Fabrication and characterization of a silicon cantilever probe with an integrated quartz-glass (fused-silica) tip for scanning near-field optical microscopy

A cantilever-based probe is introduced for use in scanning near-field optical microscopy (SNOM) combined with scanning atomic-force microscopy (AFM). The probes consist of silicon cantilevers with integrated 25-mum-high fused-silica tips. The probes are batch fabricated by microfabrication technology. Transmission electron microscopy reveals that the transparent quartz tips are completely covered with an opaque aluminum layer before the SNOM measurement. Static and dynamic AFM imaging was performed. SNOM imaging in transmission mode of single fluorescent molecules shows an optical resolution better than 32 nm.     

Near-field microscopy and lithography of light-emitting polymers

We describe the application of scanning near-field optical microscopy (SNOM) to the study of the photophysical and self-organization properties of thin films of blends of conjugated polymers, and also to the lateral nanoscale patterning of conjugated-polymer structures. Such thin-film plastic semiconductor nanostructures offer significant potential for use in opto-electronic devices. The implementation of SNOM we employ is the most established form in which a probe with a sub-wavelength aperture is scanned in close proximity to the sample surface. We consider the nature of the near-field optical distribution, which decays within the first ca. 100 nm of these semiconductor materials, and address the identification of topographic artefacts in near-field optical images. While the topographic information obtained simultaneously with optical data in any SNOM experiment enables an easy comparison with the higher-resolution tapping-mode atomic force microscopy, the spectroscopic contrast provided by fluorescence SNOM gives an unambiguous chemical identification of the different phases in a conjugated-polymer blend. Both fluorescence and photoconductivity SNOM indicate that intermixing of constituent polymers in a blend, or nanoscale phase separation, is responsible for the high efficiency of devices employing these materials as their active layer. We also demonstrate a scheme for nano-optical lithography with SNOM of conjugated-polymer structures, which has been employed successfully for the fabrication of poly(-phenylene vinylene) nanostructures with 160 nm feature sizes.     

Electrochemical assay of nonlabeled DNA chip and SNOM imaging by using streptavidin-biotin interaction

The assay of DNA biosensor-based nucleic acid recognition using microfabrication technology provides for high sensitivity, good surface coverage and reproducibility. We have achieved efficient immobilization and hybridization of nonlabeled DNA using cyclic voltammetry (CV), square wave voltammetry (SWV) and scanning near-field optical microscopy (SNOM) techniques. The increased electrochemical response observed following the immobilization of biotinlyated ssDNA probe suggests that nucleic acid is a somewhat better medium for electronic transfer. We demonstrated the high coverage of immobilized FITC-labeled biotinylated DNA probe on a streptavidin-modified surface using SNOM imaging. SNOM imaging of FITC-labeled complementary DNA also exhibited fluorescent light spots of hybridization distributed throughout. No fluorescent light was observed with the hybridization of non-complementary DNA.     

Fluorescence resonance energy transfer scanning near-field optical microscopy

The method of fluorescence resonance energy transfer scanning near-field optical microscopy (FRET SNOM) consists in the separation of a FRET pair between an SNOM tip and a sample. The donor (or acceptor) centre is located at the tip apex and scanned in the vicinity of a sample while acceptor fluorescence (or donor-fluorescence quenching) is detected. It is shown that the spatial resolution for such an approach is governed not by the aperture size but by the FRET characteristic radius (F?rster radius), and thus can attain the value of 2-7 nm with the same (or higher) sensitivity as characteristic for the aperture SNOM. The theoretical fundamentals of the method, its experimental realization and connections with other types of near-field optical microscopy are discussed. Coherent FRET SNOM, which can be realized at liquid helium temperatures, and its possible applications for quantum informatics, are briefly outlined.     

Nanoscopic imaging of human chromosomes via a scanning near-field optical/atomic-force microscopy (SNOAM)

Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographic and fluorescence images of human chromosomes. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever. Optical resolution was approximately 50–100 nm in fluorescence mode. Conventional karyotyping information was linked with SNOAM topographic analyses such as location of centromere and length of individual chromosomes. The height profile clearly indicated higher teromere regions. The SNOAM fluorescence images were different shapes from topographic images probably due to results from the combination of fluorescence dye and chromosome DNA.     

Observation of nanostructure by scanning near-field optical microscope with small sphere probe

Step and terrace structure has been observed in an area of 1 μm×1 μm on the cleaved surface of KCl-KBr solid-solution single crystal by scanning near-field optical microscope (SNOM) with a small sphere probe of 500 nm diameter. Lateral spatial resolution of the SNOM system was estimated to be 20 nm from the observation of step width and the scanning-step interval. Vertical spatial resolution was estimated to be 5-2 nm from the observation of step height and noise level of photomultiplier tube (PMT). With applying a dielectric dipole radiation model to the probe surface, the reason why such a high spatial resolution was obtained in spite of the 500 nm sphere probe, was understood as the effect of the near-field term appeared in the radiation field equations.     

Near-field microscopy: throwing light on the nanoworld

Optical microscopy with nanoscale resolution, beyond that which is possible with conventional diffraction-limited microscopy, may be achieved by scanning a nanoantenna in close proximity to a sample surface. This review will first aim to provide an overview of the basic principles of this technique of scanning near-field optical microscopy (SNOM), before moving on to consider the most widely implemented form of this microscopy, in which the sample is illuminated through a small aperture held less than 10 nm from the sample surface for optical imaging with a resolution of ca. 50 nm. As an example of the application of this microscopy, the results of SNOM measurements of light-emitting polymer nanostructures are presented. In particular, SNOM enables the unambiguous identification of the different phases present in the nanostructures, through the local analysis of the fluorescence from the polymers. The exciting new possibilities for high-resolution optical microscopy and spectroscopy promised by apertureless SNOM techniques are also considered. Apertureless SNOM may involve local scattering of light from a sample surface by a tip, local enhancement of an optical signal by a metal tip, or the use of a fluorescent molecule or nanoparticle attached to a tip as a local optical probe of a surface. These new optical nanoprobes offer the promise of optical microscopy with true nanometre spatial resolution.     

Near-Field Surface-Enhanced Raman Imaging of Dye-Labeled DNA with 100-nm Resolution

Raman chemical imaging on a scale of 100 nm is demonstrated for the first time. This is made possible by the combination of scanning near-field optical microscopy (SNOM or NSOM) and surface-enhanced Raman scattering (SERS), using brilliant cresyl blue (BCB)-labeled DNA as a sample. SERS substrates were produced by evaporating silver layers on Teflon nanospheres. The near-field SERS spectra were measured with an exposure time of 60 s and yielded good signal-to-noise ratios (25:1). The distinction between reflected light from the excitation laser and Raman scattered light allows the local sample reflectivity to be separated from the signal of the adsorbed DNA molecules. This is of general importance to correct for topographic coupling that often occurs in near-field optical imaging. The presented data show a lateral dependence of the Raman signals that points to special surface sites with particularly high SERS enhancement.     

Numerical analysis of the transmission efficiency of heat-drawn and chemically etched scanning near-field optical microscopes

The scanning near-field optical microscope (SNOM) has been tested experimentally for a wide variety of applications, but, to date, there has been little work done on the numerical or analytical modeling of the optical field as it propagates throughout the SNOM probe. Therefore, the fabrication on the probes relies more on trial and error than on clear design principles. An algorithm has been developed for the study and optimization of the geometry of SNOM probes fabricated by the heat-drawn and the one-step chemically etched methods. The algorithm uses the finite-difference beam propagation method (FD-BPM) to model the field evolution throughout the SNOM structure.     

Biological imaging with a near-field optical setup

Noncontact scanning near-field optical microscope (SNOM) systems can be used to optically resolve samples in atmospheric conditions at theoretical resolutions comparable to those of transmission electron microscope and atomic force microscope systems. SNOM systems are also increasingly used to image biological samples. In this study we custom built a SNOM system with the aim of further demonstrating the potential applications of near-field optical examination of biological material. In this study we were able to image both fixed whole-cell samples in air and liquid environments and live whole-cell samples in liquids. The images acquired were of a relatively low resolution, but this work has shown that SNOM systems can be used to monitor the dynamics of living cells at subnanometric resolutions in the z axis and for fluorescent imaging of whole cells in a liquid medium.     

High-resolution optical imaging of magnetic-domain structures

High-resolution optical techniques for imaging magnetic domains in ferromagnetic materials such as confocal microscopy and scanning near-field optical microscopy (SNOM) are reviewed. The imaging capabilities of different techniques and image formation are discussed in the case of in-plane as well as out-of-plane magnetic anisotropy in different illumination configurations. It is shown that the magnetooptical resolution of near-field measurements depends on the film thickness and is limited by the diffraction on magnetic domains throughout the film. For thin magnetic films, subwavelength resolution can be attained. In addition to well-known near-field magnetooptical effects (out-of plane magnetization sensitivity of linear near-field microscopy and in-plane magnetization sensitivity of nonlinear near-field measurements), linear SNOM imaging of in-plane magnetization in thin magnetic films as well as nonlinear imaging of out-of-plane domains has been demonstrated. Thus, linear and second-harmonic near-field imaging of both in-plane and out-of-plane oriented magnetic domains can be achieved in transparent and opaque specimens. This enables applications of SNOM for studies of all kinds of magnetic materials. High-resolution optical imaging is required for characterization of the micro-magnetic and magnetooptical properties of novel magnetic structures in order to adopt a bottom-up approach in the search for new materials with improved characteristics.     

Near-field microscopy by elastic light scattering from a tip

We describe ultraresolution microscopy far beyond the classical Abbe diffraction limit of one half wavelength (lambda/2), and also beyond the practical limit (ca. lambda/10) of aperture-based scanning near-field optical microscopy (SNOM). The 'apertureless' SNOM discussed here uses light scattering from a sharp tip (hence scattering-type or s-SNOM) and has no lambda-related resolution limit. Rather, its resolution is approximately equal to the radius a of the probing tip (for commercial tips, a < 20 nm) so that 10 nm is obtained in the visible (lambda/60). A resolution of lambda/500 has been obtained in the mid-infrared at lambda = 10 microm. The advantage of infrared, terahertz and even microwave illumination is that specific excitations can be exploited to yield specific contrast, e.g. the molecular vibration offering a spectroscopic fingerprint to identify chemical composition. S-SNOM can routinely acquire simultaneous amplitude and phase images to obtain information on refractive and absorptive properties. Plasmon- or phonon-resonant materials can be highlighted by their particularly high near-field signal level. Furthermore, s-SNOM can map the characteristic optical eigenfields of small, optically resonant particles. Lastly, we describe theoretical modelling that explains and predicts s-SNOM contrast on the basis of the local dielectric function.     

Near-field photochemical imaging of noble metal nanostructures

The sub-diffraction imaging of the optical near-field in nanostructures, based on a photochemical technique, is reported. A photosensitive azobenzene-dye polymer is spin coated onto lithographic structures and is subsequently irradiated with laser light. Photoinduced mass transport creates topographic modifications at the polymer film surface that are then measured with atomic force microscopy (AFM). The AFM images correlate with rigorous theoretical calculations of the near-field intensities for a range of different nanostructures and illumination polarizations. This approach is a first step toward additional methods for resolving confined optical near fields, which can augment scanning probe methodologies for high spatial resolution of optical near fields.     

Near-field microscopy of collapsed Langmuir–Blodgett films

Fluorescence near-field scanning optical microscopy (FL-NSOM) is used to probe the nanoscale structure in stained phospholipid monolayers deposited on glass substrates at moderate surface pressures. The FL-NSOM images reveal new liquid-expanded (LE) and liquid-condensed (LC) domains, including one-to-one correlation between fluorescence contrast and film topography. In particular, films of the phospholipid DPPC stained with DiIC₁₂ exhibit multilayer structures that are observed within the solid phase domains and have LE-like fluorescence signals. These features are attributed to clusters of dye molecules resulting from the localized collapse of the film upon compression. Such collapsed features are also observed in supported films of 100% DiIC₁₂ deposited at high surface pressure. In these films, spatially-resolved spectroscopy shows that the collapsed structures are amorphous based on the fluorescence spectrum while the molecules within the solid phase of the film have a fluorescence spectrum indicative of molecular aggregates.     

Analysis of mode coupling due to spherical defects in ideal fully metal-coated scanning near-field optical microscopy probes

We investigate the effect of defects in the metal-coating layer of a scanning near-field optical microscopy (SNOM) probe on the coupling of polarization modes using rigorous electromagnetic modeling tools. Because of practical limitations, we study an ensemble of simple defects to identify important trends and then extrapolate these results to more realistic structures. We find that a probe with many random defects will produce a small but significant coupling of energy between a linearly polarized input mode and a radial/longitudinal polarization mode, which is known to produce a strongly localized emitted optical field and is desirable for SNOM applications.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.     

DNA manipulation and retrieval from an aqueous solution with micromachined nanotweezers

We have demonstrated DNA handling with micromachined nanotweezers that consist of a pair of opposing nanoprobes and integrated thermal expansion microactuators for changing the probe gap. The probe tips coated with a thin Al layer were dipped into a droplet of a solution containing lambda-DNA molecules labeled with fluorescence dye, and then an ac electric field was applied between probes for several seconds. DNA molecules were then captured between the probe tips and retrieved from the solution to the air. The DNA capture between the probe tips could be performed more successfully on the droplet surface than in the underwater region. We also conducted an observation of the retrieved DNA molecules by transmission electron microscope and found that the thickness of the retrieved DNA molecules under the condition of this experiment was approximately 21 nm when the time of the applied ac power (1 MHz, 20 Vpp) was 20 s.     

Interference effect in apertureless near-field fluorescence imaging

Apertureless scanning near-field optical microscopy has been used to image fluorescent latex spheres with a resolution of a few tens of nanometers and good signal-to-noise ratio. The near-field fluorescence images reveal optical interference with several highly contrasted fringes located around the spheres. The origin of the interference is discussed in detail, and models are used to explain their formation. Spatial coherence is also discussed.     

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.