High-resolution imaging of chromosome-related structures by atomic force microscopy

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein.     

Reconstruction of a scanned topographic image distorted by the creep effect of a Z scanner in atomic force microscopy

We analyzed the illusory slopes of scanned images caused by the creep of a Z scanner in an atomic force microscope (AFM) operated in constant-force mode. A method to reconstruct a real topographic image using two scanned images was also developed. In atomic force microscopy, scanned images are distorted by undesirable effects such as creep, hysteresis of the Z scanner, and sample tilt. In contrast to other undesirable effects, the illusory slope that appears in the slow scanning direction of an AFM scan is highly related to the creep effect of the Z scanner. In the controller for a Z scanner, a position-sensitive detector is utilized to maintain a user-defined set-point or force between a tip and a sample surface. This serves to eliminate undesirable effects. The position-sensitive detector that detects the deflection of the cantilever is used to precisely measure the topography of a sample. In the conventional constant-force mode of an atomic force microscope, the amplitude of a control signal is used to construct a scanned image. However, the control signal contains not only the topography data of the sample, but also undesirable effects. Consequently, the scanned image includes the illusory slope due to the creep effect of the Z scanner. In an automatic scanning process, which requires fast scanning and high repeatability, an atomic force microscope must scan the sample surface immediately after a fast approach operation has been completed. As such, the scanned image is badly distorted by a rapid change in the early stages of the creep effect. In this paper, a new method to obtain the tilt angle of a sample and the creep factor of the Z scanner using only two scanned images with no special tools is proposed. The two scanned images can be obtained by scanning the same area of a sample in two different slow scanning directions. We can then reconstruct a real topographic image based on the scanned image, in which both the creep effect of the Z scanner and the slope effect of the sample have been eliminated. The slope effect of the sample should be eliminated so as to avoid further distortion after removal of the creep effect. The creep effect can be removed from the scanned image using the proposed method, and a real topographic image can subsequently be efficiently reconstructed.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

一维矩形光栅AFM图像盲探针表面重建模拟研究

探针结构参数的合理选取将直接决定扫描图像及其盲探针修正图像的失真程度。基于此,以一维矩形模拟光栅为典型案例,对该模拟光栅的原子力显微镜(AFM)扫描成像过程与盲探针修正过程进行了仿真,阐明了探针结构参数对扫描成像过程与盲探针修正过程的影响规律。通过建立线宽变化度与半高宽相结合的图像重建误差评价指标,确定了针对该模拟光栅的AFM探针建议结构参数,并取得了良好的光栅图像重建效果。研究表明,应用线宽变化度结合半高宽来综合评价光栅的AFM测量和图像重建过程,有利于提升实际光栅AFM图像盲探针重建的准确度。     

A large-sample atomic force microscope observing in both air and liquid

A large-sample atomic force microscope (AFM) that allows high resolution observation in both air and liquid has been developed. With a unique beam tracking method, laser beam is capable of reflecting off the same spot on the AFM cantilever throughout raster scan over the entire scan area, either operating in air or in liquid environment. Incorporating the stand-alone AFM probe unit with an automated large sample stage, wide-scan-range imaging can be realized with high resolution and slight distortion. In addition, an image stitching method is utilized to build a broad merged image with range up to millimeters while keeping nanometer order resolution. By using a large-volume liquid bath, large and massive sample can be observed in liquid with this AFM system. Several typical experiments have been carried out to demonstrate the imaging ability and stability of this AFM. Topographic structures of gold pattern on a glass substrate are scanned at two different places on the same specimen surface. The porosity of a sheet of filter paper is then characterized in both air and water. Finally, larger-area AFM image of anodic aluminum oxide template in oxalic acid is on spot obtained by merging several individually scanned images together. Experiments show that this AFM system can offer high resolution and wide range AFM images even for large samples with remarkable capabilities in various environments.     

Stereoscopic display of atomic force microscope images using anaglyph techniques

This paper describes the use of a standard stereo-pair image display method for presenting the three-dimensional relief information found in atomic force microscope (AFM) images. The method makes use of commercially available image processing software packages. The techniques are illustrated on AFM images of the cuticle structure of a human hair fibre.     

卧式原子力显微镜的研制

提出原子力显微镜 (AFM)的新设计 ,讨论卧式 AFM的工作原理及其性能特点 ,简要介绍 AFM的控制电路系统及其图像扫描和图像处理软件系统 ,给出 AFM扫描获得的部分样品的图像结果。     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

Correction for piezoelectric creep in scanning probe microscopy images using polynomial mapping

We describe a method for using polynomial mapping to correct scanning probe microscope images for distortion due to piezoelectric creep. Because such distortion varies from image to image, this method can be used when the actual locations of some features within an image are known absolutely, or in a series of images in which the actual locations of some features are known not to vary. While the general case of polynomial mapping of degree N requires the determination of 2(N+ 1)2 matrix elements by regression, we find that by understanding the mechanism by which piezoelectric creep distorts scanning probe microscope images, we can fix most of these coefficients at 0 or 1 a priori, leaving only 2(N+ 1) coefficients to be determined by regression. We describe our implementation of this strategy using the Interactive Data Language (IDL) programming language, and demonstrate our technique on a series of atomic force microscopy (AFM) images of diblock copolymer microdomains. Using our simplified scheme, we are able to reduce the effects of distortion in an AFM image from 5% of the scan width to a single pixel, using only five reference points.     

Mixed lagrange time delay estimation autoregressive Wiener filter application for real-time SEM image enhancement

A new filter is developed for the enhancement of scanning electron microscope (SEM) images. A mixed Lagrange time delay estimation auto-regression (MLTDEAR)-based interpolator is used to provide an estimate of noise variance to a standard Wiener filter. A variety of images are captured and the performance of the filter is shown to surpass the conventional noise filters. As all the information required for processing is extracted from a single image, this method is not constrained by image registration requirements and thus can be applied in real-time in cases where specimen drift is presented in the SEM image.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

Computation of contrasts in atomic resolution electron spectroscopic images of planar defects in crystalline specimens

The image obtained in a conventional transmission electron microscope contains contributions from elastically and from inelastically scattered electrons. The electron spectroscopic imaging mode of an energy-filtering transmission electron microscope allows us to separate these two different contributions by inserting an energy-selecting slit in the energy-dispersive plane of an imaging energy filter. Selecting a specific energy loss corresponding to the ionization of the inner shell of a particular element one can obtain information on the distribution of the element within the specimen. The contrast is then caused by inelastically scattered electrons. For crystalline specimens, however, the contrast will be influenced additionally by the elastic contrast. This elastic contrast arises from electron diffraction and increases with increasing crystal thickness. Therefore the intensity distribution in the image cannot directly be interpreted as an elemental map. For a reliable interpretation of contrast formation in elemental maps it is therefore necessary to compute theoretical energy-loss images for various crystal thicknesses and compare these images with the experimental images. As an example we discuss the influence of electron diffraction effects on energy-loss images of two crystals with planar defects. Linescans are computed for various thicknesses of these crystals. Our calculations are performed using first-order perturbation theory to describe the transitions between the Bloch-wave states of the incident electron. The computed linescans for various crystal thicknesses show clearly that the influence of the elastic contrast on an image increases when we investigate thicker specimens. Furthermore, the comparison between elastic and energy-loss images demonstrates the partial preservation of the elastic contrast as a function of thickness. We find that for specimens thicker than about one third of the extinction length (here approximately 80-100 A) it is impossible to interpret an energy-loss image directly as elemental map.     

Anti-drift and auto-alignment mechanism for an astigmatic atomic force microscope system based on a digital versatile disk optical head

In this work, an anti-drift and auto-alignment mechanism is applied to an astigmatic detection system (ADS)-based atomic force microscope (AFM) for drift compensation and cantilever alignment. The optical path of the ADS adopts a commercial digital versatile disc (DVD) optical head using the astigmatic focus error signal. The ADS-based astigmatic AFM is lightweight, compact size, low priced, and easy to use. Furthermore, the optical head is capable of measuring sub-atomic displacements of high-frequency AFM probes with a sub-micron laser spot (~570 nm, FWHM) and a high-working bandwidth (80 MHz). Nevertheless, conventional DVD optical heads suffer from signal drift problems. In a previous setup, signal drifts of even thousands of nanometers had been measured. With the anti-drift and auto-alignment mechanism, the signal drift is compensated by actuating a voice coil motor of the DVD optical head. A nearly zero signal drift was achieved. Additional benefits of this mechanism are automatic cantilever alignment and simplified design.     

Scanning image detection (SID) system for conventional transmission electron microscope (CTEM) images

A new image detection system has been developed to display transmission electron microscope (TEM) images on a CRT without a video camera system. Deflection coils placed in both the upper space of an objective lens and in the lower space of the first intermediate lens scan a small electron probe simultaneously. The electrical signal acquired through an improved scintillator and a photomultiplier is synchronized with the scanning signal and displayed in a similar fashion to a conventional scanning TEM (STEM) instrument. A preliminary system using a 100 kV conventional TEM (CTEM) equipped with a hairpin-type electron gun, produced an image with a spatial resolution of 1 nm.     

Automated image analysis of atomic force microscopy images of rotavirus particles

A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.     

Atomic force microscopy of histological sections using a new electron beam etching method

In order to examine histological sections of the rat vomeronasal epithelium with the atomic force microscope (AFM), we developed an electron beam etching method that improves the resolution of AFM images. This method results in AFM images comparable to those obtained with the transmission electron microscope (TEM). Ultrathin tissue sections embedded in epoxy resin were observed before and after the treatment with electron beam radiation. Before electron beam treatment, epithelial structures such as the microvilli surface, dendritic processes, the supporting cell layers and the neuronal cell layers were all visible using the AFM. However, only a few subcellular structures could also be resolved. The AFM images were not as clear as those obtained with the TEM. After electron beam treatment, however, the resolution of AFM images was greatly improved. Most of the subcellular structures observed in TEM images, including the inner membrane of mitochondria, ciliary-structure precursor body, junctional complexes between the neurons and supporting cells, and individual microvilli were now visible in the AFM images. The electron beam treatment appeared to melt the embedding resin, bringing subcellular structures into high relief. The result of this study suggests that electron beam etching of histological samples may provide a new method for the study of subcellular structure using the AFM.     

Three-channel false colour AFM images for improved interpretation of complex surfaces: A study of filamentous cyanobacteria

Imaging signals derived from the atomic force microscope (AFM) are typically presented as separate adjacent images with greyscale or pseudo-colour palettes. We propose that information-rich false-colour composites are a useful means of presenting three-channel AFM image data. This method can aid the interpretation of complex surfaces and facilitate the perception of information that is convoluted across data channels. We illustrate this approach with images of filamentous cyanobacteria imaged in air and under aqueous buffer, using both deflection-modulation (contact) mode and amplitude-modulation (tapping) mode. Topography-dependent contrast in the error and tertiary signals aids the interpretation of the topography signal by contributing additional data, resulting in a more detailed image, and by showing variations in the probe-surface interaction. Moreover, topography-independent contrast and topography-dependent contrast in the tertiary data image (phase or friction) can be distinguished more easily as a consequence of the three dimensional colour-space.     

MEMS-based fast scanning probe microscopes

Scanning probe microscopy is a frequently used nanometer-scale surface investigation technique. Unfortunately, its applicability is limited by the relatively low image acquisition speed, typically seconds to minutes per image. Higher imaging speeds are desirable for rapid inspection of samples and for the study of a range of dynamic surface processes, such as catalysis and crystal growth. We have designed a new high-speed scanning probe microscope (SPM) based on micro-electro mechanical systems (MEMS). MEMS are small, typically micrometer size devices that can be designed to perform the scanning motion required in an SPM system. These devices can be optimized to have high resonance frequencies (up to the MHz range) and have very low mass (10⁻¹¹ kg). Therefore, MEMS can perform fast scanning motion without exciting resonances in the mechanical loop of the SPM, and hence scan the surface without causing the image distortion from which conventional piezo scanners suffer. We have designed a MEMS z-scanner which we have integrated in commercial AFM (atomic force microscope) and STM (scanning tunneling microscope) setups. We show the first successful AFM experiments.     

Thermal, sonochemical, and mechanical behaviors of single crystal [60]fullerene nanotubes

Although related to conventional carbon nanotubes in both shape and construction, fullerene nanowhiskers and fullerene nanotubes have received far less attention. A modified liquid-liquid interfacial precipitation technique is described to produce relatively uniform batches of [60]fullerene nanotubes in high yield. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) reveal that the tubes possess approximately 100-nm inside diameters and 300-nm outside diameters. The [60]fullerene nanotubes degrade slowly at 180 degrees C, eventually collapsing into micron scale [60]fullerene discs and rods, as revealed by optical microscopy and AFM. Ultrasonic cavitation chops [60]fullerene nanotubes into smaller segments within seconds. Longer ultrasonic bathing leads to considerable structural damage in which the sidewalls rupture. Mechanical stress tests using an AFM microscope tip effectively dent and break [60]fullerene nanowhiskers, revealing a hollow interior.