Near Field Optical Investigations of Langmuir−Blodgett Monolayers in

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Near Field Optical Investigations of Langmuir-Blodgett Monolayers in Liquid Environment L. Vaccaro,*,† E. L. Schmid,‡ W.-P. Ulrich,‡ H. Vogel,‡ C. Duschl,‡ and F. Marquis-Weible† Institut d’Optique Applique´ e, Ecole polytechnique fe´ de´ rale, 1015 Lausanne, Switzerland, and Laboratoire de Chimie Physique des Polyme` res et Membranes, Ecole polytechnique fe´ de´ rale, 1015 Lausanne, Switzerland Received July 19, 1999. In Final Form: November 1, 1999 Langmuir-Blodgett monolayers are studied using near field fluorescence microscopy. The setup is equipped with a feed back system based on shear force detection of the oscillating near field probe that allows optical characterization in air as well as in aqueous environments. In the monolayer liquid condensed star-shaped domains are present in a fluorophore-doped liquid expanded phase. The boundary lines between the two phases show a width of 91 nm. Observations in water give the same quality of the optical data and allow the measurement of the boundary line width with the same optical resolution. The height data at constant amplitude of the probe vibration revealed topographical contrast inversion when scanning over the boundary lines of the monolayer in water with respect to scanning in air. This setup is suited to study optical properties of complex biological systems in an aqueous environment.

Introduction Scanning near field optical microscopy (SNOM) is suited to optically investigate objects on a scale of typically 50100 nm. In SNOM an optical probe is brought in close proximity to the object to be investigated where it interacts with the sample via the near electromagnetic field either emitted or scattered by the probe or by the sample object. The resolution of this technique is not restricted by the diffraction limit but rather through the size of the optical probe which can be fabricated with subwavelength dimensions. On top of its high resolution, the technique offers the potential to work with diverse optical contrast mechanisms (fluorescence, phase, polarization, etc.). SNOM has been successfully applied to the investigation of single molecules,1 cells,2 and ultrathin organic layers,3 and it has been used in combination with fluorescence resonance energy transfer techniques4 and even vibrational absorption in the infrared.5 Fluorescence techniques have had an enormous impact in biological sciences, both in basic research and for practical applications due to their high sensitivity (single molecule detection) and their versatility and because of their noninvasive properties with respect to biological systems. For bioindustrial screening purposes, there is a high demand for fluorescence-based assays. Therefore, it seems natural to combine the high sensitivity of fluorescence spectroscopy with the superior resolution of scanning near field microscopy in order to apply it for the investigation of biological samples. However, to obtain relevant information from biological material, the samples have to be investigated in their * To whom correspondence should be addressed. (email: [email protected]). † Institut d’Optique Applique ´ e, Ecole polytechnique fe´de´rale. ‡ Laboratoire de Chimie Physique des Polyme ` res et Membranes, Ecole polytechnique fe´de´rale. (1) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422-1425. (2) Enderle, T.; Ha, T.; Chemla, D. S.; Weiss, S. Ultramicroscopy 1998, 71, 303-309. (3) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342-353. (4) Vickery, S. A.; Dunn, R. C. Biophys. J. 1999, 76, 1812-1818. (5) Knoll, B.; Keilmann, F. Nature 1999, 399, 134-137.

native environment, which is typically an aqueous buffer solution. In addition, most biological systems are very fragile and mechanical stress which can be caused by the probe has to be avoided. These boundary conditions impose some difficulties to the approach in order to fully exploit its potential: the feedback system which controls the spacing between the probe and the sample on a subnanometer scale is based on mechanical interaction of the tip with the sample. This interaction is substantially different in air and in water, and for the use of this technique in an aqueous environment, these issues have to be addressed in detail. In most of the systems currently operational, the tip to sample distance is controlled by inducing a lateral vibration of the near field probe, with nanometer amplitude, and monitoring the damping of this vibration amplitude when the tip approaches the surface.6 Recently, it has been shown that a SNOM working with this shear force detection is in principle suited to work in liquid environment.7,8 Two main problems are encountered when working in aqueous environment. First, the mechanical properties of the near field probe are degraded in liquid, which leads to reduced sensitivity in the tip-to-sample distance control. To minimize this degradation, we apply here a simple technique described previously,7 which ensures that only the last few micrometers of the near field probe are vibrating in liquid. To maintain constant mechanical properties of the near field probe during the recording of the data, the tip is immersed in the liquid at a constant depth. Second, the decrease of the tip vibration amplitude is measured in most of the operational systems through a tuning fork, and although currently progressing, the applicability of such a piezoelectric tuning fork has not been demonstrated yet in an aqueous environment. To circumvent the problem, a simple optical design has been proposed, based on a laser beam focused on the tip (6) Betzig, E.; Finn, P. L.; Weiner, J. S. Appl. Phys. Lett. 1992, 60, 2484-2486. (7) Lambelet, P.; Pfeffer, M.; Sayah, A.; Marquis-Weible, F. Ultramicroscopy 1998, 71, 117-121. (8) Gheber, L. A.; Hwang, J.; Edidin, M. Appl. Opt. 1998, 37, 35743581.

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end and reflected off the sample surface. This approach is however limited when applied to scattering biological sample.9 Here, we exploit an optical interferometric measurement developed in our group and already reported elsewhere,10 to monitor the decrease of the tip vibration amplitude. This technique is independent of the optical quality of the sample surface and equally sensitive in air and aqueous environments. This paper presents the results of a near field fluorescence study of a thiolipid monolayer transferred on a quartz substrate, performed in air and in water.11 At the air-water interface this synthetic thiolipid undergoes a first-order phase transition between a liquid-expanded (LE) phase and a liquid-condensed phase (LC)12 with the formation of starlike domains. Introducing a small amount of a fluorescently labeled lipid into the monolayer gives rise to an optical image contrast based on the lower solubility of the labeled lipid in the LC phase. The monolayers have been successfully transferred to different solid supports by the Langmuir-Blodgett (LB) technique and extensively studied by scanning force microscopy (SFM) in topography mode and in friction mode (lateral force microscopy, LFM).13-15 These measurements reveal a substructure within the stars, with different molecular orientations characterizing the different points of the stars. On glassy substrates the height difference between the LC and LE phases is less than 1 nm. LFM shows that the transition region between the two phases may be very sharp (down to 100 nm) thus below the diffraction limit of far field microscopes. Near field fluorescence imaging of lipid membranes has shown substructures at the scale of a few tens of nanometers. Gradients in the concentration of fluorescence markers have been investigated.16 However, it is unclear how general these substructures are and to what extent they depend on different preparation conditions during the LB transfer process.17 Here for the first time, we simultaneously analyze data on the size of film features well-characterized by complementary techniques together with the instrumental limiting factors related to the probe size. The structural integrity in air and water allows for a more rigorous comparison of the imaging capabilities of SNOM in different environments compared to what has been previously possible.18 2. Materials and Methods The lipid used in this study is the amphiphilic thiolipid bis(8-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-3,6-dioxaoctyl) disulfide, whose synthesis and characterization have been described elsewhere.12 N-(6-Tetramethylrhodaminethiocarbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (TRITC-DPPE), purchased from Molecular Probes (Eugene, OR), is added to the (9) Shiku, H.; Krogmeier, J. R.; Dunn, R. C. Langmuir 1999, 15, 2162-2168. (10) Pfeffer, M.; Lambelet, P.; Marquis-Weible, F. Rev. Sci. Instrum. 1997, 68, 4478-4482. (11) This monolayer has been recently proven to be stable enough in aqueous environment to perform near field measurements. (12) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (13) Santesson, L.; Wong, T. M. H.; Taborelli, M.; Descouts, P.; Liley, M.; Duschl, C.; Vogel, H. J. Phys. Chem. 1995, 99, 1038-1045. (14) Gourdon, D.; Burnham, N. A.; Kulik, A.; Dupas, E.; Oulevey, F.; Gremaud, G.; Stamou, D.; Liley, M.; Dienes, Z.; Vogel, H.; Duschl, C. Tribol. Lett. 1997, 3, 317-324. (15) Liley, M.; Gourdon, D.; Stamou, D.; Meseth, U.; Fischer, T. M.; Lautz, C.; Stahlberg, H.; Vogel, H.; Burnham, N. A.; Duschl, C. Science 1998, 280, 273-275. (16) Hwang, J.; Tamm, L. K.; Bohm, C.; Ramalingam, T.; Betzig, E.; Edidin, M. Science 1995, 270, 610-614. (17) Shiku, H.; Dunn, R. C. J. Microsc. 1999, 194, 461-466. (18) Talley, C. E.; Lee, M. A.; Dunn, R. C. Appl. Phys. Lett. 1998, 72, 2954-2956.

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Figure 1. Schematic setup of the SNOM measuring cell used for investigation in liquid. lipid as a dye dopant for fluorescence microscopy. The monolayers are prepared and transferred on a commercial Langmuir trough (RK II, Riegler & Kirstein, Mainz, Germany), mounted on the three-directional stage of a fluorescence microscope (Zeiss Axiotron, Zu¨rich, Switzerland). The thiolipid is dissolved in chloroform together with 1 mol % TRITC-PE and spread on doubly deionized water with a resistivity of 18 MΩ cm (Barnstead Nanopure, SKAN AG, Basel Switzerland). After evaporation of the solvent, the film is compressed at a speed of approximately 0.1 Å2 molecule-1 s-1 to a final pressure of 25 mN m-1. After equilibration for 30 min, the film is transferred onto a quartz slide (Wisag, Zu¨rich, Switzerland) which has previously been prepared by repeated sonification in 2% Hellmanex detergent (Hellma, Mu¨llheim, Germany) followed by water. The freshly cleaned slides are stored under water and blown dry in a stream of nitrogen before use. Directly before the LB transfer, the slides are exposed to an oxygen plasma for 10 min. This last step is found to be crucial for the underwater stability of the transferred films. The substrate is dipped into the subphase shortly before the end of the equilibration time of the compressed monolayer. The samples are drawn out of the subphase at a transfer speed of 5 µm s-1. During the LB transfer, the applied lateral pressure is kept constant via a feedback loop. Since the transfer of the thiolipid monolayer takes place during the upstroke, the polar headgroups of the thiolipid are oriented toward the quartz surface. Near field images are taken with a homemade instrument that allows near field fluorescence measurements in air as well as in liquid. The SNOM head is based on a monolithic aluminum chassis that supports the near field probe and the distance control module. The tip-to-sample distance is controlled through interferometric detection of the shear force acting on the tip when laterally vibrating above the sample surface. The interferometric detection of the shear force, described in detail elsewhere,10 is based on a visible light emitting laser diode directly mounted onto the chassis and focused onto the tip. Part of the laser beam is retroreflected by the vibrating tip, forming an external cavity for the laser diode. The tip displacement is detected by analyzing the variation of the laser intensity along the resulting fringes. Although not as simple as alternative distance control techniques, it has the advantage of operating in liquid. Care must then simply be taken to ensure that the liquid level is kept constant around the tip, to guarantee a constant Q factor of the vibrating SNOM probe while scanning the sample. This can be done with the simple setup shown in Figure 1 by using an external glass tube of 1 mm internal diameter around the SNOM fiber, tightly sealed around the fiber holder. The trapped air column thus ensures that most of the tip length is vibrating in air and that only the last 50 µm of the tip is immersed in liquid. This height remains constant during sample scanning. In this configuration, it has routinely been observed that the mechanical quality of the tip is reduced by only a factor 5 when working in liquid.7 The SNOM head is mounted onto a standard inverted microscope to have

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Figure 2. (a) Structure of the thiolipid and pressure-molecular area isotherm at 20 °C. (b) Fluorescence microscopy image of a thiolipid monolayer at the air-water interface at a surface pressure of 25 mN/m at 20 °C. simultaneous access to far field and near field optical information. The near field probe is produced by chemical etching of monomode optical fibers and subsequent metalization with aluminum. Before being used as SNOM probes, the tips are characterized by measuring the optical transmission and the light distribution profile at the tip apex. The aperture diameter is deduced from near field imaging of a test object consisting of a Cr grating deposited on glass. The aperture diameter of the tip used in this study is 60 nm, for a light transmission of 1.6 × 10-3. The sample, transferred onto a quartz substrate, is positioned below the tip on a x-y piezo scanner (Physik Instrument), that scans the sample under the SNOM tip. Closed loop control of the scanner ensures linearity and absence of creep. The near field fluorescence is excited via the tip with a frequency doubled Nd: YAG laser emitting at 532 nm. Twenty microwatts of laser power is typically coupled into the optical probe. For a typical recording time of several milliseconds per pixel and a fluorescence excitation energy in the picojoule range, photobleaching of the monolayer is minimized. The collected images are 128 × 128 pixels in size. The mechanical quality factor of the SNOM tip is equal to 127. The emitted fluorescence is collected by a microscope objective (Zeiss 40×, N.A. 0.6). Fluorescence detection is performed with an avalanche photodiode (APD, EG&G SPCMAQ131FC), after filtering the residual excitation light with a notch filter (OD6).

3. Results and Discussion The structure of the thiolipid, together with a representative isotherm, is depicted in Figure 2a. Figure 2b shows a thiolipid monolayer observed with a far field fluorescence microscope at the air-water interface. Starshaped domains of the LC phase embedded in the bright LE phase are clearly visible. Note that rhodamine (TRITCDPPE) has been used in this study; this more stable fluorophore has been demonstrated to be less prone to photobleaching. However, this gives the same fluorescent features of the monolayer as with the previously used NBD-PE fluorescent probe (NBD-PE, 1,2-dimyristoyl-snglycero-3-phosphoethanolamine labeled with 4-chloro-7nitrobenz-2-oxa-1,3-diazole is the most used fluorescent probe in monolayer experiments). In addition, the surprising distribution of TRITC-DPPE within the domains, described later in this part, was recently also observed using NBD-PE. A near field fluorescence image of the thiolipid monolayer transferred to a quartz substrate in the LE/LC coexistence region in air is shown in Figure 3a. Star-shaped LC domains appear dark in a bright LE matrix containing most of the fluorescent dopant. The comparison with the results obtained by SFM and LFM13-15 shows that near field fluorescence microscopy nicely reproduces the key

features of the structures. However, some differences between the images are discernible. LFM images show a contrast between the different points of the stars that is due to different tilt angles of the thiolipid in the different points with respect to the scan direction. As expected, with fluorescence this contrast is not observable. The different points show the same low fluorescence intensity proving the fluorescent probe has no preference for particular subdomains. There are no differences in packing density between the different points of the stars that would show up in the dye distribution. In contrast to the previous force studies, fluorescence images reveal two distinct zones in the center of the starshaped domains: a dark nucleus is separated from the points of the star by a ring of high fluorescence intensity. The unequal distribution of the dopant within the LC phase is unexpected. A homogeneous circular region in the center of the domains has also been observed using lateral force microscopy. There, the contrast between the center and the points of the domains has been attributed to a different molecular organization in the respective subdomains.13,15 The circular fluorescence distribution may be the result of a less ordered molecular arrangement in the center of the domains with a somewhat higher solubility for the fluorescent probe. In the initial stages of domain growth, there may be a critical dye concentration in the fluid state, which may lead to the formation of a mixed LC phase (note that, upon growth of the domains, the fluorophore enriches in the LE phase). This phase may then serve as a nucleus for the formation of the highly order anisotropic subdomains constituting the points of the stars. A similar behavior has been observed on mixed monolayers containing a fatty acid and an amphiphilic cyanine chromophore.19 An interesting detail is observed in the bottom left corner of Figure 3a, where a locally very high dye concentration is discernible at the junction of two stars. This is attributed to dye molecules that are excluded from the LC phase but remain trapped in the small closed compartment that is formed by four points of the two neighboring stars. Furthermore, an asymmetry is observed when analyzing the edge of the LC domain: for each point of the star, the transition on one side appears sharper than that on the opposite side. In the SFM images this asymmetry of the two boundary lines is also present in most of the domains. (19) Duschl, C.; Kemper, D.; Frey, W.; Meller, P.; Ringsdorf, H.; Knoll, W. J. Phys. Chem. 1989, 93, 4587-4593.

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Figure 3. (a) Near field fluorescence image of a thiolipid monolayer in air. (b) Intensity profile at y ) 59.40 µm. The width of the transition zone is equal to 120 nm.

Figure 4. (a) The near field fluorescence image shows a twin defect in a LC domain in air. (b) Intensity profile at y ) 54.65 µm. The width of the transition zone is equal to 70 nm.

The origin of this inequality may be the chirality of the lipid molecules. An intensity profile of the near field fluorescence along the x-line is shown in Figure 3b, recorded at y ) 59.4 µm. This profile allows the determination of the width of the transition zone between the two subphases which is equal to 120 nm. A signal detected in the LE phase of 184.8 ( 18.1 photons/18 ms is observed on a background signal of 100 photons/18 ms in the LC phase. The low image contrast, defined by

Imax - Imin Imax + Imin

(1)

is due to the fact that a very low light level is used to excite fluorescence in order to minimize photobleaching. With this very demanding condition, the background signal generated by environmental parasite light con-

tributes in a relevant way to the detected signal. Similarly, the noise generated by the laser intensity fluctuations and the thermal noise of the avalanche photodiode are not negligible. A more accurate measurement of the extension of the transition zone is obtained from a scan of a smaller area recorded on the same sample, centered around the double point formed by a twin defect observed in the lower part of the central star. An area of 3 × 2 µm2 is recorded, displayed in Figure 4a. The double point clearly appears (central part of the image), as well as the area characterized by a higher fluorescence signal (at the left of the image), previously described as resulting from dye trapped between two stars. The intensity profile of the near field fluorescence along x, recorded at y ) 54.65 µm, is presented in Figure 4b20 and shows a width of the transition zone equal to 70 nm. An average value for this width is obtained

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Figure 5. (a) Near field fluorescence image of the thiolipid monolayer immersed in water. (b) Intensity profile at y ) 53.96 µm. The width of the transition zone is 120 nm.

Figure 6. Near field fluorescence intensity profile and z-position signal of the scanner measured (a) in air at y ) 59.36 µm and (b) in water at y ) 55.73 µm.

based on several successive lines, leading to 91 ( 12 nm. This is in agreement with the value of 100 nm obtained by SFM. The near field fluorescence image of the same sample in water is presented in Figure 5a. It shows that the morphology of the film observed in air (Figure 3a) is maintained. However, now the fluorescence intensity in the circular center part is homogeneous and on the same level as in the LE phase. The possibility of a SNOM artifact can be excluded, since the same homogeneous distribution is observed using confocal microscopy (data not shown). As of yet, we lack an explanation for this behavior. At this stage we can only state that the exposition of the hydrophobic lipid tails toward the water is thermodynamically unfavorable. This may cause a desorption of (20) Note that there seems to be a difference in sharpness of the boundary lines. The horizontal lines appear sharper than the vertical ones. The resolution was determined from the vertical boundary lines in a horizontal scan line.

the molecules from the LE phase that readsorbs in the center of the stars forming a bilayer. The different molecular organization in the circular subdomain in the center of the stars may promote the selective “staining” of the center of the stars. Further investigations addressing this point are currently under way. An intensity profile of the near field fluorescence at y ) 53.96 µm is depicted in Figure 5b and shows a width of the transition zone of 120 nm. This result demonstrates that the same resolution is obtained in liquid environment as in air. A signal in the LE phase equal to 268.1 ( 24.6 photons/27 ms on a background of 150 photons/27 ms in the LC phase is observed. This shows that the quality of the recorded images in air and in water is practically identical. Figure 6 displays the vertical position (z-axis) of the scanner, recorded in a shear force feedback loop simultaneously to the near field fluorescence, for line profiles selected from Figures 3 and 5. These shear force data, acquired in air (Figure 6a) and in water (Figure 6b), show

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some very interesting behavior. First, a clear contrast in the shear force data is observed, superimposed on the optical contrast. The width of the transition zone deduced from shear force data is equal to the value obtained from optical data. Careful analysis of the z-position signal with respect to the optical signal shows that there is a clear correlation in both cases between the optical signal and the z-position data. Furthermore an inversion of contrast is observed in the data trace recorded in water when compared to the trace recorded in air. A puzzling question is however raised when attempting to give a sensible interpretation of these results. The parameter recorded in these force curves is the displacement of the piezoelectric element necessary to maintain a constant damping of the lateral vibration of the near field probe. In a constant force interpretation which implicitly considers a constant interaction force between the near field probe and the sample surface at a given height, this displacement is proportional to the sample topography. The data presented here, however, clearly show the limitation of such a simplistic interpretation. The topographical steps deduced from the presented shear force data, when scanning over the boundary between the highly ordered condensed phase and the disordered phase, do not reflect the step height between these two phases as deduced from the dimensions of the molecule and from AFM measurements.13 From the latter, the step height between the two phases is estimated to be approximately 1 nm. However, a “topography” of 15 nm is deduced from the constant force interpretation of shear force data. Note that an accurate determination of the step height between the two phases is rather difficult to perform, using an AFM as well as a SNOM, due to the unequal compliance of the two phases and to their potentially different physical and chemical interactions with the probe. As we can conclude from Figure 6, the forces responsible for damping of the lateral vibration of the near field probe are clearly quantitatively different for the two phases. For the situation in air, Figure 6a shows that the stronger damping is observed on the LC phase. This is however reversed for the situation in water, where stronger damping is observed in Figure 6b on the LE phase. This is a clear indication that the nature of this damping mechanism is different in the two media. This phenomenon needs to be further investigated, and such a study would be very useful to further understand the nature of the shear force mechanism, in particular on soft organic surfaces. Finally, we would like to state that these results let us also conclude that our SNOM instrument is very well suited for the characterization of soft biological samples. In general, LB films are fairly robust when they are kept in their proper environment. This implies in our case that

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the polar headgroups are physisorbed to the hydrophilic glass substrate and the alkyl chains are pointing toward the air. Under these conditions, the thiolipid monolayer is stable for a few days. The situation is very different when incubating this monolayer in water. Now, the system is highly unstable since the hydrophobic alkyl chains are in contact with water. The star-shaped domains can be observed for only 1 or 2 h using our SNOM instrument (of course, also using far field optical microscopy) before they disintegrate. We are not aware of any other lipid system which is stable enough to survive such a treatment even for minutes (with the exception of probably polymerized lipid layers). But there is no chance to image the starshaped features under these conditions using AFM in contact mode. In summary, although these thiolipid monolayers are more stable than any other monolayers we are aware of, the stability in the “wrong” environment is very limited and not sufficient to be characterized by using, for example, AFM. 4. Conclusions Near field fluorescence microscopy is a useful tool to study subwavelength features of lipid monolayers on solid substrates. Star-shaped lipid domains of liquid-condensed phase are observed as dark areas in a brighter, fluorophore-containing expanded phase. The transition zone between the two phases is measured as 91 nm, which is clearly below the diffraction limit characterizing the traditional far field fluorescence microscopy. The same features are observed when the monolayer is present in water. The results presented here show that the quality of the optical near field data is similar in air and in an aqueous environment. However, the height data at constant amplitude of the probe vibration revealed topographical contrast inversion when scanning over the boundary lines of the monolayer in water in respect to scanning in air. This is a clear indication that the interactions responsible for the damping of the vibrating probe are qualitatively different in both media. These results demonstrate that this technique may be successfully employed for the investigation of more complex biological systems such as functionalized lipid bilayers or whole cells in their native environment. Combined with recent progress in the development of stable and biocompatible fluorescent nanocrystals and extremely sensitive detection schemes, this approach will become a valuable tool to investigate subwavelength structural details in biological systems. Acknowledgment. The authors are thankful to Brian O’Regan for critically reading the manuscript. LA990956R