Orientation of Hemicyanine Dye in Lipid Membrane Measured by

© Copyright 2001 by the American Chemical Society

VOLUME 105, NUMBER 2, JANUARY 18, 2001

LETTERS Orientation of Hemicyanine Dye in Lipid Membrane Measured by Fluorescence Interferometry on a Silicon Chip Armin Lambacher and Peter Fromherz* Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, D 82152 Martinsried/Mu¨ nchen, Germany ReceiVed: August 4, 2000; In Final Form: NoVember 13, 2000

Amphiphilic hemicyanine dyes are fluorescent probes for voltage transients in nerve cells. Their sensitivity is assumed to be related with an intramolecular charge shift along the oriented chromophore that interacts with the electrical field across the cell membrane. Here we report on a measurement of the molecular orientation of the hemicyanine dye Di8ANEPPS in a lecithin membrane. We took advantage of the features of dipole radiation in front of a mirror. The fluorescence intensity of a stained membrane on oxidized silicon was measured as a function of the thickness of silicon dioxide up to 1000 nm and fitted with an electromagnetic theory accounting for the interference of the exciting light, for the interference of the emitted light and for the change of fluorescence lifetime. We found an angle of 37.8 ( 1.6° between the transition dipole moment and the membrane normal for an uniaxial cone model of the angular distribution function, with an order parameter 〈P2〉 ) 0.44 similar to the hydrocarbon chains of the lipid matrix.

Introduction Fluorescent amphiphilic hemicyanine dyes are frequently used as molecular probes for fast changes of the membrane voltage in neurons.1-5 The spectral features of voltage-sensitive fluorescence were determined in neuron membranes and in lipid bilayers.6-8 The mechanism of voltage sensitivity is still unclear. Several processes were considered: The transient electrical field in the membrane may interact with the intramolecular charge shift which is connected with electronic excitation and give rise to spectral shifts of excitation and emission by electrochromism.9 It may interact also with the intramolecular charge shift connected with rotamerism around a CC single bond in the excited state and lead to a changed quantum yield of fluorescence.10-14 On the other hand, the electrical field may affect the position and orientation of the dye and its solvation * Corresponding author. Phone: +49 89 8578 2820. Fax: +49 89 8578 2822. E-mail: [email protected].

at the membrane/water interface leading to modulation of solvatochromism15 or of solvent-dependent quantum yield.10-14 For all postulated mechanisms of voltage sensitivity, the orientation of the dye molecules in the membrane is a crucial parameter. Considering the chemical structure of amphiphilic hemicyanines such as Di8ANEPPS16 (Figure 1a) it may be suspected that the molecules are oriented along the membrane normal. In fact some experiments with polarized fluorescence17 and NMR18 were interpreted in terms of a perfect orientation of the dyes perpendicular to the membrane. In the present study we used the features of dipole radiation in front of a mirror to determine the orientation of a hemicyanine dye in a lipid membrane. Light absorption and light emission of dye molecules depend on the distance and on the orientation with respect to a mirror, as studied by Kuhn and Drexhage in their elegant experiments with Langmuir-Blodgett films on thin metal layers.19-21 The observations were rationalized in terms of Sommerfeld’s theory on radio antennas above a reflective

10.1021/jp002843i CCC: $20.00 © 2001 American Chemical Society Published on Web 12/20/2000

344 J. Phys. Chem. B, Vol. 105, No. 2, 2001

Figure 1. Experimental setup for measuring the orientation of a dye molecule. (a) Hemicyanine dye Di8ANEPPS. (b) Lipid bilayer with dye molecules on oxidized silicon with a thin cleft between oxide and membrane. (c) Fundamental rays of a selected direction of excitation and of emission, respectively. The details of refraction and multiple reflection are omitted. The excitation of the dye and its emission are affected by the self-interference of the incident light and of the emitted light, respectively, which is reflected at the interface silicon/oxide and the other interfaces.

and conductive surface.22-25 Recently, the modulation of fluorescence was investigated on silicon chips which were covered by terraces of silicon dioxide26-28 and the effect was applied to measure the distance of cell membranes from the substrate in a range of 10-100 nm using a cyanine dye with a transition moment parallel to the membrane. We investigated the fluorescence of the voltage-sensitive hemicyanine Di8ANEPPS in a lipid membrane on oxidized silicon as illustrated in Figure 1b. In a supported bilayer on silica29,30 the distance of membrane and substrate is known to be around 2 nm.31,32 The fluorescence is affected by reflection of the exciting light and of the emitted light at the interface silicon/oxide and all other interfaces as indicated in Figure 1c. We observed the fluorescence as a function of the thickness of silicon dioxide up to 1000 nm and fitted the data in terms of a Sommerfeld-type theory, using the orientation of the transition dipole as a free parameter. Experimental Section Silicon Chips. Silicon wafers were used with a 100 surface (n-doped, 5-14 Ω cm; Wacker, Burghausen). They were cleaned according to the standard RCA procedure.33 The wafers were oxidized homogeneously in wet oxygen at 1000 °C up to

Letters a thickness of 1000 nm (tube oven E1200LAB, Centrotherm, Blaubeuren). We cut chips of a size 30 mm × 15 mm. The silicon dioxide was structured by photolithography and etching with ammonium fluoride (AF 87.5-12.5 LSI Selectipur, Merck) at various dilutions with Milli-Q water (1:1-1:20, depending on the depth of etching) in eight successive steps. With eight different orientations of a mask of photoresist and with eight etchings steps of about 500, 250, 125, 62, 32, 16, 8, and 4 nm depth, we obtained six unit cells of 4 mm × 4 mm on a chip, each with 256 terraces of 250 µm × 250 µm. The height of the terraces ranged from 4 to 1000 nm in steps of about 4 nm. The progress of etching was checked with an ellipsometer (SD2000, Plasmos, Munich). Lipid Bilayer. Lipid bilayers were deposited from insoluble monolayers at the air-water interface using a circular trough.34 First the chips were sonicated for 10 min at 70 °C in an acid detergent (5% Ultrax 102, KLN, Heppenheim), rinsed with a jet of Milli-Q water, sonicated for 20 min at 70 °C in an alkaline detergent (2% Tickopur RP100, Bandelin, Berlin) and rinsed with a jet of Milli-Q.35 Then they were sonicated three times in Milli-Q water for 10 min at room temperature, rinsed between each stage with a jet of water, and dried in hot air. The alkaline detergent eroded the silicon oxide at a rate of about 0.5 nm in 10 min. The actual height of the terraces was measured by ellipsometry after completing the experiment. The dye Di8ANEPPS was obtained from Molecular Probes (Eugene), palmitoyl-oleoyl-phosphatidyl-choline (POPC) from Avanti Polar Lipids (Alabaster). As a solvent we used chloroform (Uvasol, Merck) purified by chromatography on basic aluminum oxide to remove traces of hydrochloric acid. The concentrations of POPC and of the dye were 5 mM and 50 µM, respectively. We submersed the chip vertically in the trough, spread a monomolecular film, compressed it to 37 mN/m, and transferred the first monolayer at a speed of 0.8 mm/s by the LangmuirBlodgett technique.36 Then we mounted the chip horizontally on a suction pipet with the structured side facing the trough and dipped it across the monolayer at a speed of about 1.5 mm/ s.37 Finally the chip was mounted in a shallow dish without exposing it to the gas phase. After completing the fluorescence measurements, the chips were cleaned by successive dips into chloroform, propanol, and Milli-Q water. Remaining organic material was removed by a mixture of concentrated sulfuric acid and 30% hydrogen peroxide (volume ratio 1:3) and a final rinsing with Milli-Q water. The Piranhjia solution did not erode silicon dioxide. At this stage the height of the terraces was determined. Fluorescence. Fluorescence pictures were taken with a microscope (Axioskop, Zeiss) through a water immersion objective (magnification 63×, numerical aperture 0.9). We used a high-pressure mercury lamp (HBO 100W/2, Osram), a bandpass filter at 436 nm (436FS10-50, Andover, Salem, NH), and a dichroic mirror (BSP480, DELTA Light & Optics, Lyngby, Denmark) under conditions of Ko¨hler illumination. We observed the fluorescence with a CCD camera (HRC, Theta System, Munich, sensor: Sony ICX039AL) through the dichroic mirror and a long-pass filter at 550 nm (OG 550, Schott, Mainz). The intensity of the lamp was monitored by a photodiode. The exposure time (40-320 ms) and the gain were controlled by a computer. The pictures were transferred to a frame grabber (ITEX AFG, Stemmer, Munich). On each of the 256 steps of oxide we selected an area of 50 µm × 50 µm to 100 µm × 100 µm which was free of visible defects and evaluated there the average fluorescence intensity.

Letters

J. Phys. Chem. B, Vol. 105, No. 2, 2001 345

Figure 2. Fluorescence intensity of Di8ANEPPS in lipid bilayer on silicon. (a) Experimental data in arbitrary units versus the thickness of silicon dioxide. The data are fitted by a Sommerfeld-type theory with a uniaxial cone model of the angular distribution function of the transition dipole moment with an angle of θ ) 36° to the membrane normal. (b) Residuals of the fit.

Results and Discussion Data. The fluorescence intensity of Di8ANEPPS in a bilayer of POPC on 256 terraces of silicon dioxide on a silicon chip is plotted in Figure 2. It was modulated by the distance from the reflecting surface of silicon in a surprisingly irregular manner. The fluorescence was low close to silicon and highest with 100 nm oxide. The intensity leveled out at large distances. We made four measurements with almost identical results (see below). Theory. Under stationary illumination, the number of quanta per unit time Jfl emitted by a dye molecule into the detector is given by the probability of the dye to be in its emitting state and the probability per unit time Pem for emission into the detector. The probability to be in the excited state depends on the probability per unit time Pex for excitation by the lamp and on the rate constants of decay by interaction with the electromagnetic field kelmag and by intramolecular processes kic as

1 Jfl ) Pex P kelmag + kic em

(1)

With lifetime and quantum yield of fluorescence at infinite distance from the chip τ∞fl ) 1/(k∞elmag + kic) and Φ∞fl ) k∞elmagτ∞fl and with ∆kelmag ) kelmag - k∞elmag we obtain

τ∞fl Jfl ) Pex P ∆kelmag em 1 + Φ∞fl ∞ kelmag

(2)

The fluorescence intensity depends on three factors which are affected by the position and orientation of the dye molecule: (i) The excitation is modulated by the interference of each incident plane wave with the corresponding waves reflected at the interface silicon/oxide and all other interfaces. (ii) The population of the excited state depends on the relative change of decay by the electromagnetic interaction of the emitting dye with the chip, radiative through the far field and nonradiative through the near field. (iii) The detected intensity of emission is modulated by interference of each plane wave emitted into the objective with the corresponding waves reflected at the interface silicon/oxide and all other interfaces.

The probability of excitation Pex depends on the squared projection of the local electrical field strength onto the direction bex|2. The of the transition dipole moment of excitation |E Bin‚e components of the electrical field were obtained from a superposition of all waves in the multilayer system. They were expressed by the Fresnel coefficients of transmission and reflection of the TE and TM modes at each interface. We averaged over the orientations of the transition dipoles and all directions and polarizations of the incident light. We used a simple cone model for the uniaxial distribution function with a unique angle θ between all dipoles and the membrane normal. Finally we integrated over the spectrum of absorbance and the intensity spectrum of the illumination. Details of the procedure are found in refs 26,27. The rate constant of decay by interaction with the electromagnetic field kelmag is controlled by the available modes into which the excited dye molecule emits a photon. The excited dye molecule was described as a linear point dipole. We expanded the far and near field into plane waves.22-25 When the dye was placed in a stratified medium, the interfaces between the media imposed boundary conditions upon the plane waves which were described by the Fresnel’s coefficients of reflection and transmission. These boundary conditions lead to changes of the mode amplitudes at the location of the dye. As the probability of emission of a photon into a certain mode is proportional to the squared projection of the transition dipole bem|2 a moment of emission onto the mode amplitude |E Bout‚e change in mode amplitude leads to a change in emission probability. By integrating over all modes of the near and far field we obtained the rate constant of emission by interaction with the electromagnetic field kelmag. The probability for emission into the detector Pem was calculated in a similar manner. We used the plane wave expansion of the electromagnetic field generated by the excited dye molecule. For each plane wave that reached the aperture of the objective we calculated the influence of the stratified medium due to reflection, absorption, refraction, and interference between the directly emitted wave and the multiple reflections at the interfaces. By integration over all emission angles within the aperture angle we obtained the total power that reached the detection system. In the computations we took into account the known optical parameters of the system, the dispersion curves of all materials, the thickness of silicon oxide as measured by ellipsometry, the illumination spectrum, the spectral data of the dye, and the spectral response of the filters and the camera in the detection system.26,27 For the distance between the supported bilayer and the silicon oxide we used a value of 2 nm.31,32 Data on the intramolecular orientation of the transition moments in the chromophore of Di8ANEPPS are not available. So we assumed an identical orientation of the transition dipole of excitation and emission with b eex ) b eem. The result of computations with a quantum yield Φ∞fl ) 0.4 are plotted in Figure 3. The intensities were normalized with respect to an infinite distance from the chip. When the transition moments were normal to the membrane with θ ) 0°, the fluorescence was high without spacer. With increasing angle the intensity near the silicon decreased and a first maximum appeared near 100 nm of oxide. The change of the inclination affected the whole pattern of intensity up to large distances. This modification of the interference pattern was particularly sensitive for small deviations from normal orientation. Fit. We fitted the experimental data of Figure 2 with the electromagnetic theory varying the orientation θ of the transition

346 J. Phys. Chem. B, Vol. 105, No. 2, 2001

Letters recording of electrical actvity in nerve cells. In the present study we measured the orientation of a selected dye in a pure lipid membrane on the solid substrate of oxidized silicon, taking advantage of standing light modes on that particular substrate. A more detailed investigation with various membranes on various coatings may help to reveal the effect of integral membrane proteins and also the possible role of the substrate. Similar measurements with individual nerve cells will be feasible, though with lower precision. The result is a sound basis for considerations on the physical mechanism of voltagesensitive fluorescence. In that respect we may attempt to look for a change of orientation of the dye by an applied electrical field using capacitive stimulation from the silicon chip.39,40

Figure 3. Theoretical fluorescence intensity of Di8ANEPPS in lipid bilayer on silicon versus thickness of silicon dioxide. The angle θ between the transition dipole and the membrane normal is varied for an uniaxial cone model of the distribution function as indicated. All other optical parameters as defined by the experimental setup. The intensities are scaled with respect to the intensity at infinite thickness of silicon dioxide.

moments with b eex ) b eem, and the scaling factor of the intensity. In addition we used the quantum yield Φ∞fl as a free fit parameter because no data are available for the chromophore of Di8ANEPPS in a lipid membrane. The result is shown in Figure 2. We obtained an optimal fit with an angle θ ) 36.0° and a quantum yield Φ∞fl ) 0.4. The resulting relative residuals are distributed rather smoothly within a band of (0.2. In three other experiments we obtained θ ) 37.6°, θ ) 37.7°, and θ ) 40.0° at Φ∞fl ) 0.4. The average inclination was θ ) 37.8 ( 1.6°. Discussion. The comparison of the fitted experimental data in Figure 2 with the series of theoretical relations in Figure 3 shows that the measurement of inclination θ ) 37.8 ( 1.6° with the interferometrical approach is highly significant. The fitted quantum yield was higher than Φfl ) 0.2 as reported for homologous hemicyanines RH364 and BNBP in lipid vesicles.10,12 Yet the fitted orientation of the dye was quite insensitive to the assumed value of the quantum yield with a change of the angle by 0.4° at a change of the quantum yield by -0.1. The result was also quite independent of the assumed distance between membrane and silicon dioxide. A change by 1 nm affected the angle only by 0.4°. There was no statistically significant evidence for different angles of the transition dipoles for excitation and emission. Apparently the chromophore of Di8ANEPPS is not aligned perfectly normal to the membrane. Previous investigations of the orientation of voltage-sensitive dyes in lipid membranes relied on polarized fluorescence and NMR.17,18 Those data were compatible with normal orientation. However, a perfect normal orientation cannot be expected for a dye embedded in the matrix of a fluid lipid bilayer. The orientation of the molecular director of the hydrocarbon chains of the lipid has been determined in dipalmitoyl-phosphatidyl-choline (DPPC) above the phase transition to the fluid state using deuterium magnetic resonance spectroscopy.38 The order parameter 〈P2〉 ) 〈3cos2θ - 1〉/2 of the angular distribution function had a constant value around 〈P2〉 ) 0.45 up to carbon number ten. Our approach did not yield a model-independent order parameter of the dye Di8ANEPPS but a representative angle of orientation within a cone model of the distribution function. From this angle θ ) 37.8 ( 1.6° we computed an order parameter P2 ) 0.44 ( 0.04 of the fluorescent probe, similar to the value of the lipid matrix. Conclusion. The orientation of voltage-sensitive dyes in membranes is a crucial parameter for optimizing optical

Acknowledgment. The project was supported by the Bundesministerium fu¨r Bildung und Forschung and by the Fonds der Chemischen Industrie. References and Notes (1) Cohen, L. B.; Salzberg, B. M. ReV. Physiol. Biochem. Pharmacol. 1978, 83, 35. (2) Grinvald, A.; Fine, A.; Farber, I. C.; Hildesheim, R. Biophys. J. 1983, 42, 195. (3) Fluhler, E.; Burnham, V. G.; Loew, L. M. Biochemistry 1985, 24, 5749. (4) Loew, L. M.; Cohen, L. B.; Salzberg, A. L.; Obaid, A. L.; Bezanilla, F. Biophys. J. 1985, 47, 71. (5) Rohr, S.; Salzberg, B. M. Biophys. J. 1994, 67, 1301. (6) Fromherz, P.; Lambacher, A. Biochim. Biophys. Acta 1991, 1068, 149. (7) Fromherz, P.; Mu¨ller, C. O. Biochim. Biophys. Acta 1993, 1150, 111. (8) Fromherz, P.; Schenk, O. Biochim. Biophys. Acta 1994, 1191, 299. (9) Loew, L. M.; Simpson, L.; Hassner, A.; Alexanian, V. J. Am. Chem. Soc. 1979, 101, 7717. (10) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1989, 93, 6729. (11) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1991, 95, 6729. (12) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1993, 97, 4540. (13) Fromherz, P.; Heilemann, A. J. Phys. Chem. 1992, 96, 6864. (14) Ro¨cker, C.; Heilemann, A.; Fromherz, P. J. Phys. Chem. 1996, 100, 12172. (15) Fromherz, P. J. Phys. Chem. 1995, 99, 7188. (16) Loew, L. M. Pure Appl. Chem. 1996, 68, 1405. (17) Loew, L. M.; Simpson, L. L. Biophys. J. 1981, 34, 353. (18) Bammel, B. P.; Hamilton, D. D.; Haugland, R. P.; Hopkins, H. P., Schuette, J.; Szalecki, W.; Smith, J. C. Biochim. Biophys. Acta 1990, 1014, 61. (19) Kuhn, H. Verhandl. Schweiz. Naturforsch. Ges. 1965, 245. (20) Bu¨cher, H.; Drexhage, K. H.; Fleck, M.; Kuhn, H.; Mo¨bius, D.; Scha¨fer, F. P.; Sondermann, J.; Sperling, W.; Tillmann, P.; Wiegand, J. Mol. Cryst. 1967, 2, 199. (21) Drexhage, K. H. Prog. Optics 1974, 12, 63. (22) Sommerfeld, A. Ann. Physik 1909, 28, 665. (23) Tews, K. H. Ann. Physik 1973, 29, 97. (24) Chance, R. R.; Miller, A. H.; Prock, A.; Silbey, R. J. Chem. Phys. 1975, 63, 1589. (25) Sullivan, K. G.; Hall, D. G. J. J. Opt. Soc. Am. B. 1997, 14, 1149. (26) Lambacher, A.; Fromherz, P. Appl. Phys. A 1996, 63, 207. (27) Braun, D.; Fromherz, P. Appl. Phys. A 1997, 65, 341. (28) Braun, D.; Fromherz, P. Phys. ReV. Lett. 1998, 81, 5241. (29) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (30) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (31) Bayerl, T. M.; Bloom, M. Biophys J. 1990, 58, 357. (32) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam., G. W.; Rennie, A. R.; Thomas, R. K.; Sackman E. Biophys. J. 1991, 59, 289. (33) Wolf, S.; Tauber, R. N. Silicon Processing for VLSI Era. Lattice Press: Sunset Beach, CA, 1986. (34) Fromherz, P. ReV. Sci. Instrum. 1975, 46, 1380. (35) Fromherz, P.; Reinbold, G. Thin Solid Films 1988, 160, 347. (36) Blodgett, K. A. J. Am. Chem. Soc. 1935, 57, 1007. (37) Langmuir, I.; Schaefer V. J. J. Am. Chem. Soc. 1938, 57, 1007. (38) Seelig, J.; Seelig, A. Quart. ReV. Biophys. 1980, 13, 19. (39) Fromherz, P.; Stett, A. Phys. ReV. Lett. 1995, 75, 1670. (40) Fromherz, P.; Kiessling, V.; Kottig, K.; Zeck. G. Appl. Phys. A 1999, 69, 571.