Estimation of AFM tip shape and status in linewidth and profile measurement

An atomic force microscopy image is a dilation of the specimen surface with the probe tip. Tips wear or are damaged as they are used. And AFM tip shape and position status make AFM images distorted. So it is necessary to characterize AFM tip shape and position parameters so as to reconstruct AFM images. A geometric model-based approach is presented to estimate AFM tip shape and position status by AFM images of test specimens and scanning electron microscope (SEM) images of AFM tip. In this model, the AFM tip is characterized by using a dynamic cone model. The geometric relationship between AFM tip and the sample structure is revealed in linewidth and profile measurement. The method can easily calculate the tip parameters including half-cone angle, installation angle, scanning tilting angle and curvature radius, and easily estimate the position status of AFM tip when AFM tip moves on the specimen. The results of linewidth and profile measurement are amended accurately through this approach.     

Multimodal atomic force microscopy: Biological imaging using atomic force microscopy combined with light fluorescence and confocal microscopies and electrophysiologic recording

Because the atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens, it provides a unique window to the microscopic biological world. A high signal-to-noise ratio in AFM images sets them apart from the images obtained from other techniques: One does not need extensive image analyses often required by other techniques to obtain high-resolution information. AFM can provide molecular details on crystalline as well as amorphous materials. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. AFM's application for biological imaging will rely on an unambiguous identification of imaged structures. For mixed macromolecules, it may be essential to make critical comparisons of the same structural features imaged with AFM and other techniques such as light fluorescence and confocal microscopies, electron microscopy and X-ray diffraction, and biochemical, immunologic, and pharmacologic techniques and electrophysiologic recordings. Significantly, the simple design of AFM allows it to be integrated with other techniques for simultaneous multimodal imaging. Recent combined multimodal imaging include light fluorescence, confocal, and near-field optical imaging as well as electrophysiologic recordings. Preliminary studies from such multimodal imaging include 1) an independent identification of macromolecules in a complex specimen using appropriately labeled markers such as fluorescent-dye labeled antibodies or dark-field microscopy; 2) imaging real-time reorganization of surface features using laser confocal and AFM; 3) a direct correlation of structural features and ion transfer via pores in a membrane; and 4) macromolecular complexes such as receptor-ligand and antigen-antibody. These features of a multimodal imaging system will provide new and significant avenues for a direct real-time structure-function correlation studies of biological macromolecules. © 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 293–300, 1997     

Fabrication and characterization of fluorescent rare-earth-doped glass-particle-based tips for near-field optical imaging applications

Fluorescent rare-earth-doped glass particles glued to the end of an atomic force microscope tip have been used to perform scanning near-field optical measurements on nanostructured samples. The fixation procedure of the fluorescent fragment at the end of the tip is described in detail. The procedure consists of depositing a thin adhesive layer on the tip. Then a tip approach is performed on a fragment that remains stuck near the tip extremity. To displace the particle and position it at the very end of the tip, a nanomanipulation is achieved by use of a second tip mounted on piezoelectric scanners. Afterward, the particle size is reduced by focused ion beam milling. These particles exhibit a strong green luminescence where excited in the near infrared by an upconversion mechanism. Images obtained near a metallic edge show a lateral resolution in the 180-200-nm range. Images we obtained by measuring the light scattered by 250-nm holes show a resolution well below 100 nm. This phenomenon can be explained by a local excitation of the particle and by the nonlinear nature of the excitation.     

The optical detection of individual DNA-conjugated gold nanoparticle labels after metal enhancement

The detection of DNA using nanoparticles as labels is an interesting alternative to the standard fluorescence technique. It requires simpler detection equipment, resulting in higher stability and lower costs. Besides easier detection, metal enhancement allows a higher sensitivity of detection. The signal-response curve for labelled DNA before and after silver enhancement was studied, applying both atomic force microscope (AFM) and optical (reflection/transmission) measurements. The dynamic range and the sensitivity were determined for nanoparticle labelling with and without metal enhancement. Nanoparticle concentrations down to the fM range could be detected. The ultimate limit of detection, the identification of individual labels, is demonstrated for the optical readout. Therefore, AFM images of the particles were correlated with the optical signal of individual or clustered particles. We demonstrate that the optical signal allows the identification of single particles.     

Chemical imaging with a confocal scanning Fourier-transform-Raman microscope

Traditional approaches in confocal microscopy have focused on techniques to generate volumetric intensity or phase images of an object. In these different imaging modes the scattered optical-field properties depend on local refractive index and absorption, properties not unique to a given material. We report here on a confocal microscope that uses Raman scattered light to generate volumetric chemical images of a material. We designed and built a prototype instrument, called a confocal scanning laser Raman microscope, that combines a confocal scanning laser microscope with a Fourier-transform-Raman spectrometer. The high depth and lateral spatial resolution of the confocal optics design define a volume element from which the Raman scattered light is collected, and the spectrometer analyzes its spectral content. The sample is scanned through the microscope probe volume, and a chemical image isgenerated based on the content of the Raman spectrum extracted from each scan position in the sample. The results inclu e instrument characterization measurements and examples of confocal chemical imaging.     

Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.     

Photon scanning-tunneling microscopy of unstained Mammalian cells and chromosomes

The photon scanning-tunneling microscope (PSTM) yields optical topographical images of samples that are thin or that are transparent at the wavelength used. A range of sample sizes can be imaged extending to well below the diffraction limit for sufficiently flat samples. But samples of the order of several to many micrometers in size can be analyzed with less-refined resolution if total internal reflection can be made to occur in the sample. We used the PSTM to examine the optical topography of mouse and human cells and of chromosomes that are unstained. Our objectives were to demonstrate the images as an alternative to conventional microscopy and to provide a sample-preparation methodology that will later permit localized, simultaneous fluorescence or absorption spectroscopy with the signals collected by the probe tip. Furthermore, the PSTM's ability to produce optical profiles in air and in water was tested to establish the basis for future investigation of possible abnormalities in the chromosomes. That is, we considered both physical and biological objectives. To this end we utilized the 442-nm line of a He-Cd laser as well as the 633-nm line from a He-Ne laser, the resulting image quality being tested partly to ascertain the increased effects of scattering at the smaller wavelength. It is shown that adequate resolution and signal-to-noise ratio can be obtained with the shorter wavelength even in the presence of intensity fluctuations from the laser, thus showing that fluorescence and absorption studies can be expected to be practicable.     

Tunneling-stabilized magnetic force microscopy of bit tracks on ahard disk

A scanning tunneling microscope (STM) for surface magnetic force measurements on thin-film longitudinal magnetic storage media is described. The usual rigid PtIr tip of the STM was replaced by a flexible Fe-film tip and the tip position was stabilized near the surface of the sample using the STM feedback system as tunneling occurs between the tip and sample surface. Images of a CoCrTa thin-film hard disk showing 5 μm×3 μm bit tracks written by the ferrite head of a computer disk drive are presented. The images shown are comparable to images of the bit tracks on textured surfaces using either ferrofluid decoration or other magnetic force microscopy (MFM) imaging techniques. The sensitivity of the Fe-film tip was such that the influence on the image due to magnetic forces was larger than the influence due to sample surface topography     

Quantitative mechanical characterization of materials at the nanoscale through direct measurement of time-resolved tip-sample interaction forces

We introduce a new method for material characterization at the nanoscale using a recently developed atomic force microscope (AFM) probe. The FIRAT (force sensing integrated readout and active tip) probe is integrated into a commercial AFM system to obtain time-resolved interaction forces (TRIFs) between the probe tip and sample at speeds suitable for nondestructive and fast imaging of material properties. We present a basic interaction model to extract the material elasticity and surface energy. Numerical simulations are performed and compared to the experimental results for three different polymers and a silicon sample. We find that our interaction model does not completely explain the observed long-range surface forces, but it agrees fairly well with the measurements during the tip-sample contact.     

Behavior of the point-spread function in photon-limited confocal microscopy

We study the behavior of the point-spread function (PSF) of the confocal scanning optical microscope (CSOM) when the available optical energy density from the sample plane is low (<7.5 microJoule/micrometers2). The PSF profile is analyzed under three photon-limited imaging conditions: (1) reflection-type CSOM with a weak source and a perfectly reflecting sample, (2) reflection-type CSOM with a strong illumination source and a weak sample, and (3) fluorescence CSOM with a weak fluorescent sample. Linfoot's image quality criteria of fidelity, structural content, and correlation quality are used to assess the reproducibility of the PSF profile as a function of the photon number. Low photon numbers yield a PSF profile that is difficult to maintain from one location in the sample plane to another. The optical sectioning capability of the CSOM was found to deteriorate more quickly against light power reduction than its transverse resolving power. The signal-to-noise ratio of the scanned CSOM image improves exponentially with the photon number from the sample plane. The noise that is generated by an unstable PSF has an average amplitude that decreases exponentially with the photon number and is significant only at low photon numbers. The CSOM image quality deteriorates because of spurious high-frequency components, degradation in the PSF dynamic range, and varying resolving power.     

Nanoscale chemical imaging using top-illumination tip-enhanced Raman spectroscopy

We present a new top-illumination scheme for tip-enhanced Raman spectroscopy (TERS) in a gap-mode configuration with illumination and detection in a straightforward fashion perpendicular to the sample surface. This illumination focuses the light tightly around the tip end, which effectively diminishes far-field background contributions during TERS measurements. The setup maintains the entire functionality range of both the scanning probe microscopy and the confocal optical microscopy of the setup. For the first time, we show large (64 × 64 up to 200 × 200 pixels), high-resolution TERS imaging with full spectral information at every pixel, which is necessary for the chemical identification of sample constituents. With a scanning tunneling microscope tip and feedback, these TERS maps can be recorded with a resolution better than 15 nm (most likely even less, as discussed with Figure 6). An excellent enhancement (～10(7)×, sufficient for detection of few molecules) allows short acquisition times (<1 s/pixel) and reasonably low laser power (in the microwatt regime) yielding spectroscopic images with high pixel numbers in reasonable time (128 × 128 pixels in <25 min). To the best of our knowledge, no Raman maps with similar pixel numbers and full spectral information have ever been published.     

Transverse dimensions of wood pulp fibres by confocal laser scanning microscopy and image analysis

Cross-sectional images of wood-pulp fibres were generated using optical sectioning by a confocal laser scanning microscope. The technique has a distinct advantage over mechanical sectioning with a microtome, as it simplifies specimen preparation. Cross-sectional images were obtained for unbleached softwood kraft pulp fibres using epifluorescence mode. The accuracy of cross-sectional images was verified by imaging fluorescent microspheres. An image analysis procedure, in which the boundaries of fibre cross-sections were defined with a maximum-gradient edge-finding technique, was developed for measuring fibre cross-sectional area and wall thickness rapidly and accurately. The measurements were insensitive to the confocal microscope's asymmetric resolution, signal deterioration through the specimen thickness, overall image quality and operator bias.     

Spatially resolved analysis of small particles by confocal Raman microscopy: depth profiling and optical trapping

Raman microscopy is a powerful method to provide spatially resolved information about the chemical composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles, either resting on a surface or optically trapped at a laser focus, where the confocal collection volume optimizes the signal from the particle. In this work, the sensitivity and spatial selectivity of detecting Raman scattering from single particles was determined as a function of particle size. An inverted confocal Raman microscope was used to acquire spectra of individual surface-bound and optically trapped polystyrene particles with sizes ranging between 200 nm and 10 microm. The particles are in contact with aqueous solution containing perchlorate ion that served as a solution-phase Raman-active probe to detect interferences from the surrounding medium. The collection volume is scanned through single particles that are attached to the surface of the coverslip, and the sensitivity and selectivity of detection are measured versus particle size. The results compare favorably with a theoretical analysis of the excitation profile and confocal collection efficiency integrated over the volumes of the spherical particles and the surrounding solution. This analysis was also applied to the detection of particles that are optically trapped and levitated above the surface of the coverslip. The results are consistent with the optical trapping of particles at or near the excitation beam focus, which optimizes excitation and selective collection of Raman scattering from the particle.     

基于音叉式AFM测量的软件控制研究

基于音叉式原子力显微镜(AFM)的基本原理,设计了一个幅度调制模式下的测量控制系统,该控制系统的主要实现功能为针尖接近、针尖工作点位置保持和样品的表面形貌测量。实验得到针尖在不同激励电压下的接近曲线,针尖分别在100mV、200mV、500mV、1V激励下,工作区间大小为4.7nm、8.5nm、12.1nm、15.7nm,灵敏度分别为0.0423V/nm、0.0564V/nm、0.0861V/nm、0.0831V/nm。实验得到针尖工作点位置保持过程中的纳米位移台位置变化曲线,针尖工作点的正向偏移值为2.3nm,负向偏移值为1.3nm。表面形貌测量采用X轴双向扫描模式,即在纳米位移台于X轴方向上的往返过程中分别测量1次表面形貌数据,对正向扫描和反向扫描的数据进行计算处理;正向扫描的间距误差为5.2nm,高度误差为0.3nm,反向扫描的间距误差为6.4nm,高度误差为0.5nm。     

Image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope

We describe the three-dimensional (3-D) image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope with the use of two-photon excitation. The 3-D point-spread functions of the 4Pi confocal and regular confocal microscope are measured and compared. Particular emphasis is given to the data acquisition procedure. 4Pi confocal microscopy results in a point-spread function that is 4 times sharper than that of a regular confocal microscope, ultimately leading to superior 3-D imaging of translucent fluorescent specimens. For a two-photon excitation wavelength of approximately 800 nm, we obtain an axial resolution of 140 nm.     

Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Measurement of Surface Topography,Packing Density,and Surface Charge Distribution of an Electrostatically Deposited Powder Layer by Image Analysis of Fluorescent Latex Spheres

A novel method of nonintrusive measurement of surface profile, packing density, and surface charge distributions of a powder layer deposited on a substrate is reported. The method employs the deposition of electrostatically charged monodispersed fluorescent latex spheres (FLS), approximately 2 m in diameter, on the surface of: (1) the substrate before deposition, (2) the powder layer after deposition, and (3) the film formed by curing the powder layer. The surface topography in all cases was mapped using an epi-fluorescent microscope with a vertical resolution of - 2 m in the z axis and - 10 m in the x and y axes. An area of 1 cm 2 1 cm is scanned in 1 mm segments, providing approximately 100 data points per cm 2 for the surface topography. For each measurement of surface topography, the substrate was positioned on the microscope stage in a manner such that the reference points (x, y, and z) remained the same for all measurements of the substrate. The surface profiles, with respect to the same reference points, were plotted using Origin 6.0 software for 3D presentation of the topography. The method was also applied to map the surface charge density distribution of electrostatically charged surfaces. The FLS imaging method provides a new tool for examination of surface profiles, packing density, and charge distribution of powder layers on a microscopic scale not provided by optical or atomic force/electrostatic force microscopy (AFM/EFM). While AFM and EFM are very effective in providing similar information with nanometer resolution, they cannot be directly applied on a larger macroscopic scale to study powder layers and for a larger surface area (up to 1 cm 2 or greater) involving deposited particles in the range of 1-50 m in diameter. For AFM, the range in the z-axis is limited to - 3 m and the x-y scan area is limited to 100 m 2 100 m. The FLS method has a much wider range but it is operated manually; an automated scanning process is required for rapid measurement. A comparison of the FLS and EFM techniques as they apply to analyzing charge distribution on coal surfaces is presented.     

The growth and characterization of ZnO/ZnTe core-shell nanowires and the electrical properties of ZnO/ZnTe core-shell nanowire field effect transistor

Vertically aligned ZnO/ZnTe core-shell nanowires were grown on a-plane sapphire substrate by using chemical vapor deposition with gold as catalyst for the growth of ZnO core and then followed by growing ZnTe shell using metal-organic chemical vapor deposition (MOCVD). Transmission electron microscope (TEM) and Raman scattering indicate that the core-shell nanostructures have good crystalline quality. Three-dimensional fluorescence images obtained by using laser scanning confocal microscope demonstrate that the nanowires have good optical properties. The core-shell nanowire was then fabricated into single nanowire field effect transistor by standard e-beam photolithography. Electrical measurements reveals that the p-type ZnO/ZnTe FET device has a turn on voltage of -1.65 V and the hole mobility is 13.3 cm2/V s.     

High-speed, sub-15 nm feature size thermochemical nanolithography

We report a nanolithography technique that allows simultaneous direct control of the local chemistry and topography of thin polymer films. Specifically, a heated atomic force microscope (AFM) tip can write sub-15 nm hydrophilic features onto a hydrophobic polymer at the rate of 1.4 mm per s. The thermally activated chemical reactions and topography changes depend on the chemical composition of the polymer, the raster speed, the temperature at the AFM tip/sample interface, and the normal load. This method is conceptually simple, direct, extremely rapid, achievable in a range of environments, and potentially adaptable to other materials systems.     

Spatial differentiation of sub-micrometer domains in a poly(hydroxyalkanoate) copolymer using instrumentation that combines atomic force microscopy (AFM) and infrared (IR) spectroscopy

Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument (AFM-IR) capable of producing sub-micrometer spatial resolution IR spectra and absorption images. This new capability enables the spectroscopic characterization of microdomain-forming polymers at levels not previously possible. Films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were solution cast on ZnSe prisms, followed by melting and annealing to generate crystalline microdomains of different sizes. A tunable IR laser generating pulses of the order of 10 ns was used for excitation of the sample films. Short duration thermomechanical waves, due to infrared absorption and resulting thermal expansion, were studied by monitoring the resulting excitation of the contact resonance modes of the AFM cantilever. Dramatic differences in the room-temperature IR spectra are observed in the 1200-1300 cm(-1) range as a function of position on a spatial scale of less than one micrometer. This spectral region is particularly sensitive to the polymer backbone conformation. Such dramatic spectral differences have also been observed previously in bulk IR measurements, but only by comparing room-temperature spectra with ones collected at higher temperatures. Less dramatic, but significant, AFM-IR spectral differences are observed in the carbonyl stretching region around 1720 cm(-1) as a function of location on the sample. Two overlapping, but relatively sharp, carbonyl bands are observed near 1720 cm(-1) in more crystalline regions of the polymer, while a broader carbonyl stretching band appears centered at 1740 cm(-1) in the more amorphous regions. Using this spectral region, it is possible to monitor the development of polymer crystalline structures at varying distances from a nucleation site, where the site was generated by bringing a heated AFM tip close to a specific location to locally anneal the sample.