Domain formation in thin lipid films probed with near-field scanning optical microscopy

High-resolution near-field scanning optical microscopy (NSOM) fluorescence and topographic images of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air-water interface at several surface pressures using the Langmuir-Blodgett technique. Sub-diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.     

Near-field scanning optical microscopy studies of L-alpha-dipalmitoylphosphatidylcholine monolayers at the air-liquid interface

The phase structure in L-alpha-dipalmitoylphosphatidylcholine-20 mol% fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate Langmuir monolayers dispersed on a 2 M sucrose solution subphase is studied with near-field scanning optical microscopy (NSOM). Cantilevered NSOM probes operating in a tapping-mode feedback or an optical interferometric feedback mode are capable of tracking the air-sucrose solution interface. At the micrometre scale, the NSOM fluorescence images reveal lipid domain features similar to those observed previously in supported Langmuir-Blodgett (LB) monolayers. At the submicrometre scale, the small nanometric lipid islands seen in LB films are not observed at the air-sucrose interface. This supports a mechanism in which domain formation in LB films can be induced by means of the transfer process onto the solid support. Progress towards extending these studies to films at the air-water interface using the optical interferometric feedback method is also discussed.     

Near-field scanning optical microscopy of zinc-porphyrin crystals

Using a near-field scanning optical microscope (NSOM), crystals of zinc-porphyrin network materials are characterized with respect to morphology and fluorescence. Needle-shaped crystals are observed. While the topography is flat, the fluorescence intensity profile in the width direction is approximately triangular. A numerical calculation shows that differences between the topographic and optical images cannot be due to an artifact. In some needle-shaped crystals, the fluorescence emission is strongly peaked at one or both ends, possibly indicating a polar crystal structure.     

Phase evolution in cholesterol/DPPC monolayers: atomic force microscopy and near field scanning optical microscopy studies

A combination of atomic force microscopy (AFM) and near field scanning optical microscopy has been used to study domain formation in dipalmitoylphosphatidylcholine (DPPC)/cholesterol monolayers with cholesterol concentrations ranging from 0 to 50%. The results show a clear evolution from a mixture of liquid expanded and liquid condensed phases for cholesterol concentrations < 10% to a mixture of liquid expanded and two cholesterol‐containing phases at intermediate concentrations, and finally to a single homogeneous liquid ordered phase for 33% cholesterol. Mixtures of the various phases are clearly identified by height differences in AFM and in some cases by fluorescence imaging for samples containing 0.5% BODIPY dye, which localizes preferentially in the more fluid phase. Note that fluorescence imaging, at least with the dye used here, is unable to distinguish between the cholesterol‐rich and cholesterol‐poor phases detected at intermediate cholesterol concentrations. The combination of fluorescence and AFM imaging provides a more complete picture of the phase evolution for cholesterol/DPPC monolayers than could be obtained by either technique alone, and presents substantial advantages over conventional fluorescence microscopy in that submicrometre‐sized domains can be readily detected.     

A near-field microscopy study of submicron domain structure in a model lung surfactant monolayer

The submicron domain structure of coexisting liquid condensed (LC) and liquid expanded (LE) phases in monolayers composed of palmitic acid and 20 wt% of a lung surfactant protein B fragment has been investigated. Near-field microscopy was used to simultaneously measure topography and fluorescence images of monolayers that were prepared at a surface pressure of 15 mN/m and a temperature of 22 degrees C. The use of a fluorescently tagged peptide allowed for unambiguous determination of the peptide location in the two-component system. The LC and LE phases in the monolayers are measured on the submicron length scale. A 6-11 A height difference between the LC and LE phases was evident in the height images. Gradual transitions between the LC and LE domains were observed across a 1.3 microm length scale in the near-field fluorescence images, but were significantly sharper in the simultaneously collected topography images and in the separately measured AFM images. These results may reflect the occurrence of peptide encroachment into the LC domains.     

Nanoscopic observation of a gold nanoparticle-conjugated protein using near-field scanning optical microscopy

This study describes a single gold nanoparticle (AuNP)-based observation of biomolecular interaction using a near-field scanning microscope (NSOM) in transmission mode. To observe streptavidin molecules, a glass surface was first patterned with a micro-scale line of (3-aminopropyl)trimethoxysilane (APTMS) by micro-contact printing (μCP) with a subsequent reaction of N-hydroxysuccinimide (NHS)-biotin. The AuNP-conjugated streptavidin was then applied to the biotin-modified glass surface and NSOM was employed to detect the resulting specific interaction between streptavidin and biotin on the glass surface. Using the optical and topological images generated from the NSOM analysis, the interaction could be observed at the nanoscopic scale. This study demonstrates that the NSOM is a powerful tool for the detection of protein interactions at the nanoscopic level when the protein is conjugated with AuNPs.     

Nanoscale organization of CD4 molecules of human T helper cell mapped by NSOM and quantum dots

CD4 molecule, the surface marker of T helper cell, has been confirmed to be the main cellular receptor for the human immunodeficiency viruses HIV-1, HIV-2 and SIV. Recent research demonstrated the importance of the spatial arrangement of CD4 on the cell membrane to its binding efficiency to virus. In this article, the combined near-field scanning optical microscopy (NSOM) and quantum dots (QDs) fluorescent labeling technology were performed to investigate the nanoscale organization of CD4 molecules with a spatial resolution about 100 nm. Simultaneous topographic image of the T helper cell and fluorescent image of QDs have been directly gained by NSOM/QDs-based system. Intensity- and size-distribution histograms of the QDs fluorescent spots verify that approximately 80% of the CD4 molecules are organized in nanosized domains randomly distributed on the cell surface. Intensity-size correlation analysis revealed heterogeneity in the molecular packing density of the domains. Our results also illustrated the combination of NSOM imaging and QDs labeling is an ultrasensitive, high-resolution technique to probe nanoscale organization of molecules on the cell surface.     

Near-field fluorescence imaging of single molecules with a resolution in the range of 10 nm

We demonstrate fluorescence imaging of single molecules, by near-field scanning optical microscopy (NSOM), using the illumination-collection mode of operation, with an aperture probe. Fluorescence images of single dye molecules were obtained with a spatial resolution of 15 nm, which is smaller than the diameter of the aperture (20 nm) of the probe employed. Such super-resolution may be attributable to non-radiative energy transfer from the molecules to the coated metal of the probe since the resolution obtained in the case of conventional NSOM is limited to 30–50 nm due to penetration of light into the metal.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

A novel light source for SICM–SNOM of living cells

We have developed a novel light source for use in a scanning near‐field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo‐3 and calcium, respectively. This complex is then excited with an Ar⁺ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high‐resolution topographic and optical images of biological samples in physiological buffer. A resolution of ～220 nm topographic and ～190 nm optical was determined through imaging fixed sea‐urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.     

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA = 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.     

Large-area topography analysis and near-field Raman spectroscopy using bent fibre probes

We present a method for combined far‐field Raman imaging, topography analysis and near‐field spectroscopy. Surface‐enhanced Raman spectra of Rhodamine 6G (R6G) deposited on silver nanoparticles were recorded using a bent fibre aperture‐type near‐field scanning optical microscope (NSOM) operated in illumination mode. Special measures were taken to enable optical normal‐force detection for control of the tip–sample distance. Comparisons between far‐field Raman images of R6G‐covered Ag particle aggregates with topographic images recorded using atomic force microscopy (AFM) indicate saturation effects due to resonance excitation.     

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA= 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.     

Imaging streptavidin 2D crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope

Streptavidin crystals were grown on biotinylated lipid monolayers at an air/water interface and transferred onto highly oriented pyrolytic graphite (HOPG). These arrays could be imaged to a resolution below 1 nm using the atomic force microscope. The surface topographs obtained were compared with negative-stain electron microscopy images and the atomic model as determined by X-ray crystallography. The streptavidin tetramer (60 kDa) exposes two free biotin-binding sites to the buffer solution, while two are occupied by linkage to the lipid monolayer. Therefore, the streptavidin 2D crystals can be used as nanoscale matrices for binding biotinylated compounds. Furthermore, this HOPG-based preparation method provides a general novel approach to study the structure of protein arrays assembled on lipid monolayers with the AFM.     

Scanning probe microscopies applied to the study of the domain wall in a ferroelectric crystal

Scanning near‐field optical microscopy is capable of measuring the topography and optical signals at the same time. This fact makes this technique a valuable tool in the study of materials at nanometric scale and, in particular, of ferroelectric materials, as it permits the study of their domains structure without the need of chemical etching and, therefore, not damaging the surface (as will be demonstrated later). We have measured the scanning near‐field optical microscopy transmission, as well as the topography, of an RbTiOPO₄ single crystalline slab, which exhibits two different of macroscopic ferroelectric domains. A chemical selective etching has been performed to distinguish between them, obtaining areas with a noticeable roughness (C⁻ domain) in comparison with the original flat aspect of the other ones (C⁺ domain). The effects of the selective chemical etching have been quantified in topographic images obtained by means of our fibre tip probe, and have been compared to topographic images obtained by Atomic Force Microscopy, with a better resolution. The near‐field optical transmission images recorded have been obtained under different excitation wavelengths. These images are modulated by the light scattering due to the grains at the rough surface, which depends on the excitation wavelength used. In addition, they show a significant optical contrast due to the variations of the dielectric constant on the proximity of the ferroelectric domain wall.     

Phase separation in polyfluorene – polymethylmethacrylate blends studied using UV near-field microscopy

In this paper we present a near‐field microscopy study of thin films of a phase‐separated blend of the fluorescent conjugated‐polymer poly(9,9‐dioctylfluorene) [PFO] with the non‐fluorescent polymer polymethylmethacrylate [PMMA]. A scanning near‐field optical microscope (NSOM) was used to generate (blue) fluorescence from the PFO following UV excitation at 362 nm. A range of different concentrations of PFO in PMMA were studied ranging from 1 to 50% PFO in PMMA by mass. By studying both the shear force and fluorescence images we were able accurately to determine the distribution of PFO in the PMMA. We found that phase separation occurs over a number of different length‐scales between 5 µm and 250 nm. We show that at PFO concentrations of 1%, the PFO lies on top of the PMMA. At a PFO relative concentration of 50%, the PMMA phase extends through the whole thickness of the film to the underlying substrate. We use such samples to discuss the resolution of NSOM when imaging thick organic films. Furthermore, we confirm that the length‐scales of phase separation can be modified via control over spin‐casting protocols.     

Near-field fluorescence imaging and simultaneous observation of surface potentials

We present a new detection method to measure simultaneously surface potential and fluorescence intensity distributions using a combined scanning near-field optical microscope-atomic force microscope (SNOM-AFM). A surface potential image of phospholipid monolayers was obtained in non-contact mode using the SNOM-AFM with a thin-step etched optical fibre probe. For applying this technique, a phospholipid of dipalmitoylphosphatidylethanolamine labelled at the head with a nitrobenzoxadiazole group was used as a fluorescent and single component Langmuir–Blodgett film. It is well known that aggregation of the lipid molecules and their fluorescence intensities are very sensitive to its environmental conditions such as humidity and temperature. We demonstrated for the first time the near-field optical imaging and simultaneous observation of surface potentials with Maxwell stress microscopy.     

Supported metal model catalyst surfaces examined by scanning tunnelling microscopy

Topographic and/or barrier-height images of ultrafine Pt and Au metal particles supported on a vacuum-deposited carbon film or titanium oxide thin films grown on titanium metal sheets were obtained. The topographic images of colloidal Au particles (5-nm diameter) adsorbed on a titanium oxide thin film showed a structure elongated in the direction normal to the x scan, indicating their weak interaction with the support surface. The topographic images of Pt vacuum-deposited on a carbon film showed c. 4-nm diameter particles, larger than those observed in electron microscopy. The problems inherent to the STM observation of such dispersed metal systems are identified. In the case of Pt particles vacuum-deposited on titanium oxide film, its barrier-height image gave better indication of different phases on the surface than its topographic image. The significance of obtaining barrier-height images along with topographic images for such sample systems is demonstrated.     

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1–4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1–2°. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.