扫描近场光学显微镜及其在生物微结构探测上的应用

扫描近场光学显微镜（ＳＮＯＭ）可探测生物体亚波长水平的光学特性，以接近分子水平的分辨率在生物微结构成象、探测和修饰等多方面有巨大的潜力。本文对ＳＮＯＭ的技术方法及其近期的的生物学应用进行了介绍。     

A simple and accurate method for calibrating the oscillation amplitude of tuning-fork based AFM sensors

We have developed a simple and accurate method for calibrating the amplitude of vibration of quartz tuning fork sensors commonly used in atomic force- and near field optical-microscopy. Unlike interferometric methods, which require a complex optical setup, the method we present requires only a simple measurement of the electro-mechanical properties of the tuning-fork oscillator and can be performed in a matter of minutes without disturbing the experimental setup. Comparison with interferometric methods shows that an accuracy of better than few percent can be routinely achieved.     

Fibre-top atomic force microscope probe with optical near-field detection capabilities

We present a fibre-top probe fabricated by carving a tipped cantilever on an optical fibre, with the tip machined in correspondence of the fibre core. When approached to an optical prism illuminated under total internal reflection conditions, the tip of the cantilever detects the optical tunnelling signal, while the light coupled from the opposite end of the fibre measures the deflection of the cantilever. Our results suggest that fibre-top technology can be used for the development of a new generation of hybrid probes that can combine atomic force microscopy with scanning near field optical microscopy.     

Photo-deprotection patterning of self-assembled monolayers

Photo-deprotectable self-assembled monolayers (SAMs) provide a versatile platform for creating functional patterned surfaces. In this study, we present nanoscale photo-patterning, multi-component patterning, and a method for producing molecular gradients using photo-deprotectable SAMs. Nanoscale patterning of photo-deprotectable SAMs was achieved by coupling a UV laser (365 nm) through a scanning near field probe to produce nanoscale lines of ～40 nm, i.e. λ/9. Multi-component patterning was achieved by a two-stage method combining both microcontact printing and soft-UV photo-patterning. The example demonstrated in this study produced a three-component patterned surface with regions of CF₃, CH₃ and COOH/CF₃ functionality. The versatility of these photocleavable SAMs is further demonstrated by creating linear molecular gradients of two functionalities along a distance of ～25 mm. The use of ‘soft’ UV gives several advantages including the ability to pattern SAMs with micron-scale features over large areas quickly, with greater control over the photochemical reactions, and compatibility with existing lithographic facilities thus offering an effective alternative to other patterning methods such microcontact-printing or deep UV patterning.     

反射式扫描近场光学显微镜及其成像

介绍了一种光纤探针同时作为光源和探测器的反射式扫描近场光学显微镜，该装置通过采用相干探测、偏振分光等技术克服了该类显微镜所普遍面临的噪声背景干扰问题，具有较高的光学信噪比，并通过样品纵向振动调制锁相实现了对样品表面光强梯度信号的探测成像，进一步改善了成像质量。成像研究表明该装置可对近场衰逝分量进行有效探测并实现了超衍射极限成像。     

Mid-infrared scanning near-field optical microscope resolves 30 nm

We explore the performance of a scanning near-field infrared microscope, which works by scattering tightly focused CO₂ laser radiation (λ = 10  μ m) from the apex of a metallized atomic force microscope tip. The infrared images of test samples prove a spatial resolution of 30 nm and are free of topographical and inertial artefacts, thus they should be of great interest for practical applications. We also observe that the infrared contrast vanishes when the input beam polarization is orthogonal to the tip axis, in accordance with theoretical expectations for a mechanism of longitudinal field interaction.     

Photocurrent near-field microscopy of Schottky barriers

We used a combination of internal photoemission and of near-field optical microscopy (SNOM) to study the lateral variations in solid interface properties such as energy barriers and electron–hole recombination. In particular we investigated the fully formed Pt–GaP, Au–GaAs, Au–SiN _x –GaAs and PtSi–Si Schottky barriers. Our approach enabled us to measure large lateral variations in the photocurrent with spatial resolution on the nanometric scale. Due to the ability of SNOM to supply parallel topographic information, we observed photocurrent variations from zone to zone that only correlated in a few cases with local variations in surface morphology. We assigned the uncorrelated fluctuations to local variations in the interface stoichiometry, the presence of interface states induced by the metallic overlayer and to defect states at the junction. Furthermore, by tuning the photon energy and applied bias we were able to measure the surface distribution of the diffusion length.     

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin–glycoprotein complex and the vinculin–talin–integrin system constitute, together a protein machinery, called costameres. The dystrophin–glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin–talin–integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near‐field fluorescence microscopy to the spatial localization of α‐sarcoglycan and β1D‐integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near‐field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture‐SNOM the sample is excited through the nanometre‐scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.