Langmuir 2001, 17, 2013-2018
2013
Scanning Near Field Optical Microscopy of Phase Separated Regions in a Mixed Interfacial Protein (BSA)-Surfactant (Tween 20) Film A. Patrick Gunning,* Alan R. Mackie, Andrew R. Kirby, and Victor J. Morris Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. Received August 5, 2000. In Final Form: December 6, 2000 Scanning near field optical microscopy (SNOM) has been used to visualize phase-separated regions in a monolayer protein film. The film was created at an air-water interface during displacement of the protein bovine serum albumin (BSA) by the nonionic surfactant Tween 20. Contrast was generated in the SNOM images by fluorescent labeling of the protein. The validity of the SNOM methodology has been confirmed by a qualitative and quantitative comparison of the displacement process as visualized by SNOM and atomic force microscopy (AFM) on fluorescently labeled BSA displaced by Tween 20 and AFM data on native BSA displaced by Tween 20. The data suggest that SNOM studies of labeled proteins can be used to investigate the behavior of particular protein species in complex multicomponent interfacial films.
Introduction Atomic force microscopy (AFM) has recently demonstrated a new mechanism by which surfactants displace proteins from interfacial films.1 The surfactants nucleate as small domains within the protein film, and these domains then grow with increasing bulk surfactant concentration, or with time at sufficiently high bulk surfactant concentrations. The growing domains compress the protein network and cause it to thicken prior to failure and collapse of the protein film. This process has been termed “orogenic” displacement because it involves buckling of the protein layer prior to collapse. The mechanism has proved to be generic for air-water, oil-water, and solid-water interfaces, for several protein systems and for nonionic, water-soluble, and oil-soluble surfactants.1-5 In addition, recent molecular simulations of mixed interfacial layers have reproduced the experimental results.6 The use of AFM together with molecular simulation provides a means of studying mixed interfacial films at molecular resolution. The displacement mechanism discovered is new, unexpected, and could not have been discovered without techniques that allow visualization of individual protein molecules. In the future molecular simulation studies could be extended to investigate mixed protein systems, and thus experimental methods are needed to confirm such simulations. To identify particular protein species within complex mixtures, it is necessary to label particular proteins. One possible approach is to use fluorescent labels and, to achieve sufficient resolution, to compare scanning near field optical microscopy (SNOM) observations on the interfacial films containing labeled proteins with AFM studies. The AFM studies will allow (1) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (2) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Surf. Interface Anal. 1999, 27, 433. (3) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (4) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Langmuir 1999, 15, 4636. (5) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Competitive displacement of β-lactoglobulin from the air/water interface by Sodium Dodecyl Sulfate. Langmuir 2000, 16, 8176. (6) Wijmans, C. M.; Dickinson, E. Langmuir 1999, 15, 8344.
visualization at molecular resolution and the SNOM could reveal the spatial distribution of particular labeled proteins in the network with a resolution of approximately 50 nm. SNOM is a relatively new technique7,8 that has only become available in the form of commercial instruments within the last 2 to 3 years. It overcomes the diffraction limitation of traditional optical microscopy by placing the illumination source, via a tapered optical fiber, in extremely close proximity to the sample in a region known as the optical “near field”. Working in the near field ensures that the light exiting the end of the fiber does not diverge via diffraction before it interacts with the sample, so that the resolution with which it can be examined is limited only by the size of the illuminating beam of light. The size of the light beam is in turn governed by the size of the aperture from which it exits, and if the end of the fiber is coated with aluminum, the aperture can be made very small through the use of ion-milling. In practice presently available commercial fibers have apertures of approximately 50-80 nm. This resolution should be sufficient to observe phase-separated regions in protein-protein, or protein-surfactant, mixed molecular interfacial films. This article describes preliminary studies on the use of SNOM to visualize the structure of labeled bovine serum albumin (BSA) films formed at the air-water interface, during displacement of the protein by the nonionic surfactant Tween 20. The displacement of labeled BSA by Tween 20 has also been studied by AFM and compared with AFM data on the displacement of native BSA by Tween 20. The comparison of the SNOM and AFM data has been used to assess the applicability of SNOM for studying multicomponent interfacial films. Experimental Methods Fluorescent Labeled BSA. The protein used in the present study is bovine serum albumin (BSA, Sigma Chemicals Ltd, Dorset, U.K.). It is possible to buy FITC labeled BSA (BSA labeled (7) Betzig, E.; Trautman, J. K.; Harris, T. D.; Weiner, J. S.; Kostelak, R. L. Science 1991, 251, 1468. (8) Pohl, D. W. In Advances in Optical and Electron Microscopy; Mulvey, T., Sheppard, C. J. R., Eds.; Academic Press: London, 1991; Vol. 12, p 243.
10.1021/la001126c CCC: $20.00 © 2001 American Chemical Society Published on Web 02/23/2001
2014
Langmuir, Vol. 17, No. 6, 2001
with fluorescein isothiocyanate). However, the successful completion of the current study required labeling of BSA with Alexa Fluor 488 dye (protein labeling kit A-10235) as specified according to the manufacturer’s instructions (Molecular Probes Inc., Eugene, OR). The degree of labeling was determined by spectrometry to be 1.2 mol of dye per mole of protein. Langmuir-Blodgett Film Preparation. Both SNOM and AFM studies were made on samples of the interfacial film formed on a Langmuir trough and sampled by Langmuir-Blodgett (LB) methods. The water used in the study was surface pure (γ0 ) 72.6 mN m-1 at 20 °C) cleaned using an Elga Elgastat UHQ water purification system. The Tween 20 was purchased as a 10% solution (Surfactamps, Rockford, IL). Surface tension measurements were made using a Wilhelmy plate and a Langmuir trough. The PTFE trough has dimensions 255 × 112 × 16 mm (a volume of 450 mL) and has one fixed and one mobile barrier. All experiments were performed at room temperature (≈20 °C) with distilled water as the subphase. Alexa Fluor labeled BSA solutions were prepared at a concentration of 0.68 mg mL-1 in phosphate-buffered saline (pH 7.2). The labeled protein was spread at the interface by careful dropwise addition of 48 µg (71 µL) of solution from a pipet. Surface tension measurements were made as a function of time to follow the adsorption of the protein at the interface. After about 30 min the surface tension readings tended to a constant value suggesting equilibrium coverage of the interface. After this period of time the Tween 20 solution was injected into the subphase to produce a concentration of 0.5 µM. Langmuir-Blodgett films were collected at periodic intervals. These LB films were formed on cleaned glass coverslips (for SNOM and AFM studies) and freshly cleaved mica (for AFM studies). Further additions of Tween 20 were made to the subphase in order to further increase the surface pressure or to adjust the surface pressure to particular values required for data collection. LB films were prepared by dipping the substrate (glass or mica), mounted perpendicular to the interface, down through the interface and then back up and out again at a constant rate of 8.4 mm min-1. The surface tension was monitored during the dipping process and showed that film transfer only occurred on the upward stroke. LB films of native BSA were prepared on mica substrates in an identical manner. The surface rheology of the protein films was measured after equilibration for 30 min. The monolayers were compressed to one-fifth of their original surface area while the surface tension was monitored. From these data the dynamic dilational modulus E can be calculated using
E ) dγ/d ln A where γ is surface tension and A is the surface area occupied by the sample. SNOM and AFM Imaging. The LB films were imaged by AFM (East Coast Scientific, Cambridge, U.K.) and SNOM (Thermomicroscopes Lumina, Sunnyvale, CA, mounted on an Olympus IX 70 inverted optical microscope). In the AFM studies, the LB films deposited onto mica were exposed to the air after removal from the trough and then imaged in the liquid cell of the AFM under redistilled 1-butanol. The butanol dissolves the surfactant and helps to improve the contrast in the images. Images were obtained in dc contact mode using 100 µm long sharpened levers with a nominal force constant, k ) 0.38 N m-1 (Veeco Digital Instruments, Santa Barbara, CA). The imaging force was in the range 1-2 nN. LB films deposited on glass coverslips were imaged in air by tapping mode AFM, using Nanoprobe TESP levers, k ) 35 N m-1 (Digital Instruments, Santa Barbara, CA) driven at a frequency of 256.4 kHz with an amplitude of approximately 10 nm. In the case of SNOM studies the LB films were formed on glass coverslips and imaged in air. The fiber optic probes were as supplied by the manufacturer with a quoted aperture of 50 nm. The Lumina SNOM was operated in the “shear force” feedback control mode. The SNOM probes are mounted onto a quartz tuning fork that is driven at its resonance frequency by a signal from a lock-in amplifier. When the probe is close to the sample surface, the oscillation is damped by attractive van der Waals interactions. This interaction provides a basis for controlling the probe-sample separation. Operation in the near field region is achieved by monitoring the extent of damping of the
Gunning et al.
Figure 1. AFM images of partially displaced BSA LB films: (a) native BSA-Tween 20 film transferred at Π ) 18.1 mN m-1, scan size 2 × 2 µm, gray scale 0-12.1 nm; (b) native BSATween 20 film transferred at Π ) 19.4 mN m-1, scan size 4 × 4 µm, gray scale 0-6.6 nm; (c) native BSA-Tween 20 film transferred at Π ) 21.0 mN m-1, scan size 4 × 4 µm, gray scale 0-11.7 nm; (d) Alex Fluor labeled BSA-Tween 20 film transferred at Π ) 21.2 mN m-1, scan size 5 × 5 µm, gray scale 0-9.2 nm. oscillation of the probe assembly using the lock-in amplifier. The signal is rectified, and then used to control a feedback circuit, which maintains a predetermined probe-sample surface separation (typically
