Near-field optical excitation as a dipole–dipole energy transfer process

The process of fluorescence excitation in the scanning near-field optical microscope (SNOM) is considered as a dipole–dipole resonance energy transfer process between a molecule under study and a SNOM aperture, which can be treated as a magnetic-type point dipole. It is shown that such an approach satisfactorily describes the conditions of the usual SNOM fluorescence experiments. Fluorescence excitation dependence on the polarization of the incident light and medium refraction index have been obtained. The equation to calculate the resonance dipole–dipole energy transfer radius (which is a natural unit of a SNOM's longitudinal resolution) is derived. Those cases where such a radius is of the order of the SNOM aperture, and thus single dipole, can strongly influence the radiation conditions are discussed briefly.     

Near-field optical excitation as a dipole-dipole energy transfer process

The process of fluorescence excitation in the scanning near-field optical microscope (SNOM) is considered as a dipole-dipole resonance energy transfer process between a molecule under study and a SNOM aperture, which can be treated as a magnetic-type point dipole. It is shown that such an approach satisfactorily describes the conditions of the usual SNOM fluorescence experiments. Fluorescence excitation dependence on the polarization of the incident light and medium refraction index have been obtained. The equation to calculate the resonance dipole-dipole energy transfer radius (which is a natural unit of a SNOM's longitudinal resolution) is derived. Those cases where such a radius is of the order of the SNOM aperture, and thus single dipole, can strongly influence the radiation conditions are discussed briefly.     

Mechanisms of cell-cell adhesion identified by immunofluorescent labelling with quantum dots: A scanning near-field optical microscopy approach

Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy from cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The application of the technique to investigate cell-cell adhesion has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions. This has been achieved via the selective tagging of the cell adhesion protein, E-cadherin, by immunofluorescence labelling. Two labelling routes were explored; Alexa Fluor 488 and semiconductor quantum dots. The quantum dots demonstrated significantly enhanced photostability and high quantum yield making them a versatile alternative to the conventional organic fluorophores often used in such a study. Analysis of individual cells revealed that E-cadherin is predominantly located along the cell periphery but is also found to extend throughout their filopodia. We have demonstrated that with a fully optimised sample preparation methodology, quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.     

Probe design optimization for a high-resolution scattering-type scanning near-field optical microscope

The scattering-type scanning near-field optical microscope (SNOM) has a probe with a sharp tip for use in high resolution imaging. As sharp a tip as possible is generally considered ideal for the observations, but actually, a sharp tip does not always provide a high resolution SNOM image. We numerically examined the scattering property of the SNOM probe by the three dimensional finite difference time domain method. In this paper, we show the criterion for the ideal scattering probe which satisfies the simple relation between radius and taper angle of the tip.     

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.     

The tetrahedral tip as a probe for scanning near-field optical microscopy at 30 nm resolution

The tetrahedral tip is introduced as a new type of a probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30 nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30 nm.     

A silanized mica substrate suitable for high-resolution fiber FISH analysis by scanning near-field optical/atomic force microscopy

We applied a novel silanized mica substrate with an extremely flat surface constructed according to Sasou et al. (Langmuir 19, 9845-9849 (2003)) to high-resolution detection of a specific gene on a DNA fiber by scanning near-field optical/atomic force microscopy (SNOM/AFM). The interaction between the substrate and fluorescence-dye conjugated peptide nucleic acid (PNA) probes, which causes fluorescence noise signal, was minimal. By using the substrate, we successfully obtained a fluorescence in situ hybridization signal from the ea47 gene on a λphage DNA labeled with an Alexa 532-conjugated 15-base PNA probe. As the results, no fluorescence noises were observed, indicating that the surface adsorbed almost none of the PNA probe. The combination of the substrate and SNOM/AFM is an effective tool for visualizing DNA sequences at nanometer-scale resolution.     

High-Resolution Lithography with Near-Field Optical Microscopy

Scanning near-field optical microscopy (SNOM) is used for lithography to avoid the resolution limiting diffraction of conventional optical methods. We have expanded a commercial SNOM for writing even complex structures on the nanometer scale. Scanning near-field optical lithography (SNOL) has been applied to conventional resists to explore its potential and the possible combination with conventional optical lithography (mix and match technique).     

Dynamic etching method for fabricating a variety of tip shapes in the optical fibre probe of a scanning near-field optical microscope

A novel etching method for an optical fibre probe of a scanning near-field optical microscope (SNOM) was developed to fabricate a variety of tip shapes through dynamic movement during etching. By moving the fibre in two-phase fluids of HF solution and organic solvent, the taper length and angle can be varied according to the movement of the position of the meniscus on the optical fibre. This method produces both long (sharp angle) and short (wide angle) tapered tips compared to tips made with stationary etching processes. A bent-type probe for a SNOM/AFM was fabricated by applying this technique and its throughput efficiency was examined. A wide-angle probe with a 50° angle at the tip showed a throughput efficiency of 3.3 × 10⁻⁴ at a resolution of 100 nm.     

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.     

Resolution enhancing using cantilevered tip-on-aperture silicon probe in scanning near-field optical microscopy

Scanning near-field optical microscopy (SNOM) achieves a resolution beyond the diffraction limit of conventional optical microscopy systems by utilizing subwavelength aperture probe scanning. A problem associated with SNOM is that the light throughput decreases markedly as the aperture diameter decreases. Apertureless scanning near-field optical microscopes obtain a much better resolution by concentrating the light field near the tip apex. However, a far-field illumination by a focused laser beam generates a large background scattering signal. Both disadvantages are overcome using the tip-on-aperture (TOA) approach, as presented in previous works. In this study, a finite difference time domain analysis of the degree of electromagnetic field enhancement is performed to verify the efficiency of TOA probes. For plasmon enhancement, silver is deposited on commercially available cantilevered SNOM tips with 20nm thicknesses. To form the aperture and TOA in the probes, electron beam-induced deposition and focused ion beam machining were applied at the end of the sharpened tip. The results show that cantilevered TOA probes were highly efficient for improvements of the resolution of optical and topological measurement of nanostructures.     

Dynamic etching method for fabricating a variety of tip shapes in the optical fibre probe of a scanning near-field optical microscope

A novel etching method for an optical fibre probe of a scanning near-field optical microscope (SNOM) was developed to fabricate a variety of tip shapes through dynamic movement during etching. By moving the fibre in two-phase fluids of HF solution and organic solvent, the taper length and angle can be varied according to the movement of the position of the meniscus on the optical fibre. This method produces both long (sharp angle) and short (wide angle) tapered tips compared to tips made with stationary etching processes. A bent-type probe for a SNOM/AFM was fabricated by applying this technique and its throughput efficiency was examined. A wide-angle probe with a 50 degrees angle at the tip showed a throughput efficiency of 3.3 x 10(-4) at a resolution of 100 nm.     

SNOM/STM using a tetrahedral tip and a sensitive current-to-voltage converter

Scanning near-field optical microscopes (SNOM) using the tetrahedral-tip (T-tip) with scanning tunnelling microscopy (STM) distance control have been realized in transmission and reflection mode. Both set-ups used ordinary STM current-to-voltage converters allowing measurement of metallic samples. In the transmission mode, a resolution of 10 nm to 1 nm with regard to material contrast can be achieved on binary metal samples. Because of the great near-field optical potential of the T-tip with respect to the optical resolution, it is a challenging task to find out whether these results can be transferred to non-metallic sample systems as well. This paper reports on a newly designed SNOM/STM transmission mode set-up using the tetrahedral-tip. It implements a sensitive current-to-voltage converter to widen the field of measurable sample systems. Beyond this, mechanical and optical measuring conditions are substantially improved compared to previous set-ups. The new set-up provides a basis for the routine investigation of metal nanostructures and adsorbed organic monolayers at resolutions in the 10 nm range.     

Imaging of optical disc using reflection-mode scattering-type scanning near-field optical microscopy

A phase-change optical disc was observed using a reflection-mode scattering-type scanning near-field optical microscope (RS-SNOM). In an a.c.-mode SNOM image, the 1.2 μm × 0.6 μm recording marks were successfully observed although the data were recorded on the groove. In contrast, no recording marks could be resolved in a d.c.-mode SNOM image. These results are in good agreement with those from a numerical simulation using the finite difference time domain method. The resolution was better than 100 nm with a.c.-mode SNOM operation and the results indicate that recording marks in phase-change optical media can be directly observed with the RS-SNOM.     

Imaging of optical disc using reflection-mode scattering-type scanning near-field optical microscopy

A phase-change optical disc was observed using a reflection-mode scattering-type scanning near-field optical microscope (RS-SNOM). In an a.c.-mode SNOM image, the 1.2 microm x 0.6 microm recording marks were successfully observed although the data were recorded on the groove. In contrast, no recording marks could be resolved in a d.c.-mode SNOM image. These results are in good agreement with those from a numerical simulation using the finite difference time domain method. The resolution was better than 100 nm with a.c.-mode SNOM operation and the results indicate that recording marks in phase-change optical media can be directly observed with the RS-SNOM.     

Excitation energy transfer of porphyrin in polymer thin films by time-resolved scanning near-field optical microspectroscopy

Thin films of water-soluble free-base porphyrin, 5,10,15,20-tetraphenyl-21H, 23H-porphinetetrasulphonic acid (TPPS) mixed with poly(diallyldimethyl ammonium chloride) (PDDA) have been prepared by a spin-coating method, in which the monomeric species were observed in the spin-coat film, whereas dimer was formed in the cast film prepared from TPPS/PDDA solution. Mesoscopic structures and dynamics of excitation energy migration and trapping of TPPS/PDDA spin-coat film have been analysed by time-resolved scanning near-field optical microspectroscopy (SNOM) and atomic force microscope. The observed film structure can be classified roughly into two parts: one is a large, flocculated polymer part, and the other is a smooth part widely spread around the flocculated polymers. In the smooth part, the observed spindle-like structure and circular hills and dips are essentially due to PDDA. The ellipsoidal small structures with ∼2µm length and <1 µm width in the flocculated polymer part show non-exponential fluorescence decays. The non-exponential dynamics originates from the excitation energy migration among TPPS monomers and energy trapping to dimers. From the analysis of fluorescence decay curves based on the equation developed by Klafter and Blumen, the spectral dimension has been estimated to be ∼1.46 for ellipsoidal structures. These results indicate that the distribution of the chromophore is inhomogeneous and a fractal-like structure exists even in the small domains determined by the resolution of the SNOM tip.     

Near-field magneto-optical analysis in reflection mode SNOM

Observation of magnetic domains with in-plane magnetisations is demonstrated by scanning near-field optical microscopy (SNOM) in reflection mode. The longitudinal and transverse magneto-optical Kerr effects are employed as the contrast mechanisms; these are observed as either a change in the polarisation of the reflected light or reflectance, depending on magnetisation direction. SNOM images of Co and Ni thin films show magneto-optical contrast depending on polarisation of the incident and detected light. For the smooth cobalt thin films, the orientation for magnetic domains is estimated, based on the correlation between the contrasts in SNOM images obtained in different polarisation configurations and the directions of the magnetic vectors of the incident and reflected light. For the nickel films with pronounced topographic structures, the resulting near-field polarisation dependencies are more complicated, suggesting that the magneto-optical contrast in SNOM images are affected by the topographic cross-talk due to the depolarisation effects on surface topographic features.     

Fluorescence scanning near-field optical microscopy of polyfluorene composites

Fluorescence scanning near-field optical microscopy (SNOM) is used to investigate binary polyfluorene-based composites of varying composition. The samples investigated contain blends of the polymer poly(9,9'-dioctylfluorene-cobenzothiadiazole), F8BT, with similar polyfluorenes of wider band gap. Images acquired from a film containing 50% by weight F8BT exhibit a high degree of correlation between the topography and fluorescence, with an F8BT-rich phase which protrudes from the surface of the film forming isolated regions with sizes from hundreds of nanometres to several micrometres. A film containing 10% by weight F8BT also has micrometre-size F8BT-rich regions, but also present are small and locally varying proportions of F8BT in the other polyfluorene component phase, indicating a hierarchy of phases within this sample. The fluorescence and topographic images of a third sample studied, containing 90% by weight F8BT, display no correlation, demonstrating that it is not always appropriate to use topographic information to determine the phase structure within polymer blends. The fluorescence SNOM images acquired from these samples are able to assist our understanding of the photovoltaic efficiency of devices fabricated from these films, which are governed by the extent of the interfacial area between these two constituent polymers.     

Spectroscopic scanning near-field optical microscopy with a free electron laser: CH2 bond imaging in diamond films

Hydrogen chemistry in thin films and biological systems is one of the most difficult experimental problems in today's science and technology. We successfully tested a novel solution, based on the spectroscopic version of scanning near-field optical microscopy (SNOM). The tunable infrared radiation of the Vanderbilt free electron laser enabled us to reveal clearly hydrogen-decorated grain boundaries on nominally hydrogen-free diamond films. The images were obtained by SNOM detection of reflected 3.5 µm photons, corresponding to the C–H stretch absorption, and reached a lateral resolution of 0.2 µm, well below the λ/2 (λ= wavelength) limit of classical microscopy.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.