After breakthrough of the membrane, a rolling mechanism would match the given geometry. For sliding, the membrane tube would have to be inverted. (ii) The front of the spreading membrane could be observed as a subtle shadow in bright-field microscopy at small aperture. With rolling, two membranes separated by a water film slide along the pipet. The thickness of that water film is determined by the repulsion of the negatively charged lipid membrane and may be in a range of 50 nm at the low salt concentration. Such a water film is compatible with the optical effect. A sliding membrane on a thin water film of about 1 nm would not be visible. Conclusion

Figure 12. Different possible mechanisms of spreading on the surface of a micropipet after impaling a giant vesicle.

be possible when more detailed data on the dynamics of pores are available.35-37 To summarize, membrane spreading along the impaled pipet has two different faces. On one hand, it is responsible for the tight seal between pipet and vesicle; on the other hand, it gives rise to pores that lower the effective resistance of the membrane. Limitation of Membrane Spreading. To stabilize the resistance of the vesicles, we tried to limit membrane spreading by barriers. For pipets with polylysine, we used two approaches of local coating: (i) We attached a small vesicle to the pipet at a distance of 20 µm from its tip. By bursting, it locally coated the pipet with lipid. Vesicles with a diameter of about 5 µm provided the optimal amount of material. When a giant vesicle was impaled, the lipid barrier limited the fast phase of spreading and slowed the slow phase. An example is shown in Figure 11A. Seals were obtained in 8 of 12 experiments. Yet the duration of the seals was not significantly improved. (ii) We fabricated barriers of PMMA stained with the fluorescent dye DiD (see methods). After impaling of a giant vesicle (Figure 11B), spreading was observed in 13 out of 23 experiments and was stopped by the barrier. In 10 experiments, we observed an A-type signal. The average duration of high resistance increased to 243 s. Surprisingly, spreading was slowed before the visible PMMA barrier was reached. That effect may be due to a foot of polymer formed by the wetting (35) Helfrich, W.; Servuss, R. M. Nuovo Cimento 1984, 3, 137-151. (36) Rawicz, W.; Olbrich, K. C.; McIntosh, T.; Needham, D.; Evans, E. Biophys. J. 2000, 79, 328-339. (37) Evans, E.; Heinrich, V.; Ludwig, F.; Rawicz, W. Biophys. J. 2003, 85, 2342-2350.

We established a method to achieve electrically tight whole-cell configurations of unilamellar giant vesicles with impaled micropipet electrodes. The resistance was up to a gigaOhm with a lifetime up to 20 min. We assign the sealing of micropipets and vesicles to a spreading of the lipid bilayer on the outer surface of the pipets. That mechanism of sealing is to be distinguished from conventional sealing of cell membranes as achieved by impaling or patching. On the other hand, the spreading process induces a tension in the vesicle that destabilizes the system by the formation of pores. Further improvements of the pipet-vesicle contact are required in order to attain a reliable system for electrical studies of lipid bilayers and reconstituted ion channels in defined membrane matrixes. The present study suggests that optimal pipet-vesicle contacts may be developed by suitable coating of the pipets to promote membrane spreading with a high resistance and by suitable barriers to limit spreading and membrane tension with a long lifetime. Acknowledgment. We thank Helge Vogl for help with clean-room technology, Gerd Hu¨bener for advice on coating chemistry, and Robert Pabst and Walter Wagner for expert mechanical engineering. The project was supported by the Deutsche Forschungsgemeinschaft (SFB 563). Appendix Voltage Relaxation for Impaled Vesicles. When a current step IP(t) ) I0Θ(t) is applied to the circuit of Figure 6, the exact solution is given by (38) Brochard, F.; de Gennes, P. G. J. Phys. Lett. Paris 1984, 45, L597-L602. (39) de Gennes, P. G. Rev. Mod. Phys. 1985, 57, 827-863. (40) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 45394548.

11182


VP(t) ) I0(R/A - A1e-t/τ1 + R/M - A2e-t/τ2)


(A1)

The two amplitudes and time constants are defined in terms of the three time constants τ/M ) R/MCM, τ/AS ) R/ACS, τ/MS ) R/MCS and the resistances R/M ) RMRL/(RM + RL) and R/A ) RA + RI with

-k2 + k4(W ( k1) W k3 τ1/2 ) k1 ( W

A1/2 )

k1 ) τ/M + τ/AS + τ/MS

For CS , CM, eq A1 simplifies to eq 1 with A1 ) R/A, A2 )

k2 ) R/A(τ/AS + 2τ/MS) + R/M(τ/M + τ/MS) k3 ) 2 τ/Mτ/AS k4 ) 0.5(R/A + R/M) W ) x-4τ/Mτ/AS + (τ/M + τ/AS + τ/MS)2 R/M, τ1 ) τ/AS, and τ2 ) τ/M. LA048233H

GigaOhm Seal by Membrane Spreading - American Chemical Society

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