Nano-slit probes for near-field optical microscopy fabricated by focused ion beams

The near-field probes described in this paper are based on metallized non-contact atomic force microscope cantilevers made of silicon. For application in high-resolution near-field optical/infrared microscopy, we use aperture probes with the aperture being fabricated by focused ion beams. This technique allows us to create apertures of sub-wavelength dimensions with different geometries. In this paper we present the use of slit-shaped apertures which show a polarization-dependent transmission efficiency and a lateral resolution of < 100 nm at a wavelength of 1064 nm. As a test sample to characterize the near-field probes we investigated gold/palladium structures, deposited on an ultrathin chromium sublayer on a silicon wafer, in constant-height mode.     

Microfabricated silicon dioxide cantilever with subwavelength aperture

We have developed a microfabricated SiO₂ cantilever with subwavelength aperture for scanning near-field optical microscopy (SNOM), to overcome the disadvantages of conventional optical fibre probes such as low reproducibility and low optical throughput. The microcantilever, which has a SiO₂ cantilever and an aperture tip near the end of the cantilever, is fabricated in a reproducible batch process. The circular aperture with a diameter of 100–150 nm is formed by a focused ion-beam technique. Incident light is directly focused on the aperture from the rear side of the cantilever using a focusing objective, and high optical throughput (10⁻² to 10⁻³) is obtained. The microcantilever can be operated as a SNOM probe in contact mode or in dynamic mode.     

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.     

Shear force near-field optical microscope based on Q-controlled bimorph sensor for biological imaging in liquid

Shear force near‐field microscopy on biological samples in their physiological environment loses considerable sensitivity and resolution as a result of liquid viscous damping. Using a bimorph‐based cantilever sensor incorporating force feedback, as recently developed by us, gives an alternative force detection scheme for biological imaging in liquid. The dynamics and sensitivity of this sensor were theoretically and experimentally discussed. Driving the bimorph cantilever close to its resonance frequency with appropriate force feedback allows us to obtain a quality factor (Q‐factor) of up to 10³ in water, without changing its intrinsic resonance frequency and spring constant. Thus, the force detection sensitivity is improved. Shear force imaging on mouse brain sections and human skin tissues in liquid with an enhanced Q‐factor of 410 have shown a high sensitivity and stability. A resolution of about 50 nm has been obtained. The experimental results suggest that the system is reliable and particularly suitable for biological cell imaging in a liquid environment.     

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.     

Coaxial probes for scanning near-field microscopy

This paper deals with the development of coaxial aperture tips integrated in a cantilever probe for combined scanning near-field infrared microscopy and scanning force microscopy. A fabrication process is introduced that allows the batch fabrication of hollow metal aperture tips integrated on a silicon cantilever. To achieve the coaxial tip arrangement a metal rod is deposited inside the hollow tip using the focused ion beam technique. Theoretical calculations with a finite integration code were performed to study the transmission characteristics of coaxial tips in comparison with conventional aperture probes. In addition, the influence of the geometrical design parameters of the coaxial probe on its optical behaviour is investigated.     

SNOM/SFM结合的近场光学探针

介绍了近年来近场光学探针技术的进展，特别是可与扫描力显徽镜结合的探针制备技术。     

近场光学显微镜及其在生物膜方面的应用

本文首先介绍近场光学显微镜的基本原理,然后介绍近场光学显微镜与传统光学显微镜、原子力显微镜、扫描隧道显微镜相比,在生物膜研究方面的优势。并在此基础上着重介绍近场光学显微镜在生物膜方面的应用。     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

A versatile multipurpose scanning probe microscope

A combined scanning probe microscope has been developed that allows simultaneous operation as a non‐contact/tapping mode atomic force microscope, a scattering near‐field optical microscope, and a scanning tunnelling microscope on conductive samples. The instrument is based on a commercial optical microscope. It operates with etched tungsten tips and exploits a tuning fork detection system for tip/sample distance control. The system has been tested on a p‐doped silicon substrate with aluminium depositions, being able to discriminate the two materials by the electrical and optical images with a lateral resolution of 130 nm.     

Photoconductive imaging in a photon scanning tunnelling microscope capable of point-contact current sensing using a conductive fibre probe

A photoconductive photon scanning tunnelling microscope was developed to investigate the point-contact photoconductive properties of condensed matter. In order to detect the current and the optical signal at a local point on a surface, we coated the edge of a bent type fibre probe with indium tin oxide. Thus it was possible to measure both photocurrent and optical property with subwavelength resolution. The performance of the novel microscope was evaluated by analysing an organic thin film of copper phthalocyanine (CuPc), which is known to be an efficient photoconductive material. Photocurrent and current–voltage characteristics were observed at the local point on the CuPc thin films. Furthermore, photoconductive images were obtained with topography and near-field optical imaging using this system. The photoconductive PSTM shows potential in various areas of future optics and electronics.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

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.     

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.     

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.     

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.     

A compact sensor head for simultaneous scanning force and near-field optical microscopy

A compact sensor head based on scanning force microscopy (SFM) using cantilever probes has been developed. The idea is to replace the microscope objective of a conventional optical microscope by this compact module and turn the optical microscope into a scanning force and near-field optical microscope with subwavelength resolution. We describe our concept and present initial results showing images of the object’s optical properties and surface topography recorded simultaneously.     

Sensitivity and resonant frequency of an AFM with sidewall and top-surface probes for both flexural and torsional modes

The resonant frequencies and flexural sensitivities of an atomic force microscope (AFM) with assembled cantilever probe (ACP) are studied. This ACP comprises a horizontal cantilever, a vertical extension and two tips located at the free ends of the cantilever and the extension, which makes the AFM capable of simultaneous topography at top surface and sidewalls of microstructures especially microgears, which consequently leads to a time-saving swift scanning process. In this work, the effects of the sample surface contact stiffness and the geometrical parameters such as the ratio of the vertical extension length to the horizontal cantilever length and the distance of the vertical extension from clamped end of the horizontal cantilever on both flexural and torsional resonant frequencies and sensitivities are assessed. These geometrical effects are illustrated in some figures. The results show that the low-order vibration modes are more sensitive for low values of the contact stiffness, but the situation is reversed for high values.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Karyotyping of barley chromosomes by a new fluorescence banding technique combined with scanning probe microscopy

Fluorescence banding has been used to classify chromosomes, except those of barley. Four of the seven barley chromosomes are indistinguishable by length or arm ratio. C-banding has been used for classification; however, it requires a long aging period. Here, we describe a new fluorescence banding method for barley. The chromosomes are treated with warm acetate followed by staining with a fluorescent dye, YOYO-1. Using this method, all seven barley chromosomes can be clearly distinguished. Atomic force microscopy and scanning near-field microscopy analyses revealed that the surfaces of the banded chromosomes were flat, indicating that the fluorescence intensity reflected the internal DNA density or condensation of chromatin.