Scanning near-field optical/atomic force microscopy for fluorescence imaging and spectroscopy of biomaterials in air and liquid: Observation of recombinantEscherichia coli with gene coding to green fluorescent protein

We have developed a system of scanning near-field optical/atomic force microscopy (SNOM/AFM) for fluorescence imaging and spectroscopy of biomaterials in air and liquid. SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Optical resolution of SNOM images shows about 50 nm in an illumination mode for a standard sample of a patterned chromium layer of 20 nm thickness on a quartz glass plate. The SNOM/AFM system contains a photon counting system and polychrometer/ICCD (intensified charge coupled device) system for observation of the fluorescence image and spectrograph of micro areas, respectively. The gene coding to green fluorescence protein (GFP) was cloned in recombinantEscherichia coli (E. coli). Topography, fluorescence image and spectrograph of recombinantE. coli by SNOM/AFM showed a difference in fluorescence in individualE. coli. Fluorescence activity of GFP can thus be used as a convenient indicator of transformation. SNOM/AFM is also applicable to observe immobilizedE. coli on a glass plate in water with a liquid chamber and may allow the viewing of observation of floating organisms.     

Scanning Near-Field Optical Microscopy and Microspectroscopy of Green Fluorescent Protein in Intact Escherichia coli Bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.     

照明模式SNOM中样品对近场光场分布的影响

在同时考虑样品的形貌及材料光学参量和入射光偏振模式的情况下,利用基于边界元方法编写的二维矢量电磁场计算程序,对工作在照明模式下的扫描近场光学显微镜（Scanning Near-field Optical Microscope,SNOM）的近场矢量电磁场分布进行了数值计算模拟研究.结果表明,在没有表面形貌特征时,探针的光能量透射率随样品材料的折射率和损耗角的增加而增大,而样品表面光斑尺寸受折射率和损耗角的影响很小;对有形貌特征的探针扫描像研究结果表明,SNOM的分辨率随着样品的折射率和损耗角的增加而提高;对SNOM不同的工作模式的扫描成像信号进行的计算结果表明,恒定间距扫描方式比恒定高度扫描方式对样品表面的细节有较强的分辨能力.     

扫描近场光学显微镜的光耦合偶极子模型

为了研究扫描近场光学显微镜中探针和粗糙样品表面的耦合相互作用,提出了一种光耦合偶极子模型.在该模型中,探针和样品突起都由光极化偶极子表示,在准静态电磁场近似的情况下样品表面的诱导极化效应由影像偶极子表示,应用偶极子辐射理论可以得到系统的自洽场方程.此模型提供了一种直观分析扫描近场光学显微镜中探针和样品相互作用机理的方法.在此基础上,进一步讨论了金属样品的近场成像特点和其特有的局域光学共振现象.数值结果表明：不同于一般的介质样品,金属样品的近场图像与入射光频率直接相关,改变入射光的频率,获得的样品近场图像的形状和对比度都会发生变化.特别是当入射光频率处于样品极化共振范围内时,金属纳米粒子的极化率会出现光极化共振,这样就可以获得样品粒子的最大有效尺寸,为提高系统的分辨率提供了一条重要途径.     

Internal reflection mode scanning near-field optical microscopy with the tetrahedral tip on metallic samples

An internal reflection mode is introduced for scanning near-field optical microscopy (SNOM) with the tetrahedral tip. A beam of light is coupled into the tip and the light specularly reflected out of the tip is detected as a photosignal for SNOM. An auxiliary STM mode is used to control the distance during the scanning process and to record the topography of the sample simultaneously with the SNOM image. Images were obtained of different metallic samples which show a contrast in the order of 10% of the total reflected photosignal. In images of metallic samples an inverted contrast is consistently obtained compared to images previously obtained of comparable samples in a transmission mode. The contrast shows a pronounced dependence on the polarization of the incident beam with respect to the orientation of the edges of the tip. In the case of gold surfaces, the photosignal as a function of distance between the tip and the surface shows a pronounced peak in the near-field range of 0–20 nm which is tentatively attributed to the excitation of surface plasmons on the gold surface. The pronounced near-field effects and the strong contrast in the near-field images and the resolution well below 50 nm are an indication of a highly efficient coupling of the incident beam to a local excitation of the tip apex which is essential for the function of the tip as a probe for SNOM. Received: 17 May 1999 / Accepted: 18 May 1999 / Published online: 21 October 1999     

Image dipole approach and polarization effects in scanning near-field optical microscopy

A coupled-dipole approach is proposed in order to study the coupling between the probe tip and the rough sample in SNOM. In the present model both the optical probe tip and the sample protrusions are represented by polarizable dipole spheres. The induced polarization effects on the sample surface can be replaced by the image dipoles in the circumstance of quasi-static electromagnetic field approximation. Applying the radiation theory of the dipole, we have established a set of self-consistent equations to describe the field distribution at the sites of the probe tip and the sample protrusions. The results are completely the same as those obtained by means of the dyadic electromagnetic propagator formalism and also the derivation procedure is relatively simple. This method permits us to analyze the physical mechanisms of the interaction between the probe tip and the rough surface in SNOM intuitively. Based on this approach, we further discuss the influence of polarization of the incident light on the imaging quality. The calculating result shows that the shape and the contrast of the images of the sample are both sensitive to the field polarization, and the z-polarized mode is proved to give better resolution in SNOM.     

Imaging of human meiotic chromosomes by scanning near-field optical microscopy (SNOM)

Centromeres and telomeres are key structures of mitotic and meiotic chromosomes. Especially telomeres develop particular structural properties at meiosis. Here, we investigated the feasibility of scanning near-field optical microscopy (SNOM) for light-microscopic imaging of meiotic telomeres in the sub-hundred nanometer resolution regime. SNOM was applied to visualise the synaptonemal complex (SC) and telomere proteins (TRF1, TRF2) after differential immuno-fluorescent labelling. We tested and compared two different preparation protocols for their applicability in a SNOM setting using micro-fabricated silicon nitride aperture tips. Protocol I consisted of differential labelling of meiotic chromosome cores (SC) by SCP3 immuno-fluorescence and telomeres by TRF1 or TRF2 immuno-fluorescence, while protocol II combined absorption labelling with alkaline phosphatase substrates of cores with fluorescent labelling of telomeres. The results obtained indicate that protocol I reveals a better visualisation of structural (topographic) details than protocol II. By means of SNOM, meiotic chromosome cores could be visualised at a resolution overtopping that of far-field light microscopy.     

High-resolution imaging of single fluorescent molecules with the optical near-field of a metal tip

We show that a concentration of light at a metal tip allows near-field optical imaging of single fluorescent dye molecules at very high resolution, despite strong quenching effects. Details as small as 10 nm were observed in the fluorescence patterns of single Cy-3 dyes bound to the termini of DNA. Data evaluation by model fitting determines the positions of the dyes to an accuracy even better than 1 nm and also yields their 3D orientation. The metal tip simultaneously provides high-resolution topographic imaging complementing the optical signal for a detailed surface examination.     

Investigation of the Doughnut Effect in Cy3-Labeled Protein Microarrays using Scanning Near-Field Optical Microscope

We demonstrate the fluorescence mapping of protein microarrays by the technique of scanning near-field optical microscopy (SNOM) and confocal microscopy. Micron sized spots (300 μm) of human Immunoglobulin G (hIgG) protein with and without a Cy3 dye labeling have been fabricated on glass substrates by an immobilization method which makes use of calixcrown derivatives termed Prolinker. We have also tried to probe into the well-known “doughnut effect” observed in fluorescence images of proteins using the SNOM technique. The topographic and fluorescence SNOM images revealed that the number of proteins at the boundary of the spot were more than at the center in the case of the microarray spot which showed brighter luminescence at the edge than at the center in the confocal image.     

Photopatterning of a monomolecular dye film by means of scanning near-field optical microscopy

Patterned bleaching of a photolabile monomolecular dye film by means of scanning near-field optical microscopy (SNOM) is demonstrated. After exposure, the written patterns were verified by SNOM with fluorescence detection. The adsorption of appropriate material to such near-field optically created patterns seems now feasible. The ultimate resolution limit of a monomolecular dye film for patterned bleaching by SNOM is discussed on the basis of a simple model.     

SETUP AND APPLICATION OF SCANNING NEAR-FIELD OPTICAL MICROSCOPY

The research on the setup and application of scanning near-field optical microscopy (SNOM) performed in our laboratory is reviewed in this report. We have constructed a versatile low temperature scanning near-field optical microscope with the capability of near-field imaging and spectroscopy, operating at liquid nitrogen temperature. A special designed coaxial double lens was used to introduce the illumination beam through a 200μm fiber; the detected optical signal was transmitted via a fiber tip to an avalanche photon detector. The performance test shows the stability of the new design. The shear force image and optical image of a standard sample are shown. A system of SNOM working at room temperature and atmosphere was used to characterize semiconductors and bio-molecular samples. It revealed the unique features of semiconductor microdisks in the near-field that is significantly different from that of far-field. The effects of different geographic microstructures on the near-field light distribution of InGaP, GaN, and InGaN multi-quantum-well microdisk were observed.     

Scanning near-field optical microscopy-based study of local dynamics of receptor-ligand interactions at the single molecule level

A scanning near-field optical microscope (SNOM)—based modification of the method to study the dynamics of single molecule receptor—ligand interactions exploiting the fluorescence imaging by total internal reflection fluorescence microscopy is introduced. The main advantage of this approach consists in the possibility to study the single molecule interaction dynamics with a subwavelength spatial resolution and a submillisecond time resolution. Additionally, due to the much smaller irradiation area and some other technical features, such a modification enables to enlarge the scope of the receptor—ligand pairs to be investigated and to improve the temporal resolution. We briefly discuss corresponding experimental set up with a special accent on the SNOM operation in liquid and present some preliminary results of related investigations.     

用扫描近场光学显微镜技术研究金膜表面等离体子共振

表面等离体子波(SPW)可与入射光横磁波极化能量耦合并被共振激发,这种现象被称为表面等离体子共振现象(SPR)。主要利用扫描近场光学显微镜(SNOM)技术和表面等离体子共振现象技术相结合,来研究金膜表面等离体子共振。设计并建立了结构独特的新型Kretschmann型表面等离体子共振现象耦合装置,同时又设计了具有厚度梯度的表面等离体子的制备方法。在此基础上,测量了改变入射角条件下的表面等离体子共振曲线,测得该装置的等离体子共振角灵敏度为1°。并且对金膜表面进行表面等离体子共振条件下的扫描近场光学显微成像。实验结果表明,在共振时金膜表面的扫描成像比不共振时清晰,而且增加了很多细节。应用表面等离体子共振现象技术将可以明显提高扫描近场光学显微镜的信噪比、分辨力等性能。     

Near-Field Fluorescence and Topography Characterization of a Single Nanometre Fluorophore by Apertureless Tip-Enhanced Scanning Near-Field Microscopy

Tip-enhanced near-field fluorescence and topography characterization of a single nanometre fluorophore is conducted by using an apertureless scanning near-field microscopy system. A fluorophore with size 80hm is mapped with a spatial resolution of 10hm. The corresponding near-field fluorescence data shows significant signal enhancement due to the apertureless tip-enhanced effect. With the nanometre spatial resolution capability and nanometre local tip-enhanced effect, the apertureless tip-enhanced scanning near-field microscopy may be further used to characterize a single molecule by realizing the local near-field spectrum assignment corresponding to topography at nanometre scale.     

Understanding tapping-mode atomic force microscopy data on the surface of soft block copolymers

In this paper, we focus on better understanding tapping-mode atomic force microscopy (AFM) data of soft block copolymer materials with regard to: (1) phase attribution; (2) the relationship between topography and inside structure; (3) contrast-reversal artifacts; (4) the influence of annealing treatment on topography. The experiments were performed on the surface of poly(styrene–ethylene/butylene–styrene) (SEBS) triblock copolymer acting as a model system. First, by coupling AFM with transmission electron microscopy (TEM) measurements, the phase attribution for AFM images was determined. Secondly, by imaging an atomically flat SEBS surface as well as an AFM tip-scratched SEBS surface, it was confirmed that the contrast in AFM height images of soft block copolymers is not necessarily the result of surface topography but the result of lateral differences in tip-indentation depth between soft and hard microdomains. It was also found that there is an enlarging effect in AFM images on the domain size of block copolymers due to the tip-indention mechanism. Thirdly, based on the tip-indention mechanism, tentative explanations in some detail for the observed AFM artifacts (a reversal in phase image followed by another reversal in height image) as function of imaging parameters were given. Last, it was demonstrated that the commonly used annealing treatments in AFM sample preparation of block copolymers may in some cases lead to a dramatic topography change due to the unexpected order-to-order structure transition.     

Influence of the probe--sample interaction on scanning near-field optical microscopic images in the far field

We have studied the influence of probe--sample interaction in a scanning near-field optical microscopy (SNOM) in the far field by using samples with a step structure. For a sample with a step height of $\sim \lambda $/4, the SNOM image contrast between the two sides of the step changes periodically at different scan heights. For a step height of $\sim \lambda $/2, the image contrast remains approximately the same. The probe--sample interaction determines the SNOM image contrast here. The influence of different refractive indices of the sample has been also analysed by using a simple theoretical model.     

SNOM Observations of Surface Plasmon Polaritons on Metal Heterostructures

We observe surface plasmon polariton （SPP） refraction on a metal heterostructured sample with a scattered-type scanning near-field optical microscope （SNOM）. The sample consists of AI and Au in-plane whose boundary is smooth enough with proper etching time. SPPs excited on the AI film travel to the boundary and a portion of SPPs propagates into the Au film. In addition, interference fringes appear in the SNOM image bent at the boundary. The result is analysed with effective index method and the refracted angle is explained by Shell＇s law.     

近场光学用于激光解吸飞行时间质谱的亚微米级空间分辨成像

基于激光离子源的飞行时间质谱法作为一门新兴的成像方法,已经被广泛应用于材料、地质、环境、药物和生命科学领域中。但受限于光学衍射极限、聚焦透镜的焦距和数值孔径等因素,使其难以实现亚微米尺寸的高空间分辨率成像。近场技术的引入成功地解决了光学衍射极限的限制,将近场技术与激光电离技术相结合,可以实现对固体样品表面纳米级弹坑的剥蚀。此外,传统的质谱成像技术常常假设样品表面是平整的,忽略其表面形貌的高低起伏,但这往往会导致信号强度不稳定和成像假象。为此,不仅需要获得样品中的化学组成与空间分布,还需同时获得样品表面的形貌信息,才能实现多功能的原位表征。在自行研制的激光解吸/电离飞行时间质谱的基础上,采用近场纳米有孔针尖离子源代替传统的远场激光聚焦,以532 nm波长激光为第一束解析激光,355 nm波长激光为后电离激光,音叉式原子力显微镜控制系统针尖与样品之间的距离维持在近场范围内,对酞菁铜镀层样品表面进行了弹坑剥蚀实验,获得了直径为550～850 nm的弹坑点阵;并对7.5 μm×7.5 μm的标准酞菁铜网格样品进行了铜离子亚微米级的高分辨率成像;此外,纳米有孔针尖离子源作为原子力显微镜的一种变体,还可同时获得成像区域的表面形貌信息,这一结合优势大大拓展了质谱技术在微纳尺度下的原位表征能力。     

AFM and SNOM characterization of carboxylic acid terminated silicon and silicon nitride surfaces

Silicon and silicon nitride surfaces have been successfully terminated with carboxylic acid monolayers and investigated by atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). On clean Si surface, AFM showed topographical variations of 0.3-0.4 nm while for the clean Si₃N₄ surface the corrugation was around 3-4 nm. After material deposition, the corrugation increased in both samples with a value in topography of 1-2 nm for Si and 5-6 nm for Si₃N₄. The space distribution of specific chemical species was obtained by taking SNOM reflectivity at several infrared wavelengths corresponding to stretch absorption bands of the material. The SNOM images showed a constant contribution in the local reflectance, suggesting that the two surfaces were uniformly covered.     

SFM/SNOM结合的扫描探测显微镜

采用光纤探针的扫描近场光学显微镜 (SNOM)存在某些弱点 ,如探针特别脆 ,不易贴近样品表面扫描 ,探针的转输效率低等。近年来发展了将SFM /SNOM结合起来的扫描探测显微镜。利用微加工工艺技术 ,将小孔集成在悬臂探针中 ,使探针既能批量制备 ,又具有很好的重复性。探针悬臂在垂直于样品表面方向上的弹性常数较小 ,针尖不易损坏。在接触模式中利用这种SFM /SNOM组合探针可将样品的形貌像、摩擦力和光学透射像等信息同时记录下来。对于综合研究样品表面的介观性质十分有利。