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.     

散射型近场扫描光学显微镜的探针振动参数优化研究

基于原子力显微镜平台设计了可见-近红外波段的散射型近场扫描光学显微镜,通过理论模型计算和实验测量,分析了散射探针振动的调制振幅和扫描反馈幅值对近场信号的影响。研究表明:与探针针尖尺寸相近的调制振幅有利于抑制背景散射噪声及优化近场信号的信噪比;当探针扫描反馈幅值与自由空间调制振幅之比大于90%时,可基本消除探针扫描过程中非简谐振动对近场成像测量的影响。     

Femtosecond near-field scanning optical microscopy study of molecular thin films

A near-field scanning optical microscope has been combined with a two-colour time-resolved pump-probe measurement system. It has a noise-equivalent transmittance change of 5.0 × 10⁻⁵ for a probe pulse with an intensity of 30 nW. The system has been used for evaluating molecular thin films that have a domain structure, particularly for observing a gate action of the single domains. The results include key features to understand an origin of the domains and suggest that the film composition is uniform over a distance of several micrometres.     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10⁻⁵.     

Current-sensing scanning near-field optical microscopy using a metal probe for nanometre-scale observation of electrochromic films

A novel technique for scanning near‐field optical microscopy capable of point‐contact current‐sensing was developed in order to investigate the nanometre‐scale optical and electrical properties of electrochromic materials. An apertureless bent‐metal probe was fabricated in order to detect optical and current signals at a local point on the electrochromic films. The near‐field optical properties could be observed using the local field enhancement effect generated at the edge of the metal probe under p‐polarized laser illumination. With regard to electrical properties, current signal could be detected with the metal probe connected to a high‐sensitive current amplifier. Using the current‐sensing scanning near‐field optical microscopy, the surface topography, optical and current images of coloured WO₃ thin films were observed simultaneously. Furthermore, nanometre‐scale electrochromic modification of local bleaching could be performed using the current‐sensing scanning near‐field optical microscopy. The current‐sensing scanning near‐field optical microscopy has potential use in various fields of nanometre‐scale optoelectronics.     

Spectroscopy with scanning near-field optical microscopy using photon tunnelling mode

We have demonstrated Raman spectroscopy using scanning near-field optical microscopy (SNOM). Photon tunnelling mode was employed, in which the sample is illuminated using an attenuated total reflection (ATR) configuration and the evanescent wave perturbed by the sample is picked up by a sharpened optical fibre probe. By this experimental arrangement Raman scattering from the optical fibre probe was greatly reduced, therefore we were able to excite the sample using more intense laser light compared to the illumination mode SNOM. Raman spectra of copper phthalocyanine (CuPc) were obtained in the off-resonance condition and without using surface-enhanced Raman scattering (SERS).     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Fluorescence resonance energy transfer detected by scanning near-field optical microscopy

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1–10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near-field optical microscope (SNOM) operated in the shared-aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel-by-pixel energy transfer efficiency measurements using SNOM.     

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.     

Optoelectronic detector probes for scanning near-field optical microscopy

A brief explanation of the optoelectronic probe concept and a comparison between the implementation of passive waveguide probes and optoelectronic probes in scanning near-field optical microscopy (SNOM) is presented. The first probe realizations using cleaved semiconductor crystals and the work at present in progress using microfabricated Si pyramids are described. These crystals with evaporated metal electrodes forming a slit aperture with subwave-length dimensions work as metal–semiconductor–metal photodetectors. Their optical detection behaviour is investigated by measuring the intensity distribution of a laser focal point. Measurements where the external bias voltage is changed show that it is possible to modify the detection behaviour of the device because of the varying depletion widths. The last part of the article describes a concept where pyramidal probes should function simultaneously as sensors for scanning force microscopy (SFM) to measure topography and as optoelectronic probes for scanning near-field optoelectronic microscopy (SNOEM).     

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 -α-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.     

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.     

Tapping-mode tuning-fork near-field scanning optical microscopy of low power semiconductor lasers

The newly developed inverted tapping-mode tuning-fork near-field scanning optical microscopy (TMTF-NSOM) is used to study the local near-field optical properties of strained AlGaInP/Ga_0.4In_0.6P low power visible multiquantum-well laser diodes. In contrast to shear-force mode NSOM, TMTF-NSOM provides the function to acquire the evanescent wave intensity ratio | I (2ω)|/| I (ω)| image, from which the evanescent wave decay coefficient q can be evaluated for a known tapping amplitude. Moreover, we probe the near-field stimulated emission spectrum, which gives the free-space laser light wavelength λ_o and the index of refraction n _r of the laser diode resonant cavity. Once q , λ_o, and n _r are all measured, we can determine the angle of incidence θ_o of the dominant totally internally reflected waves incident on the front mirror facet of the resonator. Determination of such an angle is very important in modelling the stability of the laser diode resonator.     

Investigation of dyed human hair fibres using apertureless near-field scanning optical microscopy

We present the first studies of dyed human hair fibres performed with an apertureless scanning near‐field optical microscope. Samples consisted of 5‐µm‐thick cross‐sections, the hair fibres being bleached and then dyed before being cut. Hair dyed with two molecular probes diffusing deep inside the fibre or mainly spreading at its periphery were investigated at a wavelength of 655 nm. An optical resolution of about 50 nm was achieved, well below the diffraction limit; the images exhibited different optical contrasts in the cuticle region, depending on the nature of the dye. Our results suggest that the dye that remains confined at the hair periphery is mainly located at its surface and in the endocuticle.     

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.     

Polarization contrast in reflection near-field optical microscopy with uncoated fibre tips

Using cross-hatched, patterned semiconductor surfaces and round 20-nm-thick gold pads on semiconductor wafers, we investigate the imaging characteristics of a reflection near-field optical microscope with an uncoated fibre tip for different polarization configurations and light wavelengths. It is shown that cross-polarized detection allows one to effectively suppress far-field components in the detected signal and to realize imaging of optical contrast on the sub-wavelength scale. The sensitivity window of our microscope, i.e. the scale on which near-field optical images represent mainly optical contrast, is found to be ≈100 nm for light wavelengths in the visible region. We demonstrate imaging of near-field components of a dipole field and purely dielectric contrast (related to well-width fluctuations in a semiconductor quantum well) with a spatial resolution of ≈100 nm. The results obtained show that such a near-field technique can be used for polarization-sensitive imaging with reasonably high spatial resolution and suggest a number of applications for this technique.     

Scanning near-field optical microscopy of a cell membrane in liquid

The applications of scanning near‐field optical microscopy to biological specimens under physiological conditions have so far been very rare since common techniques for a probe–sample distance control are not as well suited for operation in liquid as under ambient conditions. We have shown previously that our own approach for a distance control, based on a short aperture fibre probe and a tuning fork as force sensor in a tapping mode, works well even on soft material in water. By means of an electronic self‐excitation circuit, which compensates for changes of the resonance frequency due to evaporation of liquid, the stability of the force feedback has now been further improved. We present further evidence for the excellent suitability of the tapping‐mode‐like distance control to an operation in liquid, for example, by force‐imaging of double‐stranded DNA. Moreover, we demonstrate that a nuclear envelope in liquid can be imaged with a high optical resolution of ～70 nm without affecting its structural integrity. Thereby, single nuclear pores in the nuclear envelope with a nearest neighbour distance of ～120 nm have been optically resolved for the first time.